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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information
Based on the results of this study it is concluded that TRANTOS is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.
Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2013-02-07 to 2013-03-21
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to GLP, OECD guideline 471 and EU Method B.13/14 with only one minor deviation in temperature which did not adversely affect the study integrity.
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
The temperature was recorded to be outside the range of 37.0 ± 1.0°C as specified in the protocol for approximately 2 hours in the dose range finding study (with a maximum of 39.8°C) and second mutation experiment (with a maximum of 39.4°C).
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
yes
Remarks:
The temperature was recorded to be outside the range of 37.0 ± 1.0°C as specified in the protocol for approximately 2 hours in the dose range finding study (with a maximum of 39.8°C) and second mutation experiment (with a maximum of 39.4°C).
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
- Type and identity of media:
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
- Type and identity of media:
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9-mix (rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone).
Test concentrations with justification for top dose:
3, 10, 33, 100, 333, 1000, 3330, 5000 μg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Untreated negative controls:
yes
Remarks:
DMSO
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
yes
Positive controls:
yes
Remarks:
See table below
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); Preparation of bacterial cultures: Samples of frozen stock cultures of bacteria were transferred into enriched nutrient broth and incubated in a shaking incubator (37°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 in Vogel-Bonner Medium E, 20 g glucose. The agar plates for the test with the Salmonella typhimurium strains also contained 12.5 μg/plate biotin and 15 μg/plate histidine and the agar plates for the test with the Escherichia coli strain contained 15 μg/plate tryptophan. Top agar: Milli-Q water containing 0.6% (w/v) bacteriological agar and 0.5% (w/v) sodium chloride 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.

DURATION
- Preincubation period: 48-72 hours
- Exposure duration: Not reported
- Expression time (cells in growth medium): 48 ± 4 h


NUMBER OF REPLICATIONS: 3 replicate plates

NUMBER OF CELLS EVALUATED: The revertant colonies (histidine independent or tryptophan independent) were counted manually if less than 40 colonies per plate were present. If more than 40 colonies were present, these were counted automatically with a Biocount 4000 Pro-S-colony counter.

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
Evaluation criteria:
Acceptability of the assay: A Salmonella typhimurium reverse mutation assay and/or Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:
a) The negative control data (number of spontaneous revertants per plate) should be within the laboratory historical range for each tester strain.
b) The positive control chemicals should produce responses in all tester strains, which are within the laboratory historical range documented for each positive control substance. Furthermore, the mean plate count should be at least three times the concurrent vehicle control group mean.
c) 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.

Data evaluation and statistical procedures: No formal hypothesis testing was done.
A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA 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 substance is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA 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.
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: (Dose range finding study) Precipitation of TRANTOS on the plates was observed at the start and at the end of the incubation period at concentrations of 1000 μg/plate and upwards. (Mutation study) Precipitation of TRANTOS on the plates was observed at the start and at the end of the incubation period at the concentration of 1000 μg/plate.
- Other confounding effects:

RANGE-FINDING/SCREENING STUDIES:
- TRANTOS was dissolved in dimethyl sulfoxide (DMSO) at concentrations of 1 mg/mL and below. At concentrations of 3.3 mg/mL and higher TRANTOS was suspended in dimethyl sulfoxide. In both mutation experiments, TRANTOS was dissolved in dimethyl sulfoxide up to the highest dose level of 10 mg/mL. The stock solution was treated with ultrasonic waves to obtain a homogeneous suspension (dose range finding) or until the test substance had completely dissolved (mutation experiments). Test substance concentrations were used within 2.5 hours after preparation.
-In the dose range finding test, TRANTOS was tested up to concentrations of 5000 μg/plate in the absence and presence of S9-mix in the strains TA100 and WP2uvrA. TRANTOS precipitated on the plates at dose levels of 1000 μg/plate and upwards. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed. Results of this dose range finding test were reported as part of the first experiment of the mutation assay.

COMPARISON WITH HISTORICAL CONTROL DATA: 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.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Summary Table of Experiment 1: Mutagenic response of TRANTOS in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay
  TA1535  TA1537  TA98  TA100  WP2uvrA
Without S9-Mix
Positive control 829 ± 11  532 ± 27  956 ± 32  903 ± 36  1151 ± 41
Solvent control  8 ± 1  4 ± 1  18 ± 2  93 ± 6  27 ± 5
3 N/A N/A N/A  93 ± 12 21 ± 2
10 9 ± 4  5 ± 2  22 ± 3  108 ± 6  26 ± 8
33 7 ± 2 5 ± 2 16 ± 2 88 ± 10 23 ± 9
100 8 ± 1 4 ± 3 26 ± 4 102 ± 20 18 ± 1
333 6 ± 3 4 ± 1 15 ± 4 98 ± 10 29 ± 8
1000 SP 5 ± 3 4 ± 2 18 ± 2 97 ± 6 20 ± 1
3330 MP N/A N/A N/A 97 ± 11 24 ± 2
5000 MP N/A N/A N/A 93 ± 12 27 ± 3
  TA1535  TA1537  TA98  TA100  WP2uvrA
With S9-Mix1
Positive control 422 ± 41  416 ± 29  927 ± 49 1198 ± 91  342 ± 20
Solvent control  8 ± 2  5 ± 3 22 ± 4  92 ± 7  24 ± 4
3 N/A N/A N/A  93 ± 13 29 ± 3
10 11 ± 1  4 ± 1 28 ± 4  109 ± 9  35 ± 9
33 9 ± 3 5 ± 1 26 ± 4 109 ± 4 32 ± 2
100 6 ± 2 5 ± 3 26 ± 4 99 ± 3 38 ± 4
333 6 ± 1 6 ± 1 17 ± 4 113 ± 12 30 ± 8
1000 SP 7 ± 2 7 ± 2 21 ± 4 117 ± 7 30 ± 9
3330 MP N/A N/A N/A 103 ± 5 36 ± 2
5000 MP N/A N/A N/A 108 ± 14 32 ± 2
Summary Table of Experiment 2: Mutagenic response of TRANTOS in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay
  TA1535  TA1537  TA98  TA100  WP2uvrA
Without S9-Mix
Positive control 979 ± 33 629 ± 22 1141 ± 42 1028 ± 25 1263 ± 25
Solvent control 10 ± 3 9 ± 3 23 ± 4 127 ± 11 24 ± 2
10 9 ± 3 10 ± 4 20 ± 4 140 ± 4 22 ± 4
33 9 ± 2 10 ± 3 21 ± 2 138 ± 38 19 ± 4
100 10 ± 2 6 ± 4 26 ± 3 127 ± 18 21 ± 5
333 11 ± 3 9 ± 2 21 ± 4 142 ± 7 19 ± 2
1000 SP 9 ± 1 6 ± 3 16 ± 2 124 ± 11 28 ± 4
  TA1535  TA1537  TA98  TA100  WP2uvrA
With S9-Mix1
Positive control 260 ± 16 516 ± 26 485 ± 63 1262 ± 56 305 ± 23
Solvent control 12 ± 4 14 ± 3 22 ± 2 101 ± 30 26 ± 9
10 10 ± 3 9 ± 3 30 ± 4 132 ± 9 26 ± 4
33 13 ± 4 9 ± 2 24 ± 3 145 ± 11 23 ± 4
100 11 ± 4 13 ± 3 28 ± 3 127 ± 14 25 ± 7
333 10 ± 5 7 ± 3 31 ± 7 133 ± 14 23 ± 1
1000 SP 7 ± 1 9 ± 3 29 ± 6 117 ± 19 28 ± 3
Notes: 1: The S9-mix contained 10% (v/v) S9 fraction
SP: Slight precipitation
MP: Moderate Precipitation
Conclusions:
Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

Based on the results of this study it is concluded that TRANTOS is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

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. Based on the results of this study it is concluded that TRANTOS is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.


Justification for selection of genetic toxicity endpoint
The study was conducted according to GLP, OECD guideline 471 and EU Method B.13/14 with only one minor deviation in temperature which did not adversely affect the study integrity.

Justification for classification or non-classification

Based on these results: - according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2011), TRANTOS is not classified as mutagenic. - according to the Regulation (EC) No 1272/2008 on classification, labelling and packaging of substances and mixtures, TRANTOS does not have to be classified and has no obligatory labelling requirement for mutagenicity.