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Toxicological information

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
Study period:
2016-09-13 to 2016-09-22
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
The study was performed in accordance with standard test protocols (OECD 471 resp. EU B.13/14) in a quality controlled laboratory. The study is valid according to criteria mentioned in the test protocols.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
powder
Details on test material:
white or almost white powder

Method

Species / strain
Species / strain / cell type:
S. typhimurium, other: TA97a, TA98, TA100, TA 102, TA1535
Metabolic activation:
with and without
Metabolic activation system:
S9-Mix, produced from the livers of male Sprague-Dawley rats which were treated with 500 mg Aroclor 1254/kg body weight intraperitoneally.
Test concentrations with justification for top dose:
First experiment: 5000 / 1500 / 500 / 150 / 50 μg/plate
Second experiment: 5000 / 2500 / 1250 / 625 / 313 / 156 μg/plate
Vehicle / solvent:
Demineralised 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 in the tested concentrations.
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
sodium azide
benzo(a)pyrene
other: 2-Amino-Anthracene (with metabolic activation), 4-Nitro-1,2-phenylene Diamine (without metabolic activation)
Details on test system and experimental conditions:
GENERAL PREPARATION

Per strain and dose, three plates with and three plates without S9 mix were used.
The test item solutions were prepared according to chapter 6.1.3
Top agar basis was melted in a microwave oven, after melting, 10 mL of histidine-biotinsolution
0.5 mM per 100 mL basis was added and the bottle was placed in the water bath
at 43 ±1 °C.


METHOD OF APPLICATION:

1. Plate incorporation method

The following materials were gently vortexed in a test tube and poured onto the selective
agar plates:
- 100 μL test solution at each dose level, solvent (negative control) or reference mutagen
solution (positive control)
- 500 μL S9 mix (see chapter 6.4.18, for test with metabolic activation) or phosphate
buffer (for test without metabolic activation).
- 100 μL bacteria suspension (see chapter 6.3.2, test system, culture of the strains)
- 2000 μL overlay agar (top agar)
The plates were closed and left to solidify for 10 minutes, then inverted and placed in the
dark incubator at 37 ±1 °C.

2. Pre-incubation method

The following materials were gently vortexed in a test tube and incubated at 37 ±1°C for
20 minutes:
- 100 μL test suspension at each dose level, solvent (negative control) or reference
mutagen solution (positive control)
- 500 μL S9 mix (see chapter 6.4.18, for test with metabolic activation) or phosphate
buffer (for test without metabolic activation).
- 100 μL bacteria suspension (see chapter 6.3.2, test system, culture of the strains)
After pre-incubation, 2000 μL overlay agar (top agar) was added, the tube was gently vortexed
and the mixture was poured onto the selective agar plate.
The plates were closed and left to solidify for 10 minutes, then inverted and placed in the
dark incubator at 37 ±1 °C.

- Cell density at seeding (if applicable):at least 10^9 cells/mL

TOXICITY CONTROL
Performed in experiment 1 only analogously to the titre control with the maximum dose of
test item with and without S9 on maximal-soft agar, two replicates with and without metabolic
activation, incubation for 48 hours at 37 ±1°C.
Evaluation criteria:
The colonies were counted visually and the numbers were recorded. A spreadsheet software
(Microsoft Excel®) was used to calculate mean values and standard deviations of
each treatment, solvent control and positive control.
The mean values and standard deviations of each threefold determination was calculated
as well as the increase factor f(l) of revertant induction (mean revertants divided by mean
spontaneous revertants) of the test item solutions and the positive controls. Additionally,
the absolute number of revertants (Rev. Abs.) (mean revertants minus mean spontaneous
revertants) was given.
A substance is considered to have mutagenic potential, if a reproducible increase of revertant
colonies per plate exceeding an increase factor of 2 in at least one strain can be
observed. A concentration-related increase over the range tested is also taken as a sign of
mutagenic activity.

Results and discussion

Test results
Species / strain:
other: S. typhimurium TA97a, TA98, TA100, TA102, TA1535
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

Any other information on results incl. tables

First Experiment

Confirmation of the Criteria and Validity

All strains met the criterion of at least 109 bacteria/mL, and no inconsistencies were found in the sterility control. All determined values for the spontaneous revertants of the negative controls were in the normal range of the test laboratory (historical data of the laboratory see chapter 15, page 36). All positive controls (diagnostic mutagens) showed mutagenic effects with and without metabolic activation and were within the historical control data ranges.

Solubility and Toxicity

In the first experiment, the test item showed no precipitates on the plates in all tested concentrations. No signs of toxicity towards the bacteria strains could be observed. The bacterial background lawn was visible and not affected. The number of revertant colonies was not reduced.

Mutagenicity

No significant increase of the number of revertant colonies in the treatments with and without metabolic activation could be observed. No concentration-related increase over the tested range was found.

Therefore, the test item is stated as not mutagenic under the test conditions. To verify this result, a further experiment was performed.

Second Experiment

Confirmation of the Criteria and Validity

All strains met the criterion of at least 109 bacteria/mL, and no inconsistencies were found in the sterility control. Nearly all determined values for the spontaneous revertants of the negative controls (only one exception) were in the normal range of the test laboratory (historical data of the laboratory see chapter 15, page 36). All positive controls (diagnostic mutagens) showed mutagenic effects with and without metabolic activation and were within the historical control data ranges.

Solubility and Toxicity

In the second experiment, the test item showed no precipitates on the plates in all tested concentrations.

No signs of toxicity towards the bacteria strains could be observed. The bacterial background lawn was visible and not affected. The number of revertant colonies was not reduced.

Mutagenicity

No significant increase of the number of revertant colonies in the treatments with and without metabolic activation could be observed. No concentration-related increase over the tested range was found.

Therefore, the test item is stated as not mutagenic under the test conditions.

Applicant's summary and conclusion

Conclusions:
Interpretation of results:
-negative with metabolic activation
-negative without metabolic activation

The test item Nicotine bitartrate dihydrate showed no increase in the number of revertants in all bacteria strains in both experiments. Nearly all negative and all 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 Nicotine bitartrate dihydrate is not mutagenic in the Salmonella typhimurium test strains TA97a, TA98, TA100, TA102 and TA1535 in the absence and presence of metabolic activation under the experimental conditions in the present study.

Acceptability of the study:
In all experiments, no precipitation of the test item was observed at any of the tested concentrations up to 5000 μg/plate.
In both experiments, the test item caused no cytotoxicity towards all bacteria strains.
The confirmation tests of the genotype did not show any irregularities. The control of the titre was above the demanded value of 109 bacteria/mL.
Nearly all of the means of all replicates of the spontaneous revertants (in negative and solvent controls) were within the range of the historical data of the test facility.
All numbers of revertant colonies of the positive controls were within the range of the historical data of the laboratory and were increased in comparison with the negative controls, which demonstrated the mutagenic potential of the diagnostic mutagens. Since all criteria for acceptability have been met, the study is considered valid.
Executive summary:

Two valid experiments were performed.

The study procedures described in this report were based on the most recent OECD and EC guidelines.

The test item Nicotine bitartrate dihydrate was tested in the Salmonella typhimurium reverse mutation assay with five strains of Salmonella typhimurium (TA97a, TA98, TA100, TA102 and TA1535). The test was performed in two experiments in the presence and absence of S9-mix (rat liver S9-mix induced by Aroclor 1254).

In the first experiment, Nicotine bitartrate dihydrate (dissolved in demin. water) was tested up to concentrations of 5000 μg/plate in the absence and presence of S9-mix (0.74 % final concentration in the treatment) in the strains TA97a, TA98, TA100, TA102 and TA1535

using the plate incorporation method.

Nicotine bitartrate dihydrate showed no precipitates on the plates at any of the concentrations and no toxicity towards any of the bacteria strains was observed.

The test item showed no increase in the number of revertants in all tested strains, in the presence and the absence of metabolic activation.

On the base of the first experiment, Nicotine bitartrate dihydrate was tested up to concentrations of 5000 μg/plate in the absence and presence of S9-mix (0.74% final concentration in the treatment) in all bacteria strains using the pre-incubation method.

Nicotine bitartrate dihydrate showed no precipitates on the plates at any of the concentrations and showed no toxicity towards any of the bacteria strains. The test item showed no increase in the number of revertants in all tested strains, in the presence and the absence of metabolic activation.

Based on the results of this study it is concluded that Nicotine bitartrate dihydrate is not mutagenic in the Salmonella typhimurium strains TA97a, TA98, TA100, TA102 and TA1535 in the absence and presence of metabolic activation under the experimental conditions in this study.