Nicotine dihydrogen ditartrate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03-June-2020 to 03-Sept-2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The study was performed in accordance with standard test protocol (OECD 487) 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:

2020

Report date:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

Nicotine dihydrogen ditartrate

EC Number:

200-607-2

EC Name:

Nicotine dihydrogen ditartrate

Cas Number:

65-31-6

Molecular formula:

C10-H14-N2.2C4-H6-O6

IUPAC Name:

nicotine dihydrogen ditartrate

Test material form:

solid: particulate/powder

Details on test material:

Lot No: 1937H039
Production Date: 17-Sept-2019
Retest Date: 17-Sept-2024

Specific details on test material used for the study:

Batch no. 1937H039
Source of test item CONTRAF-NICOTEX-TOBACCO GmbH
Production date 17. Sep. 2019
Expiry date 17. Sep. 2024

A certificate of analysis was provided by the sponsor and is attached (in copy) to this final report.

Method

Species / strain

Species / strain / cell type:

lymphocytes: human, obtained from pheripheral blood samples

Details on mammalian cell type (if applicable):

For each experiment, sufficient whole blood was drawn from the peripheral circulation of a non smoking volunteer (18-35) who had been previously screened for suitability. The volunteer had not knowingly been exposed to high levels of radiation or hazardous chemicals and had not knowingly recently suffered from a viral infection. For each experiment, one appropriate donor will be chosen.

The details of the donors used are:
Preliminary Toxicity Test: male, aged 28 years
Main Experiment: female, aged 28 years

Cytokinesis block (if used):

Cytochalasin B

Metabolic activation:

with and without

Metabolic activation system:

standard metabolizing system (liver S9 mix from male rats, treated with Phenobarbital-5,6 and Benzoflavone) + supplementation with co-factors

Test concentrations with justification for top dose:

The dose levels used in the Main Experiment were selected using data from the preliminary toxicity test where the results indicated that the maximum concentration will be based on the maximum recommended dose level according to the test guideline. The dose levels selected for the main test were as follows:
Final concentration of the test item Nicotine Bitartrate Dihydrate:

Experiment I - Exposure 4 h
With and without S9-mixture
0, 0.13, 0.25, 0.5, 1, 2 mg/mL

Experiment II - Exposure 23.5 h
Without S9-mixture
0, 0.13, 0.25, 0.5, 1, 2 mg/mL

Vehicle / solvent:

minimal culture medium (MCM)

Controls

Negative solvent / vehicle controls:

yes

Remarks:

minimal culture medium (MCM) for test item, 0.9% NaCl for positive controls

Positive controls:

yes

Positive control substance:

colchicine

cyclophosphamide

mitomycin C

Remarks:

Cyclophosphamide monohydrate (CPA) as agent requiring metabolic activation, Mitomycin C (MMC) as agent not requiring metabolic activation in short exposure period, Colchicine as agent non requiring metabolic activation in extended exposure period

Details on test system and experimental conditions:

Please see "Any other information on materials and methods incl. tables"

Evaluation criteria:

The following applies once all acceptability criteria are met.

The test item is considered unable to induce chromosome breaks and/or loss in this test system (negative result) if, in all experimental conditions, the following criteria are met:
• 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, considering also e.g. 95.5 % control limits where appropriate.
The test item is considered able to induce chromosome breaks and/or loss in this test sys-tem (positive result) if, in any of the experimental conditions, all of the following criteria are met:
• At least one test concentration shows a statistically significant increase of micronu-cleate cells compared to the concurrent solvent control.
• In at least one experimental condition a dose-related increase of micronucleate cells can be observed using trend analysis.
• Any of the results lies outside the range of the historical laboratory control data for solvent controls, considering also e.g. 95.5 % control limits where appropriate.

Statistical significance was confirmed by means of the non-parametric χ2 test or Fisher’s exact test for smaller values in the fourfold table. A linear regression (least squares) was performed to assess a possible dose dependent increase of binucleated cells with micro-nuclei. Both biological and statistical significance were considered together.

Statistics:

The number of binucleated cells with micronuclei in each treatment group was compared with the solvent control. Statistical significance was tested using Fisher’s exact test at the five per cent level (p < 0.05). For positive controls with high values of binucleated cells with micronuclei, the chi-square-test was used.

Results and discussion

Test results

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:

valid

Positive controls validity:

valid

Any other information on results incl. tables

Osmolality and pH

The osmolality and the pH values of the solvent controls, the positive controls and the test item were determined to exclude a negative influence on the assay by those parameters. The results are summarized in the following table:

Sample	Osmolality [mosmol/kg]	pH-Value
MCM (minimal culture medium)	263	7.292
MCM + 0.9% NaCl [0.5% v/v]	264	7.286
MCM + CPA [30 µg/mL]	265	7.288
MCM + MMC [0.3 µg/mL]	266	7.288
MCM + test item [2 mg/mL]	269	6.676
MCM + test item [1 mg/mL]	267	6.999
MCM + test item [0.5 mg/mL]	266	7.155
MCM + test item [0.25 mg/mL]	265	7.207
MCM + test item [0.13 mg/mL]	265	7.215

Note: Values in brackets indicate the final test concentration.

None of the tested positive controls or the test item provoked a critical change of the osmolality in comparison to the solvent controls. The pH values of the two highest test item concentrations were slightly decreased, but neither relevant cytotoxicity nor genotoxicity were observed. Therefore, a negative influence of these parameters on the assay is not to be expected.

 

 Experiment I (with and without S9-Mix)

The results of the pre-experiment (which was evaluated as exp. I) are presented in the following table.

Experiment I: Cytotoxicity

Treatment	Precipitation		Haemo-lysis			Mean CBPI		Mean Cytotoxicity [%]^#)
Without S9 mix
Solvent control MCM	no			no		1.788		--
Solvent control 0.9% NaCl 0.5% v/v	no			no		1.791		--
Positive control MMC 0.3 µg/mL	no			no		1.484		17.1
Test item 2 mg/mL	no			no		1.793		-0.3
Test item 1 mg/mL	no			no		1.756		1.8
Test item 0.5 mg/mL	no			no		1.721		3.7
Test item 0.25 mg/mL	no			no		1.796		-0.5
Test item 0.13 mg/mL	no			no		1.727		3.4
With S9 mix
Solvent control MCM		no			no		1.857	--
Solvent control 0.9% NaCl 0.5% v/v		no			no		1.801	--
Positive control CPA 30 µg/mL		no			no		1.364	24.2
Test item 2 mg/mL		no			no		1.835	1.2
Test item 1 mg/mL		no			no		1.899	-2.3
Test item 0.5 mg/mL		no			no		1.861	-0.2
Test item 0.25 mg/mL		no			no		1.875	-1.0
Test item 0.13 mg/mL		no			no		1.838	1.0

^#)negative values indicate a lower cytotoxicity as compared to concurrent controls

 The concentrations to be evaluated for genotoxicity were selected on this base.

The results of the evaluated concentrations in experiment I are presented in the following table.

Experiment I: Genotoxicity

Treatment	Mean Cytotoxicity [%]^#)	Total No. of BINC examined	Total No. of MBNC	MBNC [%]
Without S9 mix
Solvent control MCM	--	2010	10	0.50
Solvent control 0.9% NaCl 0.5% v/v	--	2008	8	0.40
Positive control MMC 0.3 µg/mL	17.1	2005	223	11.12**
Test item 2 mg/mL	-0.3	2005	5	0.25
Test item 1 mg/mL	1.8	2008	8	0.40
Test item 0.5 mg/mL	3.7	2007	7	0.35
With S9 mix
Solvent control MCM	--	2007	7	0.35
Solvent control 0.9% NaCl 0.5% v/v	--	2005	1	0.05
Positive control CPA 30 µg/mL	24.2	2014	112	5.56**
Test item 2 mg/mL	1.2	2011	11	0.55
Test item 1 mg/mL	-2.3	2008	8	0.40
Test item 0.5 mg/mL	-0.2	2013	12	0.60

Asterisks indicate statistically significant differences to solvent control, with * p < 0.05, ** p < 0.01

^#)negative values indicate a lower cytotoxicity as compared to concurrent controls

 

In experiment I without and with metabolic activation, no cytotoxicity was observed at any of the test item concentrations.

Neither a statistically significant nor a concentration-related increase of binucleated cells with micronuclei was detected at the evaluated concentrations. All values were in the range of the historical laboratory control data for the concurrent solvent control MCM. Therefore, a second experiment (experiment II without metabolic activation, extended exposure) was performed.

 Experiment II (without S9-Mix)

The results of experiment II are presented in the following tables:

Experiment II: Cytotoxicity

Treatment	Precipitation	Haemo-lysis	Mean CBPI	Mean Cytotoxicity [%]
Solvent control MCM	no	no	1.863	--
Solvent control 0.9% NaCl 0.5% v/v	no	no	1.844	--
Positive control MMC 0.3 µg/mL	no	no	1.444	21.7
Positive control Colchicine 0.035 µg/mL	no	no	1.187	35.6
Test item 2 mg/mL	no	no	1.639	12.0
Test item 1 mg/mL	no	no	1.753	5.9
Test item 0.5 mg/mL	no	no	1.701	8.7
Test item 0.25 mg/mL	no	no	1.827	2.0
Test item 0.13 mg/mL	no	no	1.750	6.1

 The concentrations to be evaluated for genotoxicity were selected on this base.

 The results of the evaluated concentrations in experiment II are presented in the following table:

 Experiment II: Genotoxicity

Treatment		Mean Cytotoxicity [%]		Total No. of BINC examined		Total No. of MBNC		MBNC [%]
Without S9 mix
Solvent control MCM		--		2010		10		0.50
Solvent control 0.9% NaCl 0.5% v/v		--		2012		9		0.45
Positive control MMC 0.3 µg/mL		21.7		2009		158		7.86**
Positive control Colchicine 0.035 µg/mL		35.6		2007		85		4.24**
Test item 2 mg/mL		12.0		2014		14		0.70
Test item 1 mg/mL		5.9		2021		18		0.89
Test item 0.5 mg/mL		8.7		2013		13		0.65

Asterisks indicate statistically significant differences to solvent control, with * p < 0.05, ** p < 0.01

In experiment II, no relevant cytotoxic effect was observed.

Neither a statistically significant nor a concentration-related increase of binucleated cells with micronuclei was detected at the evaluated concentrations. All values lay in the range of the historical laboratory control data for the concurrent solvent control MCM.

  

Discussion

This study was performed to assess the potential of Nicotine bitartrate dihydrate to induce formation of micronuclei in human lymphocytes cultured in vitro in absence and presence of an exogenous metabolic activation system (liver S9 mix from male rats, treated with Phenobarbital-5,6 and Benzoflavone).

For the analysis of the genotoxic potential of the test item, 5 concentrations were tested in duplicate cultures of human lymphocytes in two independent and valid experiments. Three concentrations were chosen for evaluation of genotoxicity. A minimum of 1000 binucleated cells was evaluated per culture and scored for the presence of micronuclei. The recorded values were compared with a negative control (solvent only, in this case serum free cell culture medium).

The study is considered acceptable for the following reasons: Micronucleus induction of the solvent controls was in the range of the historical control data/literature data. The positive control compounds Mitomycin C (0.3 µg/mL), CPA (30 µg/mL) and Colchicine (0.035 µg/mL) showed distinct increases in the number of binucleated cells with micronuclei, demonstrating the sensitivity of the test system.

In both independent experiments, no relevant cytotoxicity was observed at the evaluated test item concentrations.

Neither a statistically significant nor a concentration-related increase of binucleated cells with micronuclei was detected at the evaluated concentrations. All values were in the range of the historical laboratory control data for the concurrent solvent control MCM. Therefore, all criteria for a negative result were met.

In conclusion, under the experimental conditions reported,Nicotine bitartrate dihydratedid not induce the formation of micronuclei in human lymphocytes in vitro up to and includingthe maximum concentration prescribed by the testing guideline.

The result of the micronucleus test with the test itemNicotine bitartrate dihydrateis considered as “negative” under the conditions of the test.

Applicant's summary and conclusion

Conclusions:

In conclusion, under the experimental conditions reported, Nicotine bitartrate dihydrate did not induce the formation of micronuclei in human lymphocytes in vitro up to and including the highest test concentration prescribed by the testing guideline.
The result of the micronucleus test with the test item Nicotine bitartrate dihydrate is considered as “negative” under the conditions of the test.

Executive summary:

This study was performed to assess the potential of Nicotine bitartrate dihydrate to induce formation of micronuclei in human lymphocytes cultured in vitro in absence and presence of an exogenous metabolic activation system (liver S9 mix from male rats, treated with Phenobarbital-5,6 and Benzoflavone).

The test item was dissolved in minimal culture medium to prepare a stock solution with a concentration of 20 mg/mL, corresponding to the highest concentration (2 mg/mL) in the test. In addition, a geometric series of dilutions was prepared from the stock solution.

Two valid and independent experiments were performed.

Human peripheral blood lymphocytes in whole blood culture were stimulated to divide by addition of phytohaemagglutinin and exposed to medium control, solvent control, test item and positive control. All cell cultures were set up in duplicates. After exposure and harvesting, slides were prepared and stained. In order to assess the toxicity of the test item to the cultivated human lymphocytes, the cytokinesis-block proliferation index (CBPI) was calculated for all evaluable cultures. On the basis of these data, the appropriate concentrations were selected to determine the proportion of binucleated cells containing micronuclei.

In both independent experiments, no relevant cytotoxicity was observed up to and including the maximum concentration prescribed by the testing guideline.

Neither a statistically significant nor a concentration-related increase of binucleated cells with micronuclei was detected at the evaluated concentrations. All values lay in the range of the historical laboratory control data for the concurrent solvent control MCM. Therefore, all criteria for a negative result were met.

The study is considered acceptable: micronucleus induction of the solvent controls was in the range of the historical control data/literature data. All positive control compounds caused high, statistically significant increases in the proportion of binucleated cells with micronuclei, demonstrating the sensitivity of the test system.

In conclusion, under the experimental conditions reported, Nicotine bitartrate dihydratedid not induce the formation of micronuclei in human lymphocytes in vitro up to and including the highest test concentration prescribed by the testing guideline.

The result of the micronucleus test with the test item Nicotine bitartrate dihydrateis considered as “negative” under the conditions of the test.