Nicotine

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Two studies with Klimisch 2 (Ames tests) , one HPRT test (Klimisch 1) and one micronucleus test (Klimisch 1) are assessed as key studies for nicotine.

No aneugenic, mutagenic or clastogenic effects were observed in vitro in these tests.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23-06-2017 to 26-09-2017

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.

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

Species / strain / cell type:

human lymphoblastoid cells (TK6)

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. Based on over 20 years in house data for cell cycle times for lymphocytes using BrdU (bromodeoxyuridine) incorporation to assess the number of first, second and third division metaphase cells to calculate the average generation time (AGT) for human lymphocytes it is considered to be approximately 16 hours. Therefore using this average the in-house exposure time for the experiments for 1.5 x AGT is 24 hours.
The details of the donors used are:
Preliminary Toxicity Test: male, aged 24 years
Main Experiment: male, aged 28 years

Cytokinesis block (if used):

Cytochalasin B

Metabolic activation:

with and without

Metabolic activation system:

standard metabolizing system (S9)

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 should be limited on the maximum recommended dose level. The dose levels selected for the Main Test were as follows:
Groups & Final concentration of test item (S)-Nicotine (µg/mL):

4-hour without S9, 4-hour with S9 (2%), 24-hour without S9: 0, 202.5, 303.75, 405, 607.5, 810, 1215, 1620

Negative solvent / vehicle controls:

yes

Remarks:

MEM

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

not specified

Details on test system and experimental conditions:

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

Evaluation criteria:

Providing that all of the acceptability criteria are fulfilled, a test item is considered to be clearly negative if, in most/all of the experimental conditions examined:
1. None of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
2. There is no dose-related increase.
3. The results in all evaluated dose groups should be within the range of the laboratory historical control data.
Providing that all of the acceptability criteria are fulfilled, a test item may be considered to be clearly positive, if in any of the experimental conditions examined, there is one or more of the following applicable:
1. At least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
2. There is an increase which can be considered to be dose-related.
3. The results are substantially outside the range of the laboratory historical negative control data.
When all the criteria are met, the test item is considered able to induce chromosome breaks and/or gain or loss in this test system.
There is no requirement for verification of a clear positive or negative response.
In case the response is neither clearly negative nor clearly positive as described above or in order to assist in establishing the biological relevance of a result, the data should be evaluated by expert judgement and/or further investigations. The Study Scoring additional cells (where appropriate) or performing a repeat experiment possibly using modified experimental conditions could be useful.
Test items that induce micronuclei in the MNvit test may do so because they induce chromosome breakage, chromosome loss, or a combination of the two. Further analysis using anti-kinetechore antibodies, centromere specific in situ probes, or other methods can be used to determine whether the mechanism of micronucleus induction is due to clastogenic and/or aneugenic activity.

Statistics:

The frequency of binucleate cells with micronuclei was compared, where necessary, with the concurrent vehicle control value using the Chi-squared Test on observed numbers of cells with micronuclei. Other statistical analyses may be used if appropriate (Hoffman et al., 2003). A toxicologically significant response was recorded when the p value calculated from the statistical analysis of the frequency of binucleate cells with micronuclei was less than 0.05 and there was a dose-related increase in the frequency of binucleate cells with micronuclei which was reproducible.

Major Computerized Systems
The following Major Computerized systems were used in this study:
• Data analysis was performed using an in-house developed program
• Delta Building Monitoring System

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Preliminary Toxicity Test

The dose range for the Preliminary Toxicity Test was 6.33 to 1620 µg/mL. The maximum dose was the maximum recommended dose level.

No precipitate of the test item was observed in the parallel blood-free cultures or hemolysis at the end of the exposure at any dose level or exposure group.

Microscopic assessment of the slides prepared from the exposed cultures showed that binucleate cells were present at up to 1620 µg/mL in all three exposure groups. The test item induced no evidence of toxicity in any of the exposure groups.

Therefore, the selection of the maximum dose level for the Main Experiment was based on the maximum recommended dose level for all three exposure groups.

Micronucleus Test – Main Experiment

The qualitative assessment of the slides determined was similar to that observed in the Preliminary Toxicity Test and that there were binucleate cells suitable for scoring at the maximum dose level of test item, 1620 µg/mL, in all three exposure groups.

No precipitate of the test item was observed in the parallel blood-free cultures at the end of the exposure at any dose level or exposure group.

They confirm the qualitative observations in that no dose-related inhibition of CBPI was observed in any of the exposure groups. As there was no toxicity or precipitate observed in then cultures, the maximum dose level selected for analysis of binucleate cells was the maximum recommended dose level (1620 µg/mL).

The vehicle control cultures had frequencies of cells with micronuclei within the expected range. The positive control items induced statistically significant increases in the frequency of cells with micronuclei. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test item did not induce any statistically significant increases in the frequency of binucleate cells with micronuclei, either in the absence or presence of metabolic activation.

CBPI and Micronucleus Data – Main Experiment - 4-Hour Exposure Without Metabolic Activation (S9)

Exposure Time +/- S9	Dose Level [µg/mL]	Replicate	Nucleate cells/500 cells			CBPI	% Control CBPI	% Cytostasis	Micronuclei (MN) Per 1000 Binucleate cells			% Binucleate Cells with MN	Mean % Binucleate Cells with MN
			Mono	Bi	Multi				1MN	2MN	>2MN
4Hr-S9	0	A	186	268	46	1.72	100	0	1	0	0	0.10	0.30
		B	164	264	72	1.82			5	0	0	0.50
	810	A	206	245	49	1.69	88	12	2	1	0	0.30	0.75
		B	218	228	54	1.67			12	0	0	1.20
	1215	A	212	252	36	1.65	83	17	5	2	0	0.70	0.60
		B	227	230	43	1.63			5	0	0	0.50
	1620	A	213	244	43	1.66	94	6	8	1	0	0.90	0.60
		B	163	279	58	1.79			3	0	0	0.30
	MMC 0.2	A	287	210	3	1.43	58	42	113	9	2	12.40	9.35***
		B	282	207	11	1.46			57	6	0	6.30

CBPI and Micronucleus Data – Main Experiment - 4-Hour Exposure With Metabolic Activation (S9)

Exposure Time +/- S9	Dose Level [µg/mL]	Replicate	Nucleate cells/500 cells			CBPI	% Control CBPI	% Cytostasis	Micronuclei (MN) Per 1000 Binucleate cells			% Binucleate Cells with MN	Mean % Binucleate Cells with MN
			Mono	Bi	Multi				1MN	2MN	>2MN
4Hr+S9	0	A	352	136	12	1.32	100	0	4	1	0	0.50	0.55
		B	331	148	21	1.38			3	2	1	0.60
	810	A	330	144	26	1.39	106	0	6	0	0	0.60	0.60
		B	348	130	22	1.35			6	0	0	0.60
	1215	A	349	131	20	1.34	94	6	2	0	0	0.20	0.55
		B	358	122	20	1.32			9	0	0	0.90
	1620	A	360	123	17	1.31	93	7	2	0	0	0.20	0.40
		B	351	129	20	1.34			6	0	0	0.60
	CP 5	A	441	59	0	1.12	30	70	30	3	2	3.50	4.95***
		B	453	47	0	1.09			51	8	5	6.40

CBPI and Micronucleus Data – Main Experiment - 24-Hour Exposure Without Metabolic Activation (S9)

Exposure Time +/- S9	Dose Level [µg/mL]	Replicate	Nucleate cells/500 cells			CBPI	% Control CBPI	% Cytostasis	Micronuclei (MN) Per 1000 Binucleate cells			% Binucleate Cells with MN	Mean % Binucleate Cells with MN
			Mono	Bi	Multi				1MN	2MN	>2MN
24Hr-S9	0	A	165	270	65	1.80	100	0	4	1	0	0.50	0.60
		B	187	261	52	1.73			5	1	1	0.70
	810	A	173	302	25	1.70	92	8	8	0	0	0.80	0.90
		B	168	311	21	1.71			9	1	0	1.00
	1215	A	229	257	14	1.57	78	22	9	0	0	0.90	0.90
		B	208	274	18	1.62			8	1	0	0.90
	1620	A	229	251	20	1.58	72	28	10	4	0	1.40	0.95
		B	255	232	13	1.52			4	1	0	0.50
	DC 0.075	A	352	131	17	1.33	42	58	21	2	2	2.50	2.05***
		B	354	132	14	1.32			11	5	0	1.60

MMC   = Mitomycin C

***  = P<0.001

Conclusions:

The test item, (S)-Nicotine, did not induce a statistically significant increase in the frequency of binucleate cells with micronuclei in either the absence or presence of a metabolizing system. The test item was therefore considered to be non-clastogenic and non-aneugenic to human lymphocytes in vitro.

Executive summary:

This summary describes the results of anin vitro study for the detection of the clastogenic and aneugenic potential of (S)-Nicotineon the nuclei of normal human lymphocytes. 

Methods

Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for micronuclei in binucleate cellsatthree dose levels, together with vehicle and positive controls. Three exposure conditions in a single experiment were used for the study using a 4‑hour exposure in the presence and absence of a standard metabolizing system (S9) at a 2% final concentration and a 24-hour exposure in the absence of metabolic activation. At the end of the exposure period, the cell cultures were washed and then incubated for a further 24 hours in the presence of Cytochalasin B.

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 should be limited on the maximum recommended dose level. 

Results

All vehicle (Eagle's minimal essential medium with HEPES buffer (MEM)) controls had frequencies of cells with micronuclei within the range expected for normal human lymphocytes.

The positive control items induced statistically significant increases in the frequency of cells with micronuclei. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test item was non-toxic and did not induce any statistically significant increases in the frequency of cells with micronuclei, using a dose range that included a dose level that was the maximum recommended dose level.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-07-20 to 2017-10-05

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The study was performed in accordance with standard test protocol (OECD 476) in a quality controlled laboratory. The study is valid according to criteria mentioned in the test protocols.

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: V79 HPRT Gene Mutation Assay

Target gene:

Hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

The V79 cell stocks were obtained from Harlan CCR in 2010 and originated from Labor für Mutagenitätsprüfungen (LMP); Technical University; 64287 Darmstadt, Germany.

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

0 6.33 12.66 25.31 50.63 101.25 202.5 405 810 1620
The molecular weight of the test item was 162.2, therefore, the maximum proposed concentration level in the solubility test was set at 1620 µg/mL, the 10 mM limit dose level, and no correction for the purity of the test item was applied.

Vehicle / solvent:

-Identity: MEM
-Supplier: Sigma Aldrich
-Batch number: RNBF9655

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

Preliminary Cytotoxicity Test

Several days before starting each experiment, a fresh stock of cells was removed from the liquid nitrogen freezer and grown up to provide sufficient cells for use in the test. The preliminary cytotoxicity test was performed on cell cultures plated out at 1 x 107 cells/225 cm2 flask approximately 24 hours before dosing. This was demonstrated to provide at least 20 x 106 available for dosing in each flask using a parallel flask, counted at the time of dosing. On dosing, the growth media was removed and replaced with serum-free Minimal Essential Medium (MEM). One flask per concentration was treated for 4-hours without metabolic activation and for 4-hours with metabolic activation (2% S9). The concentrations of test item used were 6.33, 12.66, 25.31, 50.63, 101.25, 202.5, 4.5, 810, and 1620 µg/mL.
Exposure was for 4 hours at approximately 37 °C with a humidified atmosphere of 5% CO2 in air, after which the cultures were washed twice with phosphate buffered saline (PBS) before being detached from the flasks using trypsin. Cells from each flask were suspended in MEM with 10% FBS, a sample was removed from each concentration group and counted using a Coulter counter. For each culture, 200 cells were plated out into three 25 cm2 flasks with 5 mL of MEM with 10% FBS and incubated for 6 to 7 days at approximately 37 °C in an incubator with a humidified atmosphere of 5% CO2 in air. The cells were then fixed and stained and total numbers of colonies in each flask counted to give cloning efficiencies (CE).
Results from the preliminary cytotoxicity test were used to select the test item concentrations for the mutagenicity experiment.

Mutagenicity Test – Main Experiment

Several days before starting each experiment, a fresh stock of cells was removed from the liquid nitrogen freezer and grown up to provide sufficient cells for use in the test. Cells were seeded at 1 x 107 cells/225 cm2 flask approximately 24 hours being exposed to the test or control items. This was demonstrated to provide at least 20 x 106 available for dosing in each flask using a parallel flask. Duplicate cultures were set up, both in the presence and absence of metabolic activation, with eight test item concentrations, and vehicle and positive controls. Treatment was for 4 hours in serum free media (MEM) at 37 °C in an incubator with a humidified atmosphere of 5% CO2 in air.
The concentration range of test item was 50.63 to 1620 µg/mL in both the absence and presence of metabolic activation.
At the end of the treatment period the flasks were washed twice with PBS, detached from the flasks with trypsin and the cells suspended in MEM with 10% FBS. A sample of each concentration group cell suspension was counted using a Coulter counter. Cultures were plated out at 2 x 106 cells/flask in a 225 cm2 flask to allow growth and expression of induced mutants, and in triplicate in 25 cm2 flasks at 200 cells/flask to obtain the cloning efficiency, for an estimate of cytotoxicity at the end of the exposure period. Cells were grown in MEM with 10% FBS and incubated at 37 °C in an incubator with a humidified atmosphere of 5% CO2 in air.
Cytotoxicity flasks were incubated for 6 or 7 days then fixed with methanol and stained with Giemsa. Colonies were manually counted and recorded to estimate cytotoxicity.
During the 7 Day expression period the cultures were sub-cultured and maintained on days 2 and 5 to maintain logarithmic growth. At the end of the expression period the cell monolayers were detached using trypsin, cell suspensions counted using a Coulter counter and plated out as follows:
i) In triplicate at 200 cells/25 cm2 flask in 5 mL of MEM with 10% FBS to determine cloning efficiency. Flasks were incubated for 6 to 7 days, fixed with methanol and stained with Giemsa. Colonies were manually counted, counts were recorded for each culture and the percentage cloning efficiency for each dose group calculated.
ii) At 2 x 105 cells/petri dish (ten replicates per group) in MEM with 10% FBS supplemented with 11 µg/mL 6-Thioguanine (6-TG), to determine mutant frequency. The dishes were incubated for 7 days at 37 °C in an incubator with humidified atmosphere of 5% CO2 in air, then fixed with methanol and stained with Giemsa. Mutant colonies were manually counted and recorded for each dish.
The percentage cloning efficiency and mutation frequency per survivor were calculated for each dose group.
Fixation and staining of all flasks/petri dishes was achieved by aspirating off the media, washing with phosphate buffered saline, fixing for 5 minutes with methanol and finally staining with a 10% Giemsa solution for 5 minutes.

Evaluation criteria:

Calculations

The cloning efficiency (CE), % control, mutant plate counts, mutant frequency/106 (MF10-6) and mutant frequency/106 survival rate (MFSV) were calculated using the following formulae:
CE% = ( xCE counts/200)x100
% Control = (CE% of Dose IDx/CE% of Dose ID0)x100
MF 10-6 for each dose = Total mutant plate counts/2
MFS 10-6 for each dose = (MF 10^-6/Day 7 CE%)x100
Where:
Concentration ID0 = Vehicle control values
Concentration IDx = Concentration values
Small errors may occur when calculating mean cell concentrations and volumes for diluting; and in the calculation of means for cloning efficiency and mutant frequency; if these errors are ≤5% they are regarded to be within reasonable experimental error and considered not to affect the integrity of the study.

Statistics:

When there is no indication of any increases in mutant frequency at any concentration then statistical analysis may not be necessary. In all other circumstances comparisons will be made between the appropriate vehicle control value and each individual concentration, using Student’s t-test. Other statistical analysis may be used if they are considered to be appropriate.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Preliminary Cytotoxicity Test

A concentration range of 6.33 to 1620 µg/mL was used in the preliminary cytotoxicity test. The maximum concentration tested was the 10 mM limit dose level.

At the end of the exposure period, precipitate of the test item was not observed at any of the dose levels in either the absence or presence of metabolic activation.

There was no evidence of marked or concentration related reductions in cloning efficiency in either the absence or presence of metabolic activation.

The maximum concentration selected for the main mutagenicity experiment was therefore the 10 mM limit dose level of 1620 µg/mL as recommended by the OECD 476 guidelines.

Mutagenicity Test – Main Experiment

The concentrations of the controls and the test item are given in the table below:

Group	Final concentration of (S)-Nicotine (µg/mL)
4-hour without S9	0*, 50.63*, 101.25*, 202.5*, 405*, 810*, 1620*,EMS 500* and 750*
4-hour with S9 (2%)	0*, 50.63*, 101.25*, 202.5*, 405*, 810*, 1620*,DMBA 1.0* and 2.0*

At the end of the exposure period, precipitate of the test item was not observed at any of the dose levels in either the absence or presence of metabolic activation.

The Day 0 and Day 7 cloning efficiencies for the exposure groups in the absence and there were no marked concentration‑related reductions in the Day 0 cloning efficiency values in either the absence or presence of metabolic activation. There was also no evidence of any reductions in the Day 7 cloning efficiencies in either the absence or presence of metabolic activation, therefore indicating that residual toxicity had not occurred. 

The test item did not induce any toxicologically significant or concentration-related increases in the mutant frequency at any of the concentration levels in the main test (including the 10 mM limit dose level as recommended by the OECD 476 guidelines), in either the absence or presence of metabolic activation.

The vehicle control values were all considered to be within an acceptable range, and the positive controls all gave marked increases in mutant frequency, indicating the test and the metabolic activation system were operating as expected.

*           = Concentrations plated out for cloning efficiency and mutant frequency

EMS     = Ethylmethanesulphonate

DMBA      = Dimethyl benzanthracene

Conclusions:

The test item did not induce any toxicologically significant or concentration-related increases in mutant frequency per survivor in either the absence or presence of metabolic activation. The test item was therefore considered to be non-mutagenic to V79 cells at the HPRT locus under the conditions of this test.

Executive summary:

Introduction

The purpose of this study is to assess the potential mutagenicity of a test item, supplied by the Sponsor, on the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus of the V79 cell line.

Methods

Chinese hamster (V79) cells were treated with the test item at six concentrations, in duplicate, together with vehicle (MEM medium) and positive controls in both the absence and presence of metabolic activation. 

The concentrations used in the main test were selected using data from the preliminary toxicity test where the results indicated that the maximum concentration should be the 10 mM limit dose level of 1620 µg/mL, as recommended by the OECD 476 guidelines. The concentrations of test item plated for cloning efficiency and expression of mutant colonies were as follows:

Exposure Group	Final concentration of (S)-Nicotine (µg/mL)
4-hour without S9	50.63, 101.25, 202.5, 405, 810, 1620
4-hour with S9 (2%)

 Results

The vehicle (MEM medium) controls gave mutant frequencies within the range expected of V79 cells at the HPRT locus.

The positive control substances induced marked increases in the mutant frequency, sufficient to indicate the satisfactory performance of the test and of the activity of the metabolizing system.

The test item did not induce any toxicologically significant or concentration-related increases in the mutant frequency at any of the concentration levels in the main test, in either the absence or presence of metabolic activation

Conclusion

The test item was shown to be non-mutagenic to V79 cells at the HPRT locus under the conditions of the test.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Conducted according to procedures which were the basis for OECD guideline. However, not all details of the study published, therefore rated with reliability score 2.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

See "Principles of method if other than guideline"

Principles of method if other than guideline:

Not tested in E.coli strain for bacterial reverse mutation as this strain was not included in the original Ames test (Ames, B. N. et al., 1975). However, E.coli WP2 (wild-type, repair-proficient), WP67 (uvrA- pol A-) and CM871 (uvrA- recA- lex A-) were tested in a DNA-repair test using the liquid micromethod procedure.

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain / cell type:

S. typhimurium TA 1538

Metabolic activation:

with and without

Metabolic activation system:

S9 mix according to Ames, B. N. et al. (1975), 10% liver S9 fractions from Aroclor- treated Sprague-Dawley rats

Test concentrations with justification for top dose:

Concentrations not stated, but tested at various dilutions with a geometric ratio of 2 (duplicate or triplicate) starting from solubility or toxicity limit.

Vehicle / solvent:

All compounds dissolved / diluted either in bidistilled water or in dimethyl sulfoxide (DMSO).

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

3-methylcholanthrene

4-nitroquinoline-N-oxide

7,12-dimethylbenzanthracene

sodium azide

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

cyclophosphamide

other: 2-aminoanthracene

Remarks:

135 compounds tested therefore more positive controls than marked above, including recommended positive controls of OECD guideline 471 (benzo(a)pyrene, cyclophosphamide and 2-aminoanthracene) for metabolic activation system.

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation) according to the standard procedure described by Ames, B. N. et al. and Maron, D. M., and Ames, B. N. (1975 and 1983).

Evaluation criteria:

For compounds which were negative, the potency was given as a "less than" figure. Therefore an arbitrary value of 100 revertants was divided by the nmoles of the maximum dose tested for this compound.

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 nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

other: DMSO, if used, was shown to be negative

Untreated negative controls validity:

other: Spontaneous reversion rate was within the ranges indicated by Ames, B. N. et al. and Maron, D. M., and Ames, B. N. (1975 and 1983).

Positive controls validity:

other: All substances chosen as positive controls above were shown to induce genotoxicity

Species / strain:

S. typhimurium TA 1538

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:

other: DMSO, if used, was shown to be negative

Untreated negative controls validity:

other: Spontaneous reversion rate was within the ranges indicated by Ames, B. N. et al. and Maron, D. M., and Ames, B. N. (1975 and 1983).

Positive controls validity:

other: All substances chosen as positive controls above were shown to induce genotoxicity

Additional information on results:

The potency of nicotine (in 100 revertants / nmole) to induce reverse mutation in Salmonella thyphimurium strains TA1535, TA100, TA1538, TA98 and TA1537 was < 0.0004.

Conclusions:

Interpretation of results (migrated information):
negative without metabolic activation
negative with metabolic activation

Executive summary:

Nicotine was tested amongst 135 compounds in the Ames reversion test and in a bacterial DNA-repair test. The concentrations tested were not published for the Ames reversion test but were chosen at various dilutions starting from the solubility or toxicity limit. This is in accordance with the principles of OECD guideline 471. Given that 134 other compounds were tested, a high amount of positive controls were included. The metabolic activation was shown for benzo(a)pyrene, cyclophosphamide, 2-aminoanthracene and other compounds activated by S9 mix. Nicotine was stated to be nongenotoxic in the Ames reversion test and in the bacterial DNA-repair test with and without metabolic activation.

References

Ames, B.N., McCann, J. and Yamasaki, E., Methods for Detecting Carcinogens and Mutagens with the Salmonella/Mammalian-Microsome Mutagenicity Test, Mutat. Res., 1975, 31, 347-364.

Maron, D. M., Ames, B. N. Revised Methods for the Salmonella Mutagenicity Test,Mutat. Res., 1983, 113,173-215.

Reference 4

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Conducted according to procedures which were the basis for OECD guideline. Relevant data reported. Due to differences to current OECD guideline 471 and no conduction under GLP, study was rated with Klimisch score 2. Given that also the major metabolites were tested, this study was selected as key study.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

See "Principles of method if other than guideline"

Principles of method if other than guideline:

Tested up to 1000 µg/plate instead of 5000 µg/plate. Not tested in E.coli strain for bacterial reverse mutation as this strain was not included in the original Ames test.

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain / cell type:

S. typhimurium TA 1538

Metabolic activation:

with and without

Metabolic activation system:

S9 mix (5%) from male Sprague-Dawley rats given a single 500 mg/kg injection i.p. of Aroclor 1254 according to Ames, B.N. et al. (1975).

Test concentrations with justification for top dose:

0 µg/plate; 62.5 µg/plate; 125.0 µg/plate; 250.0 µg/plate; 500.0 µg/plate; 750.0 µg/plate; 1000.0 µg/plate

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

other: 2-aminoanthracene

Remarks:

Recommended by OECD guideline 471

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation according to Maron, D. M., and Ames, B. N. (1983).
The test sample, S9 mix or 0.2 M phosphate buffer and the test bacteria were added to 15x85 mm test tubes and shaken and incubated for 20 min at 37 °C. Afterwards 2 ml molten top agar containing 0.5 mM histidine-biotin was added and the mixtures were poured onto minimal glucose agar and incubated at 37 °C for 48 h.

DURATION
- Preincubation period: 20 min
- Exposure duration: 48 h
- Triplicate plates at each concentration

Evaluation criteria:

If a sample induced a concentration-dependent increase in the number of revertants (two times the solvent control) in at minimum one concentration , it was considered to be mutagenic.

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

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:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

other: data on positive controls reported and far above negative control and data on test item

Additional information on results:

Nicotine, cotinine, cotinine-N-oxide, nicotine-N'-oxide and trans-3'-hydroxycotinine did not show an increase in revertants per plate given as mean +/- S.D.. No concentration dependent increase could be seen.

Remarks on result:

other: all strains/cell types tested

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

Nicotine and four of its major metabolites (cotinine, cotinine-N-oxide, nicotine-N'-oxide and trans-3'-hydroxycotinine) were tested up to a concentration of 1000 µg/plate. This concentration was reported to be far above plasma or urine concentrations of human smokers. No increase in revertants per plate could be seen. Positive controls, as recommended in OECD guideline 471, were shown to clearly increase revertants per plate.

Executive summary:

Nicotine and four of its major metabolites were tested according to Maron, D.M. and Ames, B. N. et al. (1983), which was one of the basic procedures that resulted in OECD guideline 471. All compounds were tested up to a concentration of 1000.0 µg/plate. No increase in revertants could be seen with or without metabolic activation. Positive controls, as recommended by OECD guideline 471, led to an increase in revertants at lower concentrations.

References

Ames, B.N., McCann, J. and Yamasaki, E., Methods for Detecting Carcinogens and Mutagens with the Salmonella/Mammalian-Microsome Mutagenicity Test, Mutat. Res., 1975, 31, 347-364.

Maron, D. M., Ames, B. N. Revised Methods for the Salmonella Mutagenicity Test, Mutat. Res., 1983, 113, 173-215.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Mode of Action Analysis / Human Relevance Framework

The studies on nicotine were GLP compliant and quality was overall very high (HPRT-assay and micronucleus test=Klimisch 1, Ames tests = Klimisch 2). Therefore, there is no reason to believe that these results would not be applicable to humans.

Justification for classification or non-classification

Clearly negative in vitro studies, with and without metabolic activation. Therefore, the substance is not classified for genetic toxicity and there is no need to carry out in vivo studies in genetic toxicity. There is no reason to believe that the negative results would not be relevant to humans