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Toxicological information

Genetic toxicity: in vitro

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Administrative data

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.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2018

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian cell micronucleus test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid

Method

Species / strain
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
Controls
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

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
Vehicle controls validity:
valid
Positive controls validity:
valid

Any other information on results incl. tables

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 0.10  0.30    
 B  164 264  72  1.82  0.50 
810  A  206 245  49  1.69  88     12    0.30  0.75    
 B  218 228  54  1.67  12  1.20 
1215  A  212 252  36  1.65  83      17    0.70  0.60    
 B 227  230  43  1.63  0.50 
1620  A  213 244  43  1.66  94      6    0.90  0.60    
 B  163 279  58  1.79  0.30 
MMC 0.2  A  287 210  1.43  58      42    113  12.40      9.35***
 B  282 207  11  1.46  57  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 0.50  0.55    
 B 331  148  21  1.38  0.60 
    810  A 330 144  26  1.39  106       0   0.60  0.60    
 B 348  130  22  1.35  0.60 
   1215   A 349  131  20  1.34  94        6  0.20  0.55   
 B 358  122  20  1.32  0.90 
   1620   A 360  123  17  1.31  93        7  0.20  0.40    
 B 351  129  20  1.34  0.60 
   CP 5   A 441  59  1.12  30        70  30  3.50  4.95***    
 B 453  47  1.09  51  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     0.50  0.60    
 B  187 261  52  1.73  0.70 
   810   A  173 302  25  1.70  92     8     0.80  0.90    
 B  168 311  21  1.71  1.00 
   1215   A  229 257  14  1.57  78     22     0.90  0.90    
 B  208 274  18  1.62  0.90 
   1620   A  229 251  20  1.58  72     28     10  1.40  0.95    
 B  255 232  13  1.52  0.50 
   DC 0.075   A  352 131  17  1.33  42     58     21  2.50  2.05***    
 B  354 132  14  1.32  11  1.60 

MMC   = Mitomycin C

***  = P<0.001

Applicant's summary and conclusion

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.