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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:
10.08 - 02.11.2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Version / remarks:
adopted 29 July 2016
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Hessen Ministerium für Umwelt, Klimatschutz, Landwirtschaft und Verbraucherschutz; Wiesbaden.
Type of assay:
in vitro mammalian cell micronucleus test

Test material

Constituent 1
Chemical structure
Reference substance name:
Cinnamyl acetate
EC Number:
203-121-9
EC Name:
Cinnamyl acetate
Cas Number:
103-54-8
Molecular formula:
C11H12O2
IUPAC Name:
cinnamyl acetate

Method

Target gene:
not applicable
Species / strain
Species / strain / cell type:
lymphocytes:
Remarks:
Human lymphocytes from healthy non-smoking donors not receiving medication primary culture
Details on mammalian cell type (if applicable):
- One donor (30 years old)
- The lymphocytes of the donor have been shown to respond well to stimulation of proliferation with PHA and to positive control substances
- The donor had a previously established low incidence of micronuclei in her peripheral blood lymphocytes
Additional strain / cell type characteristics:
not applicable
Cytokinesis block (if used):
Cytochalasin B,
Metabolic activation:
with and without
Metabolic activation system:
Mammalian Microsomal Fraction S9 Mix (co-factor supplemented post-mitochondrial fraction, prepared from the livers of rats treated with Phenobarbital / ß-naphthoflavone)
Test concentrations with justification for top dose:
- Experiment IA: 4 hours without S9 mix; 107, 188 and 329 μg/mL
- Experiment IB: 4h with S9 mix; 329, 575 and 1007 μg/mL
- Experiment II: 20 hours without S9 mix; 107, 187 and 327 μg/mL
Vehicle / solvent:
DMSO
Controls
Negative solvent / vehicle controls:
yes
Remarks:
0.5 % DMSO
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
other: Demecolcin
Details on test system and experimental conditions:
CELL CULTURE CONDITIONS
- Culture medium: c Modified Eagles Medium/Ham's F12 (DMEM/F12, mixture 1:1) supplemented with 200 mM GlutaMAX™, penicillin/streptomycin (100 U/mL/100 μg/mL), the mitogen PHA (3 μg/mL), 10 % FBS (fetal bovine serum), 10 mM HEPES and the anticoagulant heparin (125 U.S.P.-U/mL).
- Temperature: 37 °C
- Humidity: 5.5 % CO2 in humidified air

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 48 hour with phytohemeagglutinine (PHA) to stimulate proliferation
- Exposure duration: 4 or 20 hours
- Washing: 2x
- Recovery period: 16 hours
- Harvesting: 40 hours after beginning of treatment
- Post-incubation: at 37 °C for 20 minutes
- Fixation time: 2 x 20 minutes
- Fixative: ico-cold mixture of methanol and glacial acetic acid (19 : 1 parts)

SPINDLE INHIBITOR (cytogenetic assays): cytochalasin B

STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: 2

METHODS OF SLIDE PREPARATION: dropping the cell suspension in fresh fixative onto a clean microscope slide

NUMBER OF CELLS EVALUATED: At least 1000 binucleate cells per culture were scored for cytogenetic damage on coded slides.
- Frequency of micronucleated cells: reported as % micronucleated cells. To describe a cytotoxic effect the CBPI was determined in 500 cells per culture and cytotoxicity is expressed as % cytostasis.
A CBPI of 1 (all cells are mononucleate) is equivalent to 100 % cytostasis.

CRITERIA FOR MICRONUCLEUS IDENTIFICATION: publication of Countryman and Heddle (1976).
The micronuclei have to be stained in the same way as the main nucleus. The area of the micro nucleus should not extend the third part of the area of the main nucleus.

DETERMINATION OF CYTOTOXICITY
- Method: Cytotoxicity is characterized by the percentages of reduction in the CBPI in comparison with the controls (% cytostasis) by counting 500 cells per culture. The pre-test was performed with 10 concentrations of the test item separated by no more than a factor of √10 and a solvent and positive control. All cell cultures were set up in duplicate. Exposure time was 4 hrs (with and without S9 mix).
The preparation interval was 40 hrs after start of the exposure. The test concentrations ranged from 11.4 to 1762 μg/mL (with and without S9 mix).

OTHER EXAMINATIONS:
- Determination of polyploidy: no
- Determination of endoreplication: no
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable): no

DETAILED INFO ON POSITIVE CONTROLS:
Without metabolic activation:
- Mitomycin C (pulse treatment), dissolved in deionized water. Concentration: 1.0 µg/mL.
- Demecolcin (continuous treatment), dissolved in deionized water. Concentration: 125 ng/mL.
With metabolic activation:
- Cyclophosphamide, dissolved in saline (0.9% NaCl (w/v)). Concentration: 17.5 µg/mL.

OTHER:
- Test Item Preparation: Stock formulations of the test item and serial dilutions were made in DMSO.
All formulations were prepared freshly before treatment and used within two hours of preparation.
The osmolarity and pH were determined by using an osmometer or a pH meter.

ACCEPTANCE CRITERIA: The micronucleus assay will be considered acceptable if it meets the following criteria:
− The concurrent solvent control will normally be within the laboratory historical solvent control data range (95% confidence interval)
− The concurrent positive controls should induce responses that are compatible with the laboratory historical positive control data and produce a statistically significant increase
− Cell proliferation criteria in the solvent control are considered to be acceptable
− All experimental conditions described in section ‘Experimental performance’ were tested unless one exposure condition resulted in a clearly positive result
− The quality of the slides must allow the evaluation of an adequate number of cells and concentrations
− The criteria for the selection of top concentration are according to the current OECD Guideline for the in vitro micronucleus test. The highest test item concentration should be 10 mM, 2 mg/mL or, 2 μL/mL, whichever is the lowest.
Rationale for test conditions:
Phase separation of the test item in the culture medium was observed at the end of treatment as follows:
- Experiment IA (with S9): 1007 μg/mL and above
- Experiment IB (without S9): 329 μg/mL and above
- Experiment II (without S9): 327 μg/mL and above

In the absence and presence of S9 mix, no cytotoxicity was observed up to the highest evaluated concentration, which showed phase separation.
Evaluation criteria:
A test item is considered to be clearly negative if, in all of the experimental conditions examined:
− None of the test item concentrations exhibits a statistically significant increase compared with the concurrent solvent control
− There is no concentration-related increase
− The results in all evaluated test item concentrations should be within the range of the laboratory historical solvent control data (95% confidence interval)

A test item is considered to be clearly positive if, in any of the experimental conditions examined:
− At least one of the test item concentrations exhibits a statistically significant increase compared with the concurrent solvent control
− The increase is concentration-related in at least one experimental condition
− The results are outside the range of the laboratory historical solvent control data

There is no requirement for verification of a clear positive or negative response. In case the response is neither clearly negative nor clearly positive and/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.
Scoring additional cells (where appropriate) or performing a repeat experiment possibly using modified experimental conditions (e.g. narrow concentration spacing, other metabolic activation conditions, i.e. S9 concentration or S9 origin) could be useful.
However, results may remain questionable regardless of the number of times the experiment is repeated. If the data set will not allow a conclusion of positive or negative, the test item will therefore be concluded as equivocal.
Statistics:
Statistical significance was confirmed by the Chi square test (α < 0.05), using a validated test script of “R”, a language and environment for statistical computing and graphics. Within this test script a statistical analysis was conducted for those values that indicated an increase in the number of cells with micronuclei compared to the concurrent solvent control.

Results and discussion

Test results
Species / strain:
lymphocytes: Human lymphocytes from healthy non-smoking donors not receiving medication primary culture
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
No cytotoxicity was observed up to the highest evaluated concentration, which showed phase separation.
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid

Any other information on results incl. tables

Summary of results

Exp.

Preparation interval

Test item concentration in μg/mL

Proliferation index
CBPI

Cytostasis
in %*

Micronucleated cells 
in %**

Exposure period 4 hrs without S9 mix

IB

40 hrs

Solvent control1

2.01

 

0.35

Positive control2

1.62

39.0

15.60S

107

1.98

3.2

0.85S

188

1.99

1.6

0.30

329PS

1.96

4.7

1.00S

Exposure period 20 hrs without S9 mix

II

40 hrs

Solvent control1

1.89

 

0.85

Positive control3

1.50

44.3

4.40S

107

1.80

9.8

0.45

187

1.83

6.4

0.55

327PS

1.70

21.9

0.55

Exposure period 4 hrs with S9 mix

IA

40 hrs

Solvent control1

2.06

 

0.55

Positive control4

1.56

46.9

8.20S

329

2.04

1.5

0.30

575

2.04

1.9

0.70

1007PS

1.96

9.1

0.80

 

* For the positive control groups and the test item treatment groups the values are related to the solvent controls

** The number of micronucleated cells was determined in a sample of 2000 binucleated cells

PSPhase separation occurred at the end of treatment

SThe number of micronucleated cells is statistically significantly higher than corresponding control values

1DMSO         0.5 % (v/v)

2MMC          1.0μg/mL

3Demecolcin    125 ng/mL

4CPA            17.5 μg/mL

Historical control data:

Solvent Control without S9 
Micronucleated cells in % 
  Pulse treatment (4/40)  Continuous treatment (20/40) 
No. of experiments  78*  79** 
Mean  0.60  0.57 
95 % Ctrl limit  0.08 – 1.12  0.12 – 1.03 
     
1x SD  0.26  0.23 
2x SD  0.52  0.46 
Min  0.15  0.05 
Max  1.65  1.35 

Solvent Control with S9 
Micronucleated cells in % 
  Pulse treatment (4/40) 
No. of experiments  96* 
Mean  0.62 
95 % Ctrl limit  0.16 – 1.08 
   
1x SD  0.23 
2x SD  0.46 
Min  0.15 
Max  1.30 

Positive Control without S9 
Micronucleated cells in % 
  Pulse treatment (4/40)  Continuous treatment (20/40) 
MMC  Demecolcin 
No. of experiments  78 81
Mean  12.48  3.72 
95 % Ctrl limit  1.44 – 23.52  1.43 – 6.01 
     
1x SD  5.52  1.15 
2x SD  11.04  2.30 
Min  4.15  2.10 
Max  30.30  7.25 

Positive Control with S9 
Micronucleated cells in % 
  Pulse treatment (4/40) 
CPA 
No. of experiments  165
Mean  5.16 
95 % Ctrl limit  0.84 – 9.49 
   
1x SD  2.16 
2x SD  4.32 
Min  2.10 
Max  11.90 

Applicant's summary and conclusion

Conclusions:
Under the experimental conditions reported, the test item did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes and should be considered non-mutagenic.
Executive summary:

In the current study the potential of the test item to induce micronuclei in human lymphocytes in vitro was assessed according to OECD 487 and according to GLP regulations.

The occurrence of micronuclei in interphase cells provides an indirect but easy and rapid measure of structural chromosomal damage and aneugenicity in cells that have undergone cell division during or after exposure to the test substance. Micronuclei arise from chromosomal fragments or whole chromosomes and are inducible by clastogens or agents affecting the spindle apparatus.

The induction of cytogenetic damage in human lymphocytes was assessed in two independent experiments and in each experimental group two parallel cultures were analyzed. Per culture 1000 binucleated cells were evaluated for cytogenetic damage. The test item was dissolved in DMSO. In Experiment I, the exposure period was 4 hours with and without S9 mix. In Experiment II, the exposure period was 20 hours without S9 mix.

The cells were stimulated for 48 hour with phytohemeagglutinine (PHA) to activate the proliferation, before exposure. After exposure, the cells were washed and left to recover for 16 hours before the cytokinesis was blocked by cytochalasin B for 20 minutes. The cells were prepared 40 hours after start of treatment with the test item.

The highest applied concentration in this study (1007 μg/mL of the test item, approx. 10 mM) was chosen with regard to the molecular weight of the test item and with respect to the current OECD Guideline 487. The chosen treatment concentrations were in Experiment I (4 hours with and without S9 mix): 107, 188, 329, 575 and 1007 μg/mL and in Experiment II (20 hours without S9 mix): 107, 187 and 327 μg/mL.

Dose selection of the cytogenetic experiment was performed considering the toxicity data and the occurrence of test item phase separation in accordance with OECD Guideline 487. In the absence and presence of S9 mix, no cytotoxicity was observed up to the highest evaluated concentration, which showed phase separation.

In the absence and presence of S9 mix, no relevant increase in the number of micronucleate cells was observed after treatment with the test item. However, in Experiment IB in the absence of S9 mix statistically significant increases in micronucleate cells were observed after treatment with 107 and 329 μg/mL (0.85 and 1.00 %). Since these values are clearly within the range of the historical solvent control data (0.08 – 1.12 % micronucleate cells), they are considered as biologically irrelevant.

Appropriate mutagens were used as positive controls. They induced statistically significant increases in cells with micronuclei.

In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes. Therefore, the test item is considered to be non-mutagenic in this in vitro micronucleus test.