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Genetic toxicity: in vitro

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in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Study was conducted similar to OECD Guideline 487, however there was no metabolic activation.
Justification for type of information:
Mutagenicity refers to the induction of permanent transmissible changes in the amount or structure of genetic material of cells or organisms. The Bacterial Reverse Mutation Test (OECD 471, EU B.13/14) is required to fulfil Annex VII information requirements on mutagenicity. Genotoxicity is a broader term to processes which alter the structure, information content or segregation of DNA, that are not necessarily associated with mutagenicity. In order capture broader mechanisms of genetic toxicity, an assessment of cytogenicity or micronucleus formation is required to fulfil REACH Annex VIII-X information requirements. However, all existing available information should be evaluated, including any in vitro and in vivo data exceeding the tonnage requirements.

Data source

Reference Type:
Inhibition by β-caryophyllene of ethyl methanesulfonate-induced clastogenicity in cultured human lymphocytes
Di Sotto A, Mazzanti G, Carbone F, Hrelia P, Maffei F
Bibliographic source:
Mutation Research, 699, 23-28

Materials and methods

Test guideline
equivalent or similar to guideline
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
No metabolic activation.
Principles of method if other than guideline:
- Principle of test: The clastogenicity test was performed by the cytokinesis-block technique using human lymphocytes.
- Short description of test conditions: Lymphocytes were cultivated for 48 hours and were supplemented with cytochalasin-B at 44 hours. At 48 hours of cultivation, the lymphocytes were treated with the test item concentrations. After 72 hours of exposure, cells were fixed and stained and analysed under a light microscope.
- Parameters analysed / observed: Nuclear mitotic index and micronucleus frequency
GLP compliance:
not specified
Study was performed in a university research laboratory.
Type of assay:
in vitro mammalian cell micronucleus test

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
Specific details on test material used for the study:
- Source of test material: Sigma-Aldrich Co (St. Louis, MO, USA)
- Purity: ≥98.5%


Species / strain
Species / strain / cell type:
lymphocytes: human
Details on mammalian cell type (if applicable):
- Suitability of cells: Peripheral lymphocytes
- Sex, age and number of blood donors: Healthy, non-smoking males, less than 40 years old
- Whether whole blood or separated lymphocytes were used: Lymphocytes were separated from whole blood by using a density gradient.
- Methods for maintenance in cell culture: Lymphocytes were cultured at a concentration of 2E6 cells in 5 mL RPMI 1640 medium supplemented with 15% foetal calf serum (FCS), 1% phytohaemagglutinin (PHA), 1% penicillin–streptomycin solution and 1 mM l-glutamine.

- Type and identity of media: The cultures were incubated in RPMI 1640 medium at 37°C in a wet, 5% CO2 atmosphere.
Additional strain / cell type characteristics:
not applicable
Cytokinesis block (if used):
Yes, 3 µg/mL cytochalasin-B was added after cultivation for 44 hours
Metabolic activation:
Test concentrations with justification for top dose:
1, 5, 10, 50, 100 and 200 µg/mL
The highest concentration at which neither necrosis nor cytotoxic or cytostatic effects were observed in the preliminary cytotoxicity test was used as the maximum concentration in the clastogenicity test.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Ethanol/distilled water (50% v/v)
- Justification for choice of solvent/vehicle: Test substance was dissolved in vehicle in order to avoid precipitation of the substance in the medium.
Untreated negative controls:
sterile DMSO
Negative solvent / vehicle controls:
True negative controls:
Positive controls:
Positive control substance:
other: colcemide
Details on test system and experimental conditions:

- Preincubation period: 48 hours, 3 µg/mL cytochalasin-B was added after cultivation for 44 hours
- Exposure duration: 72 hours
- Fixation time (start of exposure up to fixation or harvest of cells): 72 hours

STAIN: Conventional May-Grünwald–Giemsa stain

NUMBER OF REPLICATIONS: The experiments were repeated at least twice, and in each experiment, each concentration was tested in two parallel cultures; data from the two experiments were pooled.

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: At the end of the 72-h incubation period, the lymphocytes were collected, treated with a mild hypotonic solution (1:2 RPMI 1640 medium/H2O, supplemented with 2% FCS) for 2 min and then fixed in ice-cold acetic acid:methanol (1:1). After fixation, the cells were put directly onto slides by use of a cytospin centrifuge, air-dried and stained with conventional May-Grünwald–Giemsa stain.

NUMBER OF CELLS EVALUATED: At least 1000 lymphocytes were scored for each dose and at least 2000 binucleated cells (BNCs; 1000 for each culture) were examined for the presence of one, two or more micronuclei. All slides were coded and scored with a light microscope at 1000× magnification under oil immersion.

- Method: Nuclear division index (NDI)
- Any supplementary information relevant to cytotoxicity: NDI was determined by scoring at least 1000 cells per dose for the presence of one, two, three or more nuclei and calculated as follows: NDI = (1M1+2M2+3M3+4M4)/n, where M1–M4 indicates the number of cells with 1–4 nuclei and n indicates the total number of cells scored. The percent NDI of treated cells (NDIt) was calculated with respect to the control (NDIc).
Evaluation criteria:
NDI and MN were evaluated according to the criteria described by Fenech (2007).
The one-way analysis of variance (one-way ANOVA), followed by Dunnett’s Multiple Comparison Post Test, was used to verify significant differences between treatments (P<0.05).

Results and discussion

Test results
Key result
Species / strain:
lymphocytes: human
Metabolic activation:
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
not examined
Untreated negative controls validity:
Positive controls validity:
Additional information on results:
RANGE-FINDING/SCREENING STUDIES: At concentrations of 1, 5, 10, 50, and 100 µg/mL, β-caryophyllene did not induce any cytotoxic effect and did not affect the NDI, so these cultures were evaluated for the presence of micronuclei. A significant reduction (34%) in NDI with respect to the control was observed in the human lymphocyte cultures treated with β-caryophyllene at the highest concentration tested (200 µg/mL).

- Number of cells for each treated and control culture: See figure in attachment.

Applicant's summary and conclusion

No significant genotoxic effects, in terms of an increase in MN frequency, were observed at concentrations up to 100 µg/mL. β-caryophyllene did not induce any cytotoxic effects and did not affect the NDI at concentrations up to 100 µg/mL.
Executive summary:

The clastogenicity and cytotoxicity of beta-caryophyllene was determined in the in vitro micronucleus assay in human lymphocytes (2010). Peripheral lymphocytes obtained from healthy, non-smoking males under 40 years of age were cultured in supplemented RPMI 1640 culture medium, to investigate the mechanisms of action of β-Caryophyllene (1, 5, 10, 50 and 100 µg/mL) treatment before (pre-treatment), during (co-treatment) and after (post-treatment) treatment with the mutagens. The study was conducted using the cytokinesis-block technique and included a negative and positive control. Cells were stained using conventional May-Grünwald–Giemsa stain and were analysed with a light microscope. 


Up to 100 µg/mL β-Caryophyllene did not produce cytotoxicity or genotoxic effects, as demonstrated by the nuclear division index (NDI) and frequency of micronuclei (MN). A significant reduction (34%) in nuclear division index with respect to the control was observed at the highest concentration tested (200 µg/mL). Classified as a genotoxic agent, ethyl methanesulfonate (EMS) alkylates DNA and induces chromosomal aberrations. β-Caryophyllene significantly reduced the MN frequency in EMS treated cells, protecting against the clastogenic effects of EMS in both pre- and co-treatment protocols. Comparable to an OECD 487 study and published in a peer-reviewed journal, this study is considered reliable with restriction (Klimisch 2).

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