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Genetic toxicity: in vivo

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in vivo mammalian germ cell study: cytogenicity / chromosome aberration
Type of information:
experimental study
Adequacy of study:
supporting study
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Study conducted similar to EPA OPPTS 870.5915, however there are limitations in the reporting of experimental conditions and results.
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 to 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:
Antigenotoxic capacity of beta-caryophyllene in mouse, and evaluation of its antioxidant and GST induction activities
Álvarez-González I, Madrigal-Gujaidar E, Castro-Garcia S
Bibliographic source:
J. Toxicol. Sci., 39(6) 849-859

Materials and methods

Test guideline
equivalent or similar to guideline
EPA OPPTS 870.5915 (In Vivo Sister Chromatid Exchange Assay)
Principles of method if other than guideline:
- Principle of test: The genotoxicity effects on sister chromatid exchanges and chromosome aberrations and cytotoxicity potential of the test item was evaluate in vivo in male mice.
- Short description of test conditions: Mice were orally administered the test item in corn oil vehicle. A BrdU tablet was subcutaneously implanted in each mouse one hour after exposure and colchicine was injected intraperitoneally 21 hours after administration. Animals were sacrificed three hours later and bone marrow from both femurs were taken for analysis.
- Parameters analysed / observed: Proportion of metaphases in first, second and third division, average generation time, mitotic index and number and type of chromosome aberrations.
GLP compliance:
not specified
Type of assay:
sister chromatid exchange assay

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 Chemicals (St Louis, MO, USA)

Test animals

Swiss Webster
Details on test animals or test system and environmental conditions:
- Weight at study initiation: 28 g
- Housing: Polypropylene cages
- Diet (e.g. ): Rodent Laboratory chow 5001, ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 7 days

- Temperature: 22±2°C
- Humidity: 50%
- Photoperiod: 12 hr dark-light cycles

Administration / exposure

Route of administration:
oral: unspecified
- Vehicle(s)/solvent(s) used: Corn oil
Duration of treatment / exposure:
25 hours
Frequency of treatment:
Single dose administered
Doses / concentrationsopen allclose all
Dose / conc.:
20 mg/kg bw/day (nominal)
Dose / conc.:
200 mg/kg bw/day (nominal)
Dose / conc.:
2 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
Control animals:
yes, concurrent vehicle
Positive control(s):
- Justification for choice of positive control(s): Benzo(a)pyrene is known as a strong mutagen in in vitro and in vivo assays, as well as a human carcinogen.
- Route of administration: Intraperitoneal
- Doses / concentrations: 200 mg/kg


Tissues and cell types examined:
Bone marrow of femur
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: Based on a previous report that used similar experimental conditions (i.e. 'Sup_Genetic toxicity in vivo_MN_2009_RL2' supporting study by Molina-Jasso et al. 2009).

TREATMENT AND SAMPLING TIMES: One hour after test item administration, a BrdU tablet (45 mg, 60% coated with paraffin) was subcutaneously implanted in each mouse under inhaled ether anaesthesia. Twenty-one hours later, mice were injected intraperitoneally with colchicine (5 kg/kg). Three hours later, the animals were cervically dislocated and the bone marrow of both femurs were collected.

DETAILS OF SLIDE PREPARATION: Bone marrow was incubated in a solution of 0.075 M KCl for 30 minutes and then centrifuged for 10 minutes. The supernatant was discarded and the remaining cells were fixed with a solution of methanol-acetic acid (3:1). The fixation process was repeated twice. Two to three drops of the suspension were placed on a slide and flamed.

METHOD OF ANALYSIS: The chromatic differential staining was made by applying the Hoescht-Giemsa method to differentiate mitosis in the first (M1), second (M2) and third (M3) cellular division.
Evaluation criteria:
In the obtained mitotic cells, the following determinations were made per mouse:
a) the number and types of chromosome aberrations (CA) in 100 first-division metaphases;
b) the SCE frequency in 30 second-division metaphses;
c) the determination of cell proliferation kinetics in 100 cells by quantifying the rate of cells in first (M1), second (M2), and third (M3) cellular divison; and,
d) the mitotic index in 1000 cells

The average generation time (AGT) was obtained according to the formula: 21/(1)(M1)+(2)(M2)+(3)(M3) x 100

Analysis of chromosome aberrations was based on OECD 475 (2013, draft) with slight modifications.
Data were statistically analysed with the ANOVA and Tukey tests.

Results and discussion

Test results
Key result
no effects
Vehicle controls validity:
Negative controls validity:
not examined
Positive controls validity:
Additional information on results:
- Types of structural aberrations for significant dose levels: See table 2 below.
- Statistical evaluation: No statistically significant differences were observed in the number of SCE per metaphase between the test item concentrations (mean 4.1 SCE) and the control (mean 3.8 SCE).

Any other information on results incl. tables

Table 1. Cellular proliferation kinetics (CPK), average generation time (AGT) and mitotic index (MI) in mice treated with beta-caryophyllene

Treatment   M1 CPK (%)  M2 CPK (%)  M3 CPK (%)  AGT (hr)  MI (%)
 Vehicle control  29.5±2.75  62.5±3.9  8.2±1.52  12.1±0.3  4.8±0.2
 20 mg/kg beta-caryophyllene  32.4±4.5  56.2±4.2  11.4±0.9  12.1±0.5  5.3±0.4
 200 mg/kg beta-caryophyllene  38.6±2.3  51.2±2.6  10.2±0.8  12.2±0.7  4.9±0.3
 2000 mg/kg beta-caryphyllene  49.2±5.4 40.6±3.5  10.2±1.3  13.0±0.6  5.2±0.4
 Positive control  39.2±3.2  50.4±3.5  10.4±1  12.2±0.2  3.6±0.5*

Each value represents the mean ± S.D. of 100 cells (CPK) and 1000 cells (MI) per mouse.

CPK corresponds to the proportion of metaphases in the first (M1), second (M2) and third (M3) division.

* Statistically significant difference with respect to the control value, ANOVA and Student t test (P≤0.05).

Table 2. Types and amout of structural and numerical aberrations indced with beta-caryophyllene in mouse bone marrow cells

 Treatment  g  ig"  ctb  csb"  f  m  r  p
 Vehicle control  0.3±0.1  0.1±0.1  0.5±0.1  -  1.3±0.4  -  -  0.1±0.0

20 mg/kg beta-caryophyllene









 200 mg/kg beta-caryophyllene  0.6±0.1  -  0.5±0.1  0.1±0.1  1.3±0.3  -  -  0.3±0.1
 2000 mg/kg beta-caryophyllene  0.6±0.1  0.3±0.1  0.8±0.1  0.2±0.1  1.5±0.4  -  -  0.5±0.1
 Positive control  7.5±0.3*  2.8±0.5*  11.6±0.3*  6.5±0.7*  28.0±7.6*  4.6±0.7*  2.8±0.6*  3.5±0.1*

Each value represents the mean ± S.D. of 100 metaphases per mouse.

g = gaps, ig = isogaps, ctb = chromatidic break, csb = chromosome brea, f = fragrments, m = minutes, r = rings, p = polyploidy.

* Statistically significant difference with respect to the control value, ANOVA and Tukey test (P≤0.05).

Table 3. Effect of beta-caryophyllene on the induction of chromosomal abberations (CA) in mouse

 Treatment  CA without gaps (%)  CA with gaps (%)  Cells with CA (%)
 Vehicle control  1.8±0.7  2.3±0.9  0.6±0.1
 20 mg/kg beta-caryophyllene  1.7±0.0  2.0±0.2  0.6±0.1
 200 mg/kg beta-caryophyllene  1.9±0.5  2.6±0.9  1.2±0.5
 2000 mg/kg beta-caryophyllene  2.5±0.8  3.5±0.8  1.0±0.3
 Positive control  53±10.2*  64±11.9*  42.3±6.2*

Each value represents the mean ± S.D. of 100 metaphases per mouse.

* Statistically significant difference with respect to the control value, ANOVA and Tukey test (P≤0.05).

Applicant's summary and conclusion

No genotoxic effect or cytotoxic potential was observed up to 2000 mg/kg.
Executive summary:

The effects of beta-caryophyllene on the number of sister chromatid exchanges (SCE) and the number of chromosome aberrations was determined in two separate experiments in male mice (2014). In the first experiment, Male Swiss-Webster mice (6 per group) were orally administered β-Caryophyllene (20, 200 and 2000 mg/kg) or the vehicle control (10% w/v corn oil). Positive control mice received Benzo-a-pyrene (200 mg BaP/kg). In a second experiment, mice orally administered β-Caryophyllene (20, 200 and 2000 mg/kg) were intraperitoneally injected with 200 mg/kg of BaP 30 minutes later. Mice were sacrificed 25 hours after administration and bone marrow was analysed by chromatid differential staining.


No genotoxic effects were observed up to 2000 mg/kg β-Caryophyllene, as the number of SCE were not statistically significantly different between the control (mean 3.8 SCE) and test item concentrations (mean 4.1 SCE). No statistically significant differences were observed for the average generation time, numbers/types of chromosome aberrations or mitotic index were identified between the test item treatments and the control. Furthermore, treatment with β-Caryophyllene ameliorated the genotoxicity of BaP at the high dose, suggesting the substance may be geno-protective. The study was considered to be reliable with restriction (Klimisch 2), despite minor limitations in the reporting of the experimental conditions and results.

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