Caryophyllene

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key 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. The Salmonella typhimurium histidine (his) reversion system is a microbial assay which measures mutation in the histidine operon. Gain of function his- to his+ mutation is induced by chemicals which cause base changes or frameshift mutations in the genome of this organism. Two Ames tests were available on the test item using Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and TA102 (2006) and S. typhimurium strains TA98 and TA100 and Escherichia coli strain WP2uvrA (2008). The test item did not significantly increase the number of revertant colonies in any of the strains tested, both in the presence or absence of S9 metabolic activation. Comparable to an OECD Guideline 471 study, both studies were considered reliable with restriction (Klimisch 2) and sufficient to fulfil the Annex VII mutagenicity information requirements.

The mutagenicity of β-Caryophyllene was also determined in vitrousing the mouse lymphoma assay (2006), however, according to the authors the results were equivocal. In all positive/equivocal cases highly toxic concentrations with a relative total growth of < 10% have been evaluated. When excluding those doses from evaluation according to current OECD 476 guideline, only concentrations from 15-20 µg/mL (without S9-mix) and 75- 120 µg/mL (with S9-mix) can be evaluated leading to negative results.

Comparable to an OECD 476 study and published in a journal, the study is considered to be reliable with restriction (Klimisch 2).

 

However, genotoxicity is a broader term to encapsulate processes which alter the structure, information content or segregation of DNA, which are not necessarily associated with mutagenicity. An assessment of cytogenicity or micronucleus formation is required to evaluate potential genotoxicity, and fulfil REACH Annex VIII-X information requirements. 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. Up to 100 µg/mL β-Caryophyllene did not produce cytotoxicity or genotoxic effects. β-Caryophyllene significantly reduced the MN frequency in ethyl methanesulfonate (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, the study is considered to be reliable with restriction (Klimisch 2). No evidence of a genotoxicity was identified in the available in vitro studies.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

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.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Deviations:

yes

Remarks:

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

Remarks:

Study was performed in a university research laboratory.

Type of assay:

in vitro mammalian cell micronucleus test

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source of test material: Sigma-Aldrich Co (St. Louis, MO, USA)
- Purity: ≥98.5%

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

CELLS USED
- 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.

MEDIA USED
- 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:

without

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:

yes

Remarks:

sterile DMSO

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

other: colcemide

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- 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.

DETERMINATION OF CYTOTOXICITY
- 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).

Statistics:

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).

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

Positive controls validity:

valid

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

Conclusions:

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).

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

Study was conducted similar to OECD Guideline 471, however there were some limitations in the reporting of experimental conditions.

Justification for type of information:

Column 1 of REACH Annex VII informs on the standard mutagenicity information requirements, for substances produced or imported in quantities >1 tpa. The Bacterial Reverse Mutation Test (OECD 471, EU B.13/14) detects gain of function point mutations and frameshifts in vitro, and is required to fulfil Annex VII information requirements.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

The Salmonella typhimurium histidine (his) reversion system is a microbial assay which measures mutation in the histidine operon. Gain of function his- to his+ mutation is induced by chemicals which cause base changes or frameshift mutations in the genome of this organism. S. typhimurium strains TA98 and TA1537 primarily respond to frameshift mutations at the histidine gene locus his D 3052 and his C 3076, respectively. Strains TA100, TA102 and TA1535 respond to base-pair substitutions in the his G 46, his G 428 and his G 46 locus.

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

Liver S9 homogenate was prepared from male Sprague-Dawley rats and Syrian golden hamsters.

Test concentrations with justification for top dose:

100, 333, 1000, 3333 and 10000 µg/plate
Doses selected based on the levels of cytotoxicity observed in a preliminary dose range-finding study using strain TA100.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation on all strains) and preincubation methodology under reductive conditions (strains TA98 and TA100)

DURATION
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: Triplicate

Evaluation criteria:

For a test article to be considered positive, it had to induce at least a doubling (TA98, TA100, and TA1535) in the mean number of revertants per plate of at least one tester strain. This increase in the mean revertants per plate had to be accompanied by a dose response to increasing concentrations of the test chemical. If the study showed a dose response with a less than 3-fold increase on TA1537, the response had to be confirmed in a repeat experiment.

Statistics:

Not reported

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Table 1. S. typhimurium test data

S. typhimurium strain	S9	Solvent control	Positive control	Test item concentrations (µg/plate)
				100	333	1000	3333	10000
TA98	Without	17±3	348±27	13±2	13±3	14±3	28±6	27±12
	With (rat)	19±6	393±231	19±3	18±8	17±7	16±2	19±8
	With (hamster)	13±4	935±219	20±5	16±5	19±10	15±2	20±2
TA100	Without	92±10	695±136	97±3	133±7	104±10	130±18	116±9
	With (rat)	141±18	1016±102	114±10	108±9	131±12	129±12	122±15
	With (hamster)	111±11	1039±455	114±20	120±3	101±10	88±12	105±15
TA102	Without	344±15	1372±16	397±64	403±13	396±16	373±23	376±60
	With (rat)	370±53	2305±329	406±57	410±77	415±37	415±43	427±48
	With (hamster)	374±25	1212±88	414±4	377±17	405±34	416±64	409±56
TA1535	Without	19±6	370±61	21±5	16±2	19±4	16±4	16±3
	With (rat)	9±3	92±13	16±5	12±3	12±5	12±4	12±4
	With (hamster)	13±4	160±70	15±3	10±3	11±5	16±6	16±5
TA1537	Without	8±2	1226±399	14±4	12±4	8±3	9±2	9±4
	With (rat)	14±4	88±56	20±10	13±6	11±4	13±4	13±6
	With (hamster)	10±2	197±37	13±3	12±4	13±3	13±6	9±3

Conclusions:

The test item was not mutagenic to Salmonella typhimurium strains TA1535, TA1537, TA98, TA100, and TA102 in the presence and absence of a metabolising system.

Executive summary:

An Ames test was conducted on the test item using Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and TA102 (2006). The test was performed by the plate incorporation assay at test item concentrations of 100, 333, 1000, 3333 and 10000 µg/plate in the presence and absence of S9 (rat and hamster), alongside negative, solvent and positive controls. The test item was incubated for 48 and mutagenicity was assessed based on the number of revertant colonies. The test item was not mutagenic to Salmonella typhimurium strains TA1535, TA1537, TA98, TA100, and TA102 in the presence and absence of a metabolising system. Comparable to an OECD Guideline 471 study, the study was considered reliable with restriction (Klimisch 2), despite minor limitations in the reporting of experimental conditions.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

Study is similar to OECD Guideline 476, however there are limitations in the reporting of 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.

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

yes

Remarks:

Single replicate of solvent control tested, cytotoxicity not reported.

GLP compliance:

not specified

Type of assay:

other: Mouse lymphoma assay

Target gene:

Thymidine kinase (TK)

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
- Methods for maintenance in cell culture if applicable: The cells were grown in Fischer’s medium for leukemic cells of mice supplemented with 10% horse serum and 0.02% pluronic F-68. New cultureswere initiated at approximately 3 month intervals from cells stored in liquid nitrogen.

MEDIA USED
- Type and identity of media including CO2 concentration: Fischer's growth medium, incubated at 37±1 °C in 5% CO2 in air
- Periodically checked for Mycoplasma contamination: Yes

Metabolic activation:

with and without

Metabolic activation system:

Liver S9 prepared from Aroclor 1254-induced male Sprague- Dawley rats

Test concentrations with justification for top dose:

15, 17.5, 20, 22.5 and 25 µg/mL without activation
75, 100, 110, 120, 130 and 140 µg/mL with activation
The doses of chemical selected for testing were within the range yielding approximately 0-90% cytotoxicity.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

3-methylcholanthrene

ethylmethanesulphonate

methylmethanesulfonate

other: dimethylbenz[a]anthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)
- Cell density at seeding: 6E5 cells/mL, corresponding to 6E6 cells per plate. Cells in the cultures were adjusted to 3E5 cells/mL at 24 h intervals and cloned for mutant selection (1E6 cells/plate) and viable count determinations (200 cells/plate).

DURATION
- Exposure duration: 4 hours
- Expression time (cells in growth medium): 48 hours
- Selection time (if incubation with a selection agent): 10 to 12 days

SELECTION AGENT (mutation assays): Trifluorothymidine, 3 µg/mL

NUMBER OF REPLICATIONS: Duplicates

NUMBER OF CELLS EVALUATED: 200 cells/plate

Evaluation criteria:

Only colonies larger than approximately 0.2 mm in diameter were counted. Mutant frequencies were expressed as mutants per 1E6 surviving cells.

Under the old evaluation criteria, results were interpreted using a doubling of the mutant frequency over the concurrent solvent-treated control value as an indication of a positive effect, together with evidence of a dose-related increase. Doubling of the mutant frequency is representative of a positive effect. Only doses yielding total growth values of 10% were used in the analysis of induced mutant frequency. Doses yielding less than 10% total growth were used in determining dose response.

Under the new evaluation criteria, a compound is considered positive if a concentration-related increase in mutant frequency is observed and one or more dose levels with 10% or greater total growth exhibit mutant frequencies of ≥100 mutants per 1E6 clonable cells over the background level. The term LED refers to the lowest dose at which a positive result (increase in mutant frequency of ≥100 mutants per 1E6 clonable cells over the background level) was achieved and stands for lowest effective dose.

The size of mutant mouse lymphoma colonies was also determined using a colony counter. An internal discriminator was set to step sequentially to exclude increasingly larger colonies in approximate increments of 0.1 mm in colony diameter. The size range used was from approximately 0.2 to 1.1 mm.

Statistics:

Not reported

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with

Genotoxicity:

other: see applicant´s remarks

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

other: see applicant´s remarks

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Table 1. Mouse lymphoma test data

Metabolic activation	Test item concentration (µg/mL)	Average TFT resistance	Average viable count	Mutation frequency	Relative total growth
Without	Solvent control	40	179	0.44	-
Without	15	43	180	0.48	104
Without	15	37	158	0.47	94
Without	17.5	38	154	0.5	63
Without	17.5	38	162	0.47	85
Without	20	38	120	0.64	36
Without	20	46	155	0.59	59
Without	22.5	38	76	1.02	9
Without	22.5	42	120	0.71	27
Without	25	33	45	1.48	4
Without	25	26	36	1.46	4
Without	Positive control	150	77	3.88	3.44
With	Solvent control	59	165	0.72	-
With	75	62	150	0.83	86
With	75	72	158	0.92	93
With	100	60	160	0.75	73
With	100	69	162	0.86	91
With	110	50	140	0.71	43
With	110	58	159	0.73	52
With	120	51	131	0.78	27
With	120	51	127	0.81	21
With	130	36	36	2.04	3
With	130	34	64	1.07	7
With	140	59	83	1.41	10
With	Positive control	185	120	3.09	61

Conclusions:

Cytotoxicity and mutagenicity in response to β-Caryophyllene (15- 140 µg/mL) was evaluated both in the presence and absence of S9-metabolic activation, in L5178Y TK+/- 3.7.C mouse lymphoma cells cultures. Authors of the publication assessed test item as positive for genotoxic effects at 25 µg/mL without metabolic activation when evaluated under the old criteria (traditional criteria), and negative for genotoxic effects when evaluated under the new criteria (international harmonisation recommendations). Equivocal results were obtained with activation under both sets of criteria. In all positive cases highly toxic concentrations with a relative total growth of < 10% have been considered. According to current OCED guideline 476, the highest concentration should aim to achieve between 20 and 10% RS and positive results that are only found at 10% RS or below are not recommended to be used for interpretation of results.
When following evaluation criteria of the OECD 476, only concentrations from 15-20 µg/mL (without S9-mix) and 75- 120 µg/mL (with S9-mix) can be evaluated leading to negative results. As not enough valid concentrations are left for evaluation according to OECD 476, the first trial (without S9-mix) should have been repeated.

Executive summary:

The mutagenicity of β-Caryophyllene was determined in vitro using the mouse lymphoma assay (2006). L5178Y TK+/- 3.7.C mouse lymphoma cells were treated with β-Caryophyllene at concentrations of 15, 17.5, 20, 22.5 and 25 µg/mL without S9-activation and 75, 100, 110, 120, 130 and 140 µg/mL with S9-activation. Cells were incubated with the test item for 4 hours, followed by a 48 -hour incubation period in growth medium to allow recovery and mutant expression. Trifluorothymidine was added as a selection agent and the cells were incubated for a further 10 to 12 days. The study included a negative and positive control. Colonies were counted and the size of mutant colonies was determined. According to the authors, the test item was positive for genotoxic effects at 25 µg/mL without activation when evaluated under the old criteria (traditional criteria), and negative for genotoxic effects when evaluated under the new criteria (international harmonisation recommendations). Equivocal results were obtained with activation under both sets of criteria. In all positive cases highly toxic concentrations with a relative total growth of < 10% have been evaluated. When excluding those doses from evaluation, only concentrations from 15-20 µg/mL (without S9-mix) and 75- 120 µg/mL (with S9-mix) can be evaluated leading to negative results.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

In support of the in vitro data, no genotoxicity was observed in two in vivo genotoxicity studies (2009; 2014). The effects of β-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 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. In addition, no genotoxicity or bone marrow cytotoxicity were observed up to 2000 mg/kg β-Caryophyllene in a mouse erythrocyte micronucleus assay (2009). The studies were considered reliable with restriction (Klimisch 2) and sufficient to fulfil the REACH information requirement. The in vivo data further support the conclusion that β-Caryophyllene is not genotoxic.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian germ cell study: cytogenicity / chromosome aberration

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

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.

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OPPTS 870.5915 (In Vivo Sister Chromatid Exchange Assay)

Deviations:

no

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

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source of test material: Sigma Chemicals (St Louis, MO, USA)

Species:

mouse

Strain:

Swiss Webster

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- 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

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

Route of administration:

oral: unspecified

Vehicle:

- Vehicle(s)/solvent(s) used: Corn oil

Duration of treatment / exposure:

25 hours

Frequency of treatment:

Single dose administered

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:

6

Control animals:

yes, concurrent vehicle

Positive control(s):

benzo(a)pyrene
- 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.

Statistics:

Data were statistically analysed with the ANOVA and Tukey tests.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS OF DEFINITIVE STUDY
- 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).

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	0.3±0.1	-	0.5±0.1	-	1.2±0.3	-	-	0.2±0.0
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).

Conclusions:

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.

Reference 2

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

Study conducted similar to OECD Guideline 474, however there are some limitations in the reporting of the 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.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Principles of method if other than guideline:

- Principle of test: In the first test, mice were administered a single dose of the test item. In the second test, mice were administered doses of the test item on 3 consecutive days. Blood samples were analysed by fluorescence microscope.
- Short description of test conditions: The test item was administered at 20, 200 and 2000 mg/kg concentrations in corn oil vehicle. The test included a vehicle control and positive control. Blood samples were taken from the tail of each mouse before the test start and at 24, 48, 72 and 96 hours after administration.
- Parameters analysed / observed: Rate of micronucleated polychromatic erythrocytes and ratio of polychromatic erythrocytes to normochromatic erythrocytes

GLP compliance:

not specified

Type of assay:

other: Mammalian erythrocyte micronucleus test

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source of test material: Sigma Chemicals (St. Lous, MO, U.S.A.)
- Purity: 97%

Species:

mouse

Strain:

other: NIH

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 6 weeks
- Weight at study initiation: 25 g
- Housing: Polypropylene cages
- Diet: Rodent Laboratory chow 5001, ad libitum
- Water: ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature: 23°C
- Photoperiod (hrs dark / hrs light): 12 h dark-light schedule

Route of administration:

oral: unspecified

Vehicle:

- Vehicle(s)/solvent(s) used: Corn oil

Duration of treatment / exposure:

96 hours

Frequency of treatment:

Single administration (first assay) or over three consecutive days (second assay)

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:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

Adriamycin
- Justification for choice of positive control(s): Widely used topoisomerase inhibitor antineoplastic agent known to cause reactive oxygen species, DNA damage and apoptosis. Has also been reported to induce severe chromosome damage in mice by measuring sister chromatic exchanges and micronuclei.
- Route of administration: Intraperitoneal
- Doses / concentrations: 2.5 mg/kg (first assay) and 1.0 mg/kg (second assay)

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: Based on acute oral toxicity test; no mortality or signs of toxicity observed up to 5000 mg/kg.

TREATMENT AND SAMPLING TIMES: Blood samples from the tail of each mouse were obtained before administration and at 24, 48, 72 and 96 hours post-administration.

DETAILS OF SLIDE PREPARATION: Blood was fixed in methanol for 5 minutes and stained for 1 minute with a 0.1% solution of acridine orange made in phosphate buffered saline (PBS), pH 6.8. The slides were rinsed three times with PBS before analysis. Two blood smears per mouse were prepared.

METHOD OF ANALYSIS: Fluorescence microscope with a 420 nm filter. Micronuclei showed green-yellowish fluorescence, polychromatic erythrocytes (PE) have an orange colour and normochromatic erythrocytes show very low fluorescence.

Evaluation criteria:

The capacity of the test item to induce micronuclei was determined in 1000 PE per mouse for each sampling time point. Bone marrow cytotoxicity was determined by calculating the proportion of PE to NE in 1000 erythrocytes per mouse.

Statistics:

Results were statistically evaluated with the ANOVA and the Student t test.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Remarks:

No genotoxic effects up to 2000 mg/kg

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei: See tables 1 and 3.
- Ratio of PE/NE: See tables 2 and 4.
- Statistical evaluation: No statistically significant differences in the rate of micronucleated polychromatic erythrocytes or PE/NE ratio were observed between any of the test item doses and the control.

Table 1. Effect of a single administration of beta-caryophyllene on the mean rate of Micronucleated Polychromatic Erythrocytes (MNPE) in mouse

Time (h)	Corn oil 0.4 mL	Beta-caryophyllene 20 mg/kg	Beta-caryophyllene 200 mg/kg	Beta-caryophyllene 2000 mg/kg	Adriamicin 2.5 mg/kg
0	1.7±0.22	1.6±0.39	1.8±0.37	1.8±0.66	2.8±0.37
24	2.0±0.31	1.4±0.39	1.2±0.48	3.0±0.31	8.6±1.02*
48	1.6±0.26	1.6±0.24	1.4±0.51	2.0±0.70	40.4±1.77*
72	2.3±0.26	1.4±0.6	2.6±0.68	2.2±0.58	50.0±3.18*
96	2.2±0.37	2.4±0.68	1.8±0.48	1.4±0.39	30.6±1.86*

The indicated results correspond to the mean±S.E.M. of 5 mice per dose. 1000 polychromatic erythrocytes scored per mouse.

* Statistically significant difference with respect to the control value ANOVA and Student t tests, p=0.001.

Table 2. Effect of a single administration of beta-caryophyllene on the ratio of Polychromatic Erythrocytes to Normochromatic Erythrocytes in mouse

Time (h)	Corn oil 0.4 mL	Beta-caryophyllene 20 mg/kg	Beta-caryophyllene 200 mg/kg	Beta-caryophyllene 2000 mg/kg	Adriamicin 2.5 mg/kg
0	2.88±0.26	2.90±0.07	2.88±0.07	2.64±0.07	3.12±0.13
24	2.76±0.06	2.58±0.08	3.06±0.22	3.08±0.09	3.12±0.11
48	2.83±0.10	2.60±0.13	2.74±0.17	3.22±0.13	2.50±0.10
72	2.77±0.02	2.60±0.15	2.80±0.11	3.02±0.21	0.28±0.18*
96	2.94±0.06	2.68±0.13	2.94±0.13	3.66±0.15	0.82±0.08*

The indicated results correspond to the mean±S.E.M. of 5 mice per dose. 1000 polychromatic erythrocytes scored per mouse.

* Statistically significant difference with respect to the control value ANOVA and Student t tests, p=0.001.

Table 3. Effect of three administrations of beta-caryophyllene on the mean rate of Micronucleated Polychromatic Erythrocytes (MNPE) in mouse

Time (h)	Corn oil 0.4 mL	Beta-caryophyllene 20 mg/kg	Beta-caryophyllene 200 mg/kg	Beta-caryophyllene 2000 mg/kg	Adriamicin 1.0 mg/kg
0	2.4±0.24	2.4±0.68	2.2±0.37	2.2±0.58	2.5±0.25
24	2.2±0.48	2.2±0.37	1.4±0.39	2.2±0.56	18.0±0.81
48	2.4±0.39	0.8±0.48	1.8±0.37	4.2±0.42	24.0±0.96*
72	2.2±0.48	2.6±0.74	1.6±0.51	4.5±0.77	23.0±1.15*
96	3.0±0.54	2.2±0.37	1.8±0.37	4.7±0.31	11.2±1.05*

The indicated results correspond to the mean±S.E.M. of 5 mice per dose. 1000 polychromatic erythrocytes scored per mouse.

* Statistically significant difference with respect to the control value ANOVA and Student t tests, p=0.001.

Table 4. Effect of three administrations of beta-caryophyllene on the ratio of Polychromatic Erythrocytes to Normochromatic Erythrocytes in mouse

Time (h)	Corn oil 0.4 mL	Beta-caryophyllene 20 mg/kg	Beta-caryophyllene 200 mg/kg	Beta-caryophyllene 2000 mg/kg	Adriamicin 1.0 mg/kg
0	2.46±0.18	2.80±0.15	3.14±0.10	2.78±0.11	2.80±0.09
24	2.58±0.11	2.84±0.18	3.26±0.17	2.32±0.20	2.50±0.13
48	2.78±0.13	2.92±0.16	3.02±0.19	2.80±0.20	2.67±0.08
72	2.76±0.13	3.08±0.20	3.04±0.13	2.85±0.18	2.35±0.05
96	2.66±0.06	3.16±0.18	2.88±0.09	3.12±0.22	1.77±0.11*

The indicated results correspond to the mean±S.E.M. of 5 mice per dose. 1000 polychromatic erythrocytes scored per mouse.

* Statistically significant difference with respect to the control value ANOVA and Student t tests, p=0.001.

Conclusions:

No genotoxicity or bone marrow cytotoxicity were observed up to 2000 mg/kg β-Caryophyllene.

Executive summary:

In the first assay, six-week old male mice (n=5/group) were orally administered β-Caryophyllene (20, 200 or 2000 mg/kg) or a vehicle control (0.1 mg/g of corn oil). Positive control animals received an intraperitoneal injection of Adriamycin (2.5 mg/kg Adriamycin). Blood samples from the tail of each mouse were obtained prior to and 24, 48 and 72 hours post-administration. A second experimental assay was conducted in a comparable manner to investigate the effect on administration of doses on three consecutive days. The capacity of β-caryophyllene to induce micronuclei was determined in 1000 polychromatic erythrocytes (PE) per mouse in each of the five time points of the assays.

 

No genotoxic effect and no bone marrow cytotoxicity was observed up to 2000 mg/kg beta-caryophyllene. Comparable to an OECD Guideline 474 study and published in a peer-reviewed journal, the study was considered to be reliable with restriction (Klimisch 2). However, there are limitations in the reporting of the experimental conditions and results.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

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. However, genotoxicity is a broader term to encapsulate processes which alter the structure, information content or segregation of DNA, which are not necessarily associated with mutagenicity. Covering an array of mechanisms, four in vitro studies and two in vivo studies have been conducted to evaluate the genotoxicity of β-Caryophyllene. All studies were considered sufficiently reliable (Klimisch 2) and sufficient for classification. None of studies indicated any potential for mutagenicity, clastogenicity or cytogenicity. Consequently, in accordance with the CLP Regulation (EC) No. 1272/2008, the test item is not classified as a genetic toxicant.