Star anise, Illicium verum, ext.

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2019

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted 21. Jul. 1997

Deviations:

not specified

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

adopted 30. May 2008

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

(E)-anethole

EC Number:

224-052-0

EC Name:

(E)-anethole

Cas Number:

4180-23-8

Molecular formula:

C10H12O

IUPAC Name:

1-methoxy-4-prop-1-en-1-ylbenzene

Constituent 2

Reference substance name:

4-allylanisole

EC Number:

205-427-8

EC Name:

4-allylanisole

Cas Number:

140-67-0

Molecular formula:

C10H12O

IUPAC Name:

1-allyl-4-methoxybenzene

1

Reference substance name:

Caryophyllene

EC Number:

201-746-1

EC Name:

Caryophyllene

Cas Number:

87-44-5

Molecular formula:

C15H24

IUPAC Name:

4,11,11-trimethyl-8-methylenebicyclo[7.2.0]undec-4-ene

Constituent 3

Reference substance name:

2,6-dimethyl-6-(4-methyl-3-pentenyl)bicyclo[3.1.1]hept-2-ene

EC Number:

241-702-9

EC Name:

2,6-dimethyl-6-(4-methyl-3-pentenyl)bicyclo[3.1.1]hept-2-ene

Cas Number:

17699-05-7

Molecular formula:

C15H24

IUPAC Name:

2,6-dimethyl-6-(4-methylpent-3-en-1-yl)bicyclo[3.1.1]hept-2-ene

Test material form:

liquid

Method

Target gene:

Salmonella typhimurium TA97a: 4997D
Salmonella typhimurium TA98: 5011D
Salmonella typhimurium TA100: 4996D
Salmonella typhimurium TA102: 4982D
Salmonella typhimurium TA1535: 5012D

Species / strain

Species / strain 1

Species / strain / cell type:

other: Salmonella typhimurium LT2 TA97a

Additional strain / cell type characteristics:

not specified

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 98

Additional strain / cell type characteristics:

not specified

Species / strain 3

Species / strain / cell type:

S. typhimurium TA 100

Additional strain / cell type characteristics:

not specified

Species / strain 4

Species / strain / cell type:

S. typhimurium TA 102

Additional strain / cell type characteristics:

not specified

Species / strain 5

Species / strain / cell type:

S. typhimurium TA 1535

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

metabolic activation by microsomal enzymes (e.g., benzo(a)pyrene)

Test concentrations with justification for top dose:

Experiment 1a: 5 / 1.5 / 0.5 / 0.15 / 0.05 µL/plate for TA97a, TA98, TA100, TA102, TA1535;
Second Experiment: 1.5 / 0.5 / 0.15 / 0.05 / 0.015 / 0.005 µL/plate for TA97a, TA100; 0.5 / 0.15 / 0.05 / 0.015 / 0.005 / 0.0015 µL/plate for TA98.

Vehicle / solvent:

dimethyl sulfoxide (DMSO)
Justification: because the test item was sufficiently soluble and this solvent does not have any effects on the viability of the bacteria or the number of spontaneous revertants in the tested con-centrations.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive control substance:

sodium azide

benzo(a)pyrene

other: 4-Nitro-1,2-phenylene Diamine (CAS#99-56-9) and 2-Amino-Anthracene (CAS#613-13-8)

Details on test system and experimental conditions:

METHOD OF APPLICATION: In this study the plate incorporation method was applied. The study was divided into 2 main phases: 1) Preliminary cytotoxicity; 2) Ames test.

DURATION
- Preincubation period: 8 hours at 37 ± 1 °C
- Exposure duration: Experiment 1a (48 hours at 37 ±1 °C), Second Experiment (48 hours at 37 ±1 °C)
- Expression time (cells in growth medium): 24 hours at 37 ±1 °C

SELECTION AGENT (mutation assays): mutagenic substances: 4-Nitro-1,2-phenylene diamine (CAS#99-56-9), Sodium azide (CAS#26628-22-8), 2-Amino-anthracene(CAS#613-13-8) and Benzo-a-pyrene(CAS#50-32-8)

NUMBER OF REPLICATIONS: 3

Rationale for test conditions:

All negative and nearly all strain-specific positive control values (only one marginal ex-ception) were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

other: Salmonella typhimurium LT2 TA97a

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

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:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

Based on the results of this study it is concluded that Essential oil of Star Anise Oil (Illicium verum) obtained from the leaves and fruits by steam distillation is mutagenic in the Salmonella typhimurium strain TA1535 in the absence and presence of meta-bolic activation under the experimental conditions in this study.

Executive summary:

Two valid experiments were performed.

 

The study procedures described in this report were based on the most recent OECD and EC guidelines.

The test itemEssential oil of Star Anise Oil (Illicium verum) obtained from the leaves and fruits by steam distillationwas tested in theSalmonella typhimuriumreverse mutation assay with five strains ofSalmonella typhimurium(TA97a, TA98, TA100, TA102 and TA1535).

The test was performed in two experiments in the presence and absence of metabolic activation, with +S9 standing for presence of metabolic activation, and –S9 standing for absence of metabolic activation.

 

Experiment 1a:

In the first experiment,the test item (dissolved in DMSO) was tested up to concentrations of 5 µL/plate in the absence and presence of S9-mix in the strains TA97a, TA98, TA100, TA102 and TA1535 using the plate incorporation method.

The test item showed no precipitates on the plates at any of the concentrations.

Signs of toxicity were observed towards the following bacteria strains in the respective concentrations:

·        Bacteria strain TA97a: 5 and 1.5 µg/plate (decrease in the number of revertants)

·        Bacteria strain TA98: 5, 1.5 and 0.5 µg/plate (decrease in the number of revertants)

·        Bacteria strain TA100: 5 and 1.5 µg/plate (decrease in the number of revertants)

 

The bacterial background lawn was not reduced at any of the concentrations.

The results of this experiment showed that two of the tested concentrations showed an increase in the number of revertants in the tested strain TA1535, in the presence and the absence of metabolic activation. In the treatment with metabolic activation of the third concentration (0.5 µL/plate), an increase in the number of revertants in the tested strain TA1535 was observed, too.

The test item induced a dose-related increase in the number of revertants colonies in the bacteria strain TA1535, in the presence and absence of metabolic activation.

 

 

Experiment 1b:

Based on the toxicity results of the experiment 1a,the test item was tested up to concentrations of 1.5 µL/plate in the absence and presence of S9-mix in the bacteria strains TA97a and TA100 and up to concentrations of 0.5 µL/plate in the absence and presence of S9-mix in the bacteria strain TA98 using the plate incorporation method.

The test item showed no precipitates on the plates at any of the concentrations.

Signs of toxicity were observed in the following concentrations:

·        Bacteria strain TA97a: 1.5 µL/plate (decrease in the number of revertants)

·        Bacteria strain TA98: 0.5 µL/plate (decrease in the number of revertants)

Bacteria strain TA100: 1.5 µL/plate (decrease in the number of revertants)

 

The results of this experiment showed that the test item caused no increase in the number of revertants in these three bacteria strains compared to the solvent control, in both the absence and presence of metabolic activation. The test item did not induce a dose-related increase in the number of revertants colonies in all strains, in the presence and absence of metabolic activation in experiment 1b.

 

Based on the results of this study it is concluded thatEssential oil of Star Anise Oil (Illicium verum) obtained from the leaves and fruits by steam distillationis mutagenic in theSalmonella typhimuriumstrain TA1535 in the absence and presence of metabolic activation under the experimental conditions in this study.