Star anise, Illicium verum, ext.

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study planned

Study period:

2021.1.1 - 2022.1.1

Justification for type of information:

Public substance name: Star anise, Illicium verum, ext.
EC Number: 283-518-1
CAS Number: 84650-59-9

Date of considerations: 29 June 2020

Hazard endpoint for which vertebrate testing was proposed:

In vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Considerations that the general adaptation possibilities of Annex XI of the REACH Regulation were not adequate to generate the necessary information
(instruction: please address all points below):

Available GLP studies

No available GLP studies on the substance for the endpoint ‘Genetic toxicity in vivo’.

Available non-GLP studies

No available non-GLP studies on the substance for the endpoint ‘Genetic toxicity in vivo’.

Historical human data

No human data suggesting Genetic toxicity in vivo are available for this substance.

(Q)SAR

No validated (Q)SAR exists for this endpoint. There is no known mode of action for Star anise, Illicium verum, ext. causing Genetic toxicity in vivo.

In vitro methods

In accordance with ECHA’s guidance on the information requirements and chemical safety assessment. With regards to studies for Genetic toxicity in vivo, the regulatory acceptance of these studies and approaches to replace the animal testing for Genetic toxicity in vivo has not been achieved as they do not provide equivalent information and thus, cannot be used alone for classification and labeling and/or risk assessment.

Weight of evidence

No available ‘weight of evidence’ data on the substance for the endpoint ‘Genetic toxicity in vivo’.

Grouping and read-across

No data exists on analogous substances. It is the intention to use test data from Genetic toxicity in vivo for further read-across to similar substances thus avoiding the need to perform further testing.

Substance-tailored exposure driven testing [if applicable]

Not applicable

Approaches in addition to above [if applicable]

Not applicable

Other reasons [if applicable]

Not applicable

Considerations that the specific adaptation possibilities of Annexes VI to X (and column 2 thereof) were not applicable (instruction: free text):

Adaptation options as defined in Annexes VI to X were not applicable for this substance and this endpoint.

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes

Type of assay:

mammalian erythrocyte micronucleus test

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

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Administration / exposure

Route of administration:

oral: gavage

Details on exposure:

All treated animals were exposed to test or control materials via the oral dose route.
The rats were weighed immediately before each exposure event and the dose volume adjusted using a graded syringe fitted with a gavage needle.
Treated animals were exposed to dosing formulations at a dose volume of 10 mL/kg/day.

Duration of treatment / exposure:

48h

Doses / concentrations

Dose / conc.:

2 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

7M+7F

Control animals:

yes

Positive control(s):

cyclophosphamide

Examinations

Details of tissue and slide preparation:

Rats were killed by increasing levels of carbon dioxide. The marrow was flushed out using a 1:1 mixture of foetal calf serum and trisodium citrate (0.8 %, w/v) in Sorenson's buffer (pH 6.8). Routine tissue culture antibiotics were included to prevent microbial growth. This mildly hypotonic treatment served to make the micronuclei clearly visible and to distinguish them from surrounding artefacts. Following completion of the sampling procedure the contents of the tubes were briefly agitated on a vortex mixer to allow separation of the cells.
The tubes were centrifuged to pellet the cells. The supernatant fluid was discarded and the cells were then resuspended on a vortex mixer in the residual liquid.
Clean slides were assigned numbers corresponding to the tube numbers. A drop of the suspension was placed at one end of the slide and a smear made by drawing the top of a Pasteur pipette horizontally along the slide. Two slides were prepared from each tube (animal). The smear was left to air dry, fixed in methanol (for at least 5 min) and then stained with Giemsa stain (15%, v/v, in water) to give optimum erythrocyte discrimination. Permanent slide preparations were made by sealing cover slips onto the glass slides using mounting medium.

Evaluation criteria:

One of the prepared slides was selected for examination and the coded slides assessed blind by the same operator. At least two thousand polychromatic erythrocytes (PCE) per animal were scored for micronuclei and the frequency of micronucleated cells (MN-PCE) determined.
As a control against inclusion of artefacts, or action of a mutagen on the G2 and/or mitotic phase of the cell cycle, the numbers of micronucleated normochromatic erythrocytes (MN-NCE) in mature red blood corpuscles were also recorded (Maier and Schmid, 1976; Hamoud et al, 1989). In addition, scored micronuclei were assigned on the basis of size into small or large categories, historically defined as micronuclei occupying less or more than 25% of the visible cellular area. This classification provided a non-specific measure of compound induced spindle dysfunction, as large micronuclei appear to derive from lagging chromosomes caused by damage to the mitotic apparatus during bone marrow erythropoiesis (Yamamoto and Kikuchi, 1980; Vanderkerken et al, 1989). Any anomalies observed, such as large micronuclei, would be reported in the Appendix of the report.
The PCE/NCE ratio, a measure of any induced systemic toxicity, was determined by counting a minimum total of 1000 erythrocytes (PCE + NCE) per marrow preparation.
A suitable binocular microscope was used for the assessment. The scoring was done under a nominal magnification of x1250 using x12.5 magnification eye pieces and a x100 oil immersion objective.