Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information
Ames test: negative (±S9 mix) HPRT test: negative (±S9 mix) MNT test: negative (± S9 mix)
Link to relevant study records
Reference
Endpoint:
in vitro cytogenicity / micronucleus study
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 2012-09-11 to 2013-
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study according to guideline
Qualifier:
according to
Guideline:
other: OECD 487
Deviations:
yes
Remarks:
To get proper responses of statistical significance when using the specified positive controls the test design, specifically for the treatment, the expression phase and harvest time, was slightly modified compared to the OECD Guideline 487.
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian cell micronucleus test
Species / strain / cell type:
lymphocytes: human
Details on mammalian cell type (if applicable):
- Type and identity of media: DMEM:F12 (Dulbecco's modified eagle medium/Ham's F12 medium, mixture 1:1) supplemented with 200 mM GlutaMax
- Properly maintained: yes
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Mammalian Microsomal Fraction S9 Mix
Test concentrations with justification for top dose:
pre-test on cytotoxicity: up to 1983.0 µg/mL (approx. 10 mM)

Further concentrations please see Table 1
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol; final concentration of ethanol in the culture medium was 0.5 % (v/v)
- Justification for choice of solvent/vehicle: The solvent was chosen due to its solubility properties and its relative non-toxicity to the cell cultures.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
Without metabolic activation
Positive control substance:
mitomycin C
other: Demecolcin
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
With metabolic activation
Positive control substance:
cyclophosphamide
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 48 h
- Exposure duration: 4 h with and without S9 mix (experiment I) or 4 h with S9 mix and 20 h without S9 mix (experiment II: A and B) (see Table 1)
- Expression time (cells in growth medium): cells exposed for 4 h have 16 h recovery period before fixation (expression phase), no recovery period for 20 h exposure cells
- Selection time (if incubation with a selection agent): 20 h with Cytochalasin B (4 µg/mL)
- Cells were prepared 40 h after start of the exposure

SPINDLE INHIBITOR (cytogenetic assays): Cytochalasin B (4 µg/mL)
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED:
cytotoxic effect the CBPI: ca 500 cells per culture and cytotoxicity is expressed as % cytostasis
micronuclei effects: 1000 - 2000 binucleate cells per culture. The frequency of micronucleated cells was reported as % micronucleated cells

DETERMINATION OF CYTOTOXICITY
- percentages of reduction in the CBPI (cytokinesis-block proliferating index) in comparison with the controls (% cytostasis) by counting 500 cells per culture in duplicate
Evaluation criteria:
A test item can be classified as non-clastogenic and non-aneugenic if:
- the number of micronucleated cells in all evaluated dose groups is in the range of the laboratory historical control data and/or
- no statistically significant or concentration-related increase in the number of micronucleated cells is observed.

A test item can be classified as clastogenic or aneugenic if:
- the number of micronucleated cells is not in the range of the historical laboratory control data and
- either a concentration-related increase of micronucleated cells in three test groups or a statistically significant increase of the number of micronucleated cells is observed.
Statistics:
Statistical significance was confirmed by means of the Chi square test.
Species / strain:
lymphocytes: human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
lymphocytes: human
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at and above 211.4 µg/mL
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
lymphocytes: human
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
lymphocytes:
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at and above 180.0 µg/mL
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no
- Effects of osmolality: no
- Evaporation from medium: no data
- Water solubility: phase separation in Experiment I and IIA at and above 211.4 µg/mL with and without S9 mix; in Experiment IIB at and above 220.0 µg/mL without S9 mix.
- Precipitation: no
- Other confounding effects: no

RANGE-FINDING/SCREENING STUDIES: yes

COMPARISON WITH HISTORICAL CONTROL DATA: yes
Remarks on result:
other:
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
negative

Under the experimental conditions reported, the test item did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes. Therefore, Menthyl Acetate Racemic is considered to be non-aneugenic and non-clastogenic when tested upto cytotoxic, the highest evaluable or the highest required concentration.
Executive summary:

The study was conducted according to OECD Guideline 487. The expression phase and harvest time were slightly modified compared to the OECD Guideline 487. The test item Menthyl Acetate Racemic, dissolved in ethanol, was assessed for its potential to induce micronuclei in human lymphocytes in vitro in three independent experiments. The following study design was performed:

 

Without S9-Mix

With S9-Mix

 

Exp. I

Exp. IIA & IIB

Exp. I and IIA

Exposure period

 4 hrs

20 hrs

 4 hrs

Recovery

16 hrs

-

16 hrs

Cytochalasin B exposure

20 hrs

20 hrs

20 hrs

Preparation interval

40 hrs

40 hrs

40 hrs

Total culture period

88 hrs

88 hrs

88 hrs

In each experimental group two parallel cultures were analysed. 1000 binucleate cells per culture were evaluated for cytogenetic damage. The highest applied concentration in this study (1983.0 µg/mL of the test item, approx. 10 mM) was chosen with regard to the molecular weight of the test item and with respect to the guideline. Dose selection of the cytogenetic experiment was performed considering the toxicity data in accordance with the guideline. The evaluated experimental points and the results are summarised in Table 2 (see Attached document). In Experiment I the absence and presence of S9 mix and in Experiment IIA in the presence of S9 mix, no cytotoxicity was observed up to the highest applied concentration. In Experiment IIA in the absence of S9 mix concentrations showing clear cytotoxicity were not evaluable for cytogenetic damage. In Experiment IIB in the absence of S9 mix clear cytotoxicity was observed at the highest evaluated concentration. In the absence and the presence of S9 mix, no increase in the number of micronucleated cells was observed after treatment with the test item. One single statistically significant increase in micronucleated cells was observed in Experiment IIA after continuous treatment with 120.8 µg/mL in the absence of S9 mix (0.85 %). The value was clearly within the laboratory historical control data range (0.05 – 1.45 % micronucleated cells) and thus considered as being biologically irrelevant. Appropriate mutagens were used as positive controls. They induced statistically significant increases in cells with micronuclei. Under the experimental conditions reported, the test item did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes. Therefore, Menthyl Acetate Racemic is considered to be non-aneugenic and non-clastogenic in this in vitro micronucleus test, when tested up to cytotoxic, the highest evaluable or the highest required concentration.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

Three genetic toxicity in vitro assays were conducted with the substance covering different modes of action of genetic toxicity. The substance did not produce significant genetic toxicity in a reliable bacterial reverse mutation assay with S. typhimurium and E. coli, in an in vitro Gene Mutation Assay in Chinese Hamster V79 Cells (V79/HPRT) and in an in vitro micronucleus assay with human lymphocytes. It is therefore concluded that the substance is not genotoxic. Further testing on the genetic toxicity of Menthyl Acetate is not required.


Justification for selection of genetic toxicity endpoint
GLP and guideline study performed in mammalian cells to detect the activity of clastogenic and aneugenic chemicals.

Justification for classification or non-classification

Based on the above stated assessments on genotoxicity the test substance Menthyl Acetate is not considered to be genotoxic and does not need to be classified according to Council Directive 2001/59/EC (28th ATP of Directive 67/548/EEC) and according to CLP (Regulation (EC) No 1272/2008 of the European Parliament and of the Council) as implementation of UN-GHS in the EU.