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Genetic toxicity in vitro

Description of key information

Ames test (according and equivalent to OECD 471): negative (±S9 mix)


HPRT test (according to OECD 476): negative (±S9 mix)


MNT test (according to OECD 487): negative (± S9 mix)

Link to relevant study records

Referenceopen allclose all

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 2012-09-25 to 2013-
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
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. QA statement)
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 1360.0 µg/mL (approx. 10 mM)

Further concentrations please see Table 1
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO; final concentration of DMSO 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 (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: at least 1000 binucleate cells per culture were scored for cytogenetic damage on coded slides. 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
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 was observed in Experiment I at 1360.0 µg/mL in the absence and at 444.1 µg/mL and above in the presence of S9 mix and in Experiment II at 777.1 µg/mL and above in the presence of S9 mix.
- Precipitation: was observed in Experiment II at 777.1 µg/mL and above following 20 h exposure in the absence of S9 mix

RANGE-FINDING/SCREENING STUDIES: yes

COMPARISON WITH HISTORICAL CONTROL DATA: yes
Conclusions:
Under the experimental conditions reported, the test item did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes. Therefore, Methyl Benzoate is considered to be non-clastogenic and non-aneugenic in this in vitro micronucleus test, when tested up to 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 Methyl Benzoate, dissolved in DMSO, was assessed for its potential to induce micronuclei in human lymphocytes in vitro in two independent experiments. The following study design was performed:


 
















































 



Without S9-Mix



With S9-Mix



 



Exp. I



Exp. II



Exp. I and II



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 (1360.0 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 and the occurrence of test item precipitation in accordance with OECD Guideline 487. The evaluated experimental points and the results are summarised in Table 2 (see Attached document). In both cytogenetic experiments, in the absence and presence of S9 mix, no cytotoxicity indicated as cytostasis was observed up to the highest applied concentration. In both independent experiments, neither a statistically significant nor a biologically relevant increase in the number of micronucleated cells was observed after treatment with the test item. Appropriate mutagens were used as positive controls. They induced statistically significant increases in cells with micronuclei. It can be stated that under the experimental conditions reported, the test item did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes. Therefore, Methyl Benzoate is considered to be non-clastogenic and non-aneugenic in this in vitro micronucleus test, when tested up to the highest required concentration.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 2013-01-11 to 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
yes
Remarks:
Deviations are considered to have no impact on the purpose or integrity of the study.
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
yes
Remarks:
Deviations are considered to have no impact on the purpose or integrity of the study.
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Deviations:
yes
Remarks:
Deviations are considered to have no impact on the purpose or integrity of the study.
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and identity of media: Minimal Essential Medium (MEM) supplemented with 10 % foetal bovine serum
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
4- or 24-hour exposure group (-S9 mix): 85, 170, 340, 680, 906.67, 1133.33, 1360
4-hour exposure group (+S9 mix, 2 %): 85, 170, 340, 680, 906.67, 1133.33, 1360
4-hour exposure group (+S9 mix, 1 %): 42.5, 85, 170, 340, 680, 1020, 1360
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: no data
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without S9 mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
Remarks:
with S9 mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: no data
- Exposure duration: 4 h (±S9 mix), 24 h (-S9 mix)
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): 7 days
- Fixation time (start of exposure up to fixation or harvest of cells): 6 or 7 days

SELECTION AGENT (mutation assays): 6-Thioguanine
SPINDLE INHIBITOR (cytogenetic assays): not applicable
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 2x10^5

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency
Evaluation criteria:
The test item is classified as mutagenic if there is a reproducible dose-related increase in the mutation frequency where at least a threefold increase in the mutant frequency over the vehicle control value is observed. Such evaluation may be considered also in the case that a threefold increase of the mutant frequency is not observed.
A test item producing neither a dose-related increase of the mutant frequency nor a reproducible positive response at any of the test points is considered to be non-mutagenic in this system.
A single dose level that meets the minimum criterion for a positive response within a range of assayed concentrations is not sufficient to evaluate the test item as a mutagen.
Statistics:
If a test item gives a marked and dose-related increase in the mutant frequency over the vehicle controls it will be designated as mutagenic and statistical analysis will not be required. However, if weaker responses are observed then statistical analysis will be performed using the SPSS program or a suitable alternative.
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at and above 1133.33 µg/mL and precipitate at and above 906.67 µg/mL
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 1360 µg/mL and precipitate at and above 680 µg/mL
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at and above 906.67 µg/mL and precipitate at and above 680 µg/mL
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at and above 680 µg/mL and precipitate at and above 340 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: no data
- Precipitation: at and above 906.67 µg/mL (4-hour exposure, -S9 mix), at and above 680 µg/mL (4-hour exposure, +S9 mix 2% and 24-hour exposure, -S9 mix), at and above 340 µg/mL (4-hour exposure, +S9 mix 1 %)

RANGE-FINDING/SCREENING STUDIES: yes

COMPARISON WITH HISTORICAL CONTROL DATA: yes
Conclusions:
The test item was shown to be non-mutagenic to V79 cells at the HPRT locus under the conditions of the test.
Executive summary:

The purpose of this study is to assess the potential mutagenicity of a test item on the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus of the V79 cell line. The test methods described are designed to be compatible with the OECD Guidelines for Testing of Chemicals No. 476 "In Vitro Mammalian Cell Gene Mutation Tests", Method B17 of Commission Regulation (EC) No 440/2008 of 30 May 2008 and US EPA OPPTS 870.5300 Guideline.


Chinese hamster (V79) cells were treated with the test item at up to seven dose levels, in duplicate, together with vehicle (solvent) and positive controls in the presence and absence of an S9 metabolic activation system. Four treatment conditions were used for the test, i.e. In Experiment 1, a 4-hour exposure in the presence of an induced rat liver homogenate metabolizing system (S9), at a 2% final concentration and a 4-hour exposure in the absence of metabolic activation (S9). In Experiment 2, the 4-hour exposure with addition of S9 was repeated (using a 1% final S9 concentration); whilst in the absence of metabolic activation the exposure time was increased to 24 hours. The dose range of the test item was selected based on the results of a preliminary cytotoxicity test and were as follows:


















































Exposure Group



Final concentration of test item (µg/mL)



4-hour


(-S9 mix)



85



170



340



680



906.67



1133.33



1360



4-hour (+S9mix, 2 %)



85



170



340



680



906.67



1133.33



1360



24-hour (-S9 mix)



85



170



340



680



906.67



1133.33



1360



4-hour (+S9mix, 1 %)



42.5



85



170



340



680



1020



1360



The vehicle (solvent) controls gave mutant frequencies within the range expected of V79 cells at the HPRT locus. The positive control treatments, both in the presence and absence of metabolic activation, gave significant increases in the mutant frequency indicating the satisfactory performance of the test and of the metabolizing system. The test item demonstrated no significant increases in mutant frequency at any dose level, either with or without metabolic activation, in either the first or second experiment. The test item was shown to be non-mutagenic to V79 cells at the HPRT locus under the conditions of the test.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2022-03-04 to 2022-03-21
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
June 26, 2020
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
dated May 30, 2008
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Target gene:
Trp
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
other: deficiency in the DNA repair process (excision repair damage)
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- source of S9: Phenobarbital/Beta-naphthoflavone induced rat liver
- method of preparation of S9 mix: An appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution, to result in a final concentration of approx. 10% (v/v) in the S9 mix. Cofactors were added to the S9 mix to reach the following concentrations in the S9 mix: 8 mM MgCl2, 33 mM KCl, 5 mM glucose-6-phosphate, 4 mM NADP in 100 mM sodium-ortho-phosphate-buffer, pH 7.4. During the experiment, the S9 mix was stored in an ice bath. The S9 mix preparation was performed according to Ames et al. (1977).
- volume concentration of S9 mix and S9 in the final agar mixture: 10% (v/v) S9 mix at volume of 500 µL
- quality controls of S9: Each batch of S9 was routinely tested for its capability to activate the known mutagens benzo[a]pyrene and 2-aminoanthracene in the Ames test.
Test concentrations with justification for top dose:
Pre-Experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
Experiment II: 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
(top concentration according to OECD TG 471)
Vehicle / solvent:
- Vehicle/solvent used:
DMSO for test item and 2-aminoanthracene, 2-AA
Deionised water for methyl methane sulfonate, MMS

- Justification for choice of solvent/vehicle: The solvents were chosen because of their solubility properties and its relative nontoxicity to bacteria.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
other: 2-aminoanthracene, 2-AA
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments: 2

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: 10E8-10E9 cells/mL
- Test substance added in agar (plate incorporation, preincubation)

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: 60 min
- Exposure duration/duration of treatment: 48 hours at 37°C ± 1.5°C in the dark


METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: background growth inhibition


METHODS FOR MEASUREMENTS OF GENOTOXICITY
The colonies were counted using a validated computer system and compared to the spontaneous reversion rates.

ACCEPTABILITY CRITERIA
The Salmonella typhimurium and Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:
• regular background growth in the negative and solvent control;
• the spontaneous reversion rates in the negative and solvent control are in the range of our historical data;
• the positive control substances should produce an increase above the threshold of twofold the colony count of the corresponding solvent control;
• a minimum of five analysable dose levels should be present with at least three dose levels showing no signs of toxic effects, evident as a reduction in the number of revertants below the indication factor of 0.5.

Evaluation criteria:
A test item is considered as a mutagen if a biologically relevant increase in the number of revertants of twofold or above the spontaneous mutation rate of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is reached or exceeded at more than one concentration.
An increase of revertant colonies equal or above the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.
Statistics:
According to the OECD TG 471, a statistical analysis of the data is not mandatory.
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
not applicable
Genotoxicity:
not determined
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
not examined
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
not examined
Species / strain:
S. typhimurium TA 98
Metabolic activation:
not applicable
Genotoxicity:
not determined
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
not examined
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
not examined
Species / strain:
S. typhimurium TA 100
Metabolic activation:
not applicable
Genotoxicity:
not determined
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
not examined
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
not examined
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
not applicable
Genotoxicity:
not determined
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
not examined
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
not examined
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
In the pre-experiment the concentration range of the test item was 3 – 5000 µg/plate. The pre-experiment is reported as experiment I. Since minor toxic effects were observed in experiment I, seven concentrations were tested in experiment II. 5000 µg/plate were chosen as maximal concentration. The concentration range included two logarithmic decades.
The following concentrations were tested in experiment II: 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

HISTORICAL CONTROL DATA
Please refer to Table 3 in "Any other information on results incl. tables"

MAIN STUDY
The test item precipitated in the overlay agar in the test tubes in experiment I at 5000 µg/plate in the presence of S9 mix only and in experiment II at 5000 µg/plate in the presence and absence of S9 mix. Precipitation of the test item in the overlay agar on the incubated agar plates was observed in experiment I at 5000 µg/plate in the presence and absence of S9 mix and in experiment II at 5000 µg/plate in the absence of S9 mix and from 2500 to 5000 µg/plate in the presence of S9 mix. The undissolved particles had no influence on the data recording.
The plates incubated with the test item showed reduced background growth at the following concentrations (µg/plate):
Experiment I Experiment II
without S9 mix with S9 mix without S9 mix with S9 mix
/ 5000 5000 2500 – 5000
/ = normal background growth
Toxic effects, evident as a reduction in the number of revertants (below the induction factor of 0.5), were observed at the following concentrations (µg/plate):

Experiment I Experiment II
without S9 mix with S9 mix without S9 mix with S9 mix
5000 5000 5000 2500 – 5000

No substantial increase in revertant colony numbers was observed following treatment with Methyl Benzoate at any concentration level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.
Appropriate reference mutagens were used as positive controls. They showed a distinct in-crease in induced revertant colonies.

Table 1   Summary of Experiment I


 






















































































































































































Metabolic


Activation



Test


Group



Dose Level


(per plate)



 



Revertant Colony Counts (Mean ±SD)



 



 



 



 



 



 



 



 



 



WP2 uvrA



 



 



 



 



 



Without Activation



DMSO



 



 



50 ± 2



Untreated



 



 



51 ± 15



Methyl Benzoate



3 µg



 



48 ± 6



 



10 µg



 



52 ± 8



 



33 µg



 



49 ± 5



 



100 µg



 



50 ± 7



 



333 µg



 



50 ± 6



 



1000 µg



 



49 ± 5



 



2500 µg



 



31 ± 1



 



5000 µg



 



22 ± 1 P



MMS



2.0 µL



 



971 ± 43



 



 



 



 



 



With Activation



DMSO



 



 



62 ± 7



Untreated



 



 



64 ± 5



Methyl Benzoate



3 µg



 



62 ± 3



 



10 µg



 



56 ± 3



 



33 µg



 



54 ± 13



 



100 µg



 



61 ± 2



 



333 µg



 



65 ± 2



 



1000 µg



 



58 ± 4



 



2500 µg



 



33 ± 8



 



5000 µg



 



18 ± 3 P R



2-AA



10.0 µg



 



375 ± 54



 



 



 



 



 



 


























Key to Positive Controls



Key to Plate Postfix Codes



 



 



MMS


2-AA



methyl methane sulfonate


2-aminoanthracene



P


R



Precipitate


Reduced background growth


    

 


 


Table 2  Summary of Experiment II


 










































































































































































Metabolic


Activation



Test


Group



Dose Level


(per plate)



 



Revertant Colony Counts (Mean ±SD)



 



 



 



 



 



 



 



 



 



WP2 uvrA



 



 



 



 



 



Without Activation



DMSO



 



 



51 ± 2



Untreated



 



 



43 ± 3



Methyl Benzoate



10 µg



 



50 ± 10



 



33 µg



 



43 ± 1



 



100 µg



 



55 ± 8



 



333 µg



 



53 ± 4



 



1000 µg



 



37 ± 3



 



2500 µg



 



23 ± 4



 



5000 µg



 



18 ± 5 P R



MMS



2.0 µL



 



690 ± 71



 



 



 



 



 



With Activation



DMSO



 



 



56 ± 4



Untreated



 



 



52 ± 8



Methyl Benzoate



10 µg



 



50 ± 10



 



33 µg



 



52 ± 7



 



100 µg



 



46 ± 5



 



333 µg



 



60 ± 14



 



1000 µg



 



44 ± 6



 



2500 µg



 



19 ± 2 P R



 



5000 µg



 



3 ± 1 P R



2-AA



10.0 µg



 



258 ± 26



 



 



 



 



 



 


























Key to Positive Controls



Key to Plate Postfix Codes



 



 



MMS


2-AA



methyl methane sulfonate


2-aminoanthracene



P


R



Precipitate


Reduced background growth


    

 


Table 3 Historical Data


These data represent the laboratory´s historical control data from February 2020 until February 2022 representing approx. 600 experiments (WP2 uvrA the historical data are based on approx. 300 experiments).












































































































































































































Strain



 



without S9 mix



with S9 mix



 



 



Mean



SD



Min



Max



Mean



SD



Min



Max



 



Solvent control



12



2.3



7



21



12



2.3



7



23



TA 1535



Untreated control



12



2.8



7



21



12



2.6



7



22



 



Positive control



1130



127.7



356



1612



288



49.7



166



428



 



Solvent control



12



2.3



7



20



14



2.9



7



27



TA 1537



Untreated control



12



2.7



6



21



14



3.0



7



28



 



Positive control



90



22.0



52



400



363



82.7



145



589



 



Solvent control



28



4.8



17



49



38



7.1



19



62



TA 98



Untreated control



30



4.7



15



47



40



7.1



19



64



 



Positive control



554



228.5



217



1277



3092



659.3



364



5290



 



Solvent control



115



19.0



72



212



116



21.1



73



213



TA 100



Untreated control



120



20.8



66



225



121



23.8



68



211



 



Positive control



1736



234.4



542



2437



3541



795.0



1092



5335



 



Solvent control



45



6.7



28



63



51



7.8



30



71



WP2 uvrA



Untreated control



46



6.4



27



66



54



7.7



28



72



 



Positive control



858



163.7



382



1279



298



84.5



177



985



Mean = mean value of revertants/plate
SD = standard deviation
Min = minimal value
Max = maximal value


 

Conclusions:
In the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes in the genome of the Escherichia coli strain WP2 uvrA.
Therefore, the test item is considered to be non-mutagenic in this bacterial reverse mutation assay.
Executive summary:

This study was performed to investigate the potential of the test item to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Escherichia coli strain WP2 uvrA.


The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations:


Pre-Experiment/Experiment I:        3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate


Experiment II:                                 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate


The test item precipitated in the overlay agar in the test tubes in experiment I at 5000 µg/plate in the presence of S9 mix only and in experiment II at 5000 µg/plate in the presence and absence of S9 mix. Precipitation of the test item in the overlay agar on the incubated agar plates was observed in experiment I at 5000 µg/plate in the presence and absence of S9 mix and in experiment II at 5000 µg/plate in the absence of S9 mix and from 2500 to 5000 µg/plate in the presence of S9 mix. The undissolved particles had no influence on the data recording.


The plates incubated with the test item showed reduced background growth in experiment I in the presence of S9 mix only and in experiment II in the presence and absence of S9 mix.


Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in the presence and absence of S9 mix in both experiments.


No substantial increase in revertant colony numbers strains was observed following treatment with the test item at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.


Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.


 


In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes in the genome of the Escherichia coli strain WP2 uvrA.


Therefore, the test item is considered to be non-mutagenic in this bacterial reverse mutation assay.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Genetic toxicity in-vitro


Five genetic toxicity in-vitro assays were conducted with the test substance covering different modes of action for genetic toxicity.



  1. AMES (gene mutation in bacteria)


A weight-of-evidence approach was carried out for the assessment of gene mutation in bacterial cells using three AMES tests in different Escherichia coli and Salmonella typhimurium strains.


Szybalski, 1958


The in-vitro genotoxicity of the test substance methyl benzoate was studied in a non-GLP study in a bacterial reverse mutation assay using Escherichia coli. Assays were conducted by a paper-disk method in which filter paper disks saturated with the test substance (0.01 to 0.025 mL) were placed on the surface of streptomycin-free nutrient agar seeded with a streptomycin-dependent parental population. This population consisted of E. coli strain Sd-4 -73 grown overnight at 36 °C in aerated nutrient broth containing excess streptomycin. Cultures were then centrifuged, washed and re-suspended to obtain cultures with an initial cell density of 10E09 cells/mL, or diluted suspensions (one-fifth or one twenty-fifth of the initial cell concentration). Aliquots of 0.1 mL of the cell suspensions were mixed with streptomycin-free agar before the experiments. The increase in the frequency of reversion from streptomycin dependence to independence in E. coli strain Sd-4-73 was used as a measure of mutagenicity. Mutagenicity was manifested as a zone of streptomycin-independent mutant, colonies around the filter paper disk saturated with the test substance. No such zones were found in the experiments conducted with methyl benzoate and the substance was considered non-mutagenic under the conditions of the test.


US National Toxicology Programm, 1987 (Zeiger et al. 1992)


The genotoxic potential of the test substance methyl benzoate was studied in a bacterial reverse mutation assay (Ames test) under GLP and according to principles similar to those of OECD TG 471. Five bacterial strains (Salmonella typhimurium) including TA 97, TA 98, TA 100, TA 1535 and TA 1537 were tested. However, the study deviated from the guideline in that TA 102 or one other bacterial strain (e.g. Escherichia coli, strain WP2 uvrA) was not included and thus certain mutagen detection possibilities were omitted. The tests were conducted in the absence and presence of a metabolic activation system (S9 mix containing 10% or 30% metabolic activation enzymes and cofactors from Aroclor 1254-induced male Sprague-Dawley rat or Syrian hamster liver). In each test, a tube containing a suspension of one bacterial strain plus S9 mix or plain buffer without S9 mix was incubated for 20 minutes at 37 °C with the test substance. Control cultures, with all the ingredients except the test substance, were also incubated. Additionally, positive control cultures were prepared containing a known potent mutagen. After the pre-incubation period, agar was added to the cultures and the contents of the tubes were mixed and poured onto the surface of Petri dishes containing standard bacterial culture medium. The plates were then further incubated and colonies were usually counted after two days. The substance methyl benzoate did not induce increases in the number of revertant colonies in the absence or presence of the S9 mix in any of the tester strains. It was concluded that the substance was non-genotoxic under the conditions of this test.


Chang, 2022


This study was performed to investigate the potential of the test item to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Escherichia coli strain WP2 uvrA. The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations: Pre-Experiment/Experiment I:  3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate; Experiment II: 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate.


The test item precipitated in the overlay agar in the test tubes in experiment I at 5000 µg/plate in the presence of S9 mix only and in experiment II at 5000 µg/plate in the presence and absence of S9 mix. Precipitation of the test item in the overlay agar on the incubated agar plates was observed in experiment I at 5000 µg/plate in the presence and absence of S9 mix and in experiment II at 5000 µg/plate in the absence of S9 mix and from 2500 to 5000 µg/plate in the presence of S9 mix. The undissolved particles did not influence the data recording. The plates incubated with the test item showed reduced background growth in experiment I in the presence of S9 mix only and in experiment II in the presence and absence of S9 mix. Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in the presence and absence of the S9 mix in both experiments.


No substantial increase in revertant colony numbers strains was observed following treatment with the test item at any dose level neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency for higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies. In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes in the genome of the Escherichia coli strain WP2 uvrA. Therefore, the test item is considered to be non-mutagenic in this bacterial reverse mutation assay.


Taken together, the test item did not show mutagenic properties in any of the tested Escherichia coli and Salmonella typhimurium strains with and without metabolic activation. Therefore, Methyl benzoate was considered non-mutagenic in bacterial cells.



  1. HPRT (gene mutation in mammalian cells)


Morris, 2013


This study aims to assess the potential mutagenicity of a test item on the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus of the V79 cell line. The test methods described are designed to be compatible with the OECD Guidelines for Testing of Chemicals No. 476 "In-vitro Mammalian Cell Gene Mutation Tests", Method B17 of Commission Regulation (EC) No 440/2008 of 30 May 2008 and US EPA OPPTS 870.5300 Guideline.


Chinese hamster (V79) cells were treated with the test item at up to seven dose levels, in duplicate, together with vehicle (solvent) and positive controls in the presence and absence of an S9 metabolic activation system. Four treatment conditions were used for the test, i.e. In Experiment 1, a 4-hour exposure in the presence of an induced rat liver homogenate metabolizing system (S9), at a 2% final concentration and a 4-hour exposure in the absence of metabolic activation (S9). In Experiment 2, the 4-hour exposure with the addition of S9 was repeated (using a 1% final S9 concentration); whilst in the absence of metabolic activation the exposure time was increased to 24 hours. The dose range of the test item was selected based on the results of a preliminary cytotoxicity test. The vehicle (solvent) controls gave mutant frequencies within the range expected of V79 cells at the HPRT locus. The positive control treatments, both in the presence and absence of metabolic activation, showed significant increases in the mutant frequency, indicating the satisfactory performance of the test and the metabolizing system. The test item demonstrated no significant increases in mutant frequency at any dose level, either with or without metabolic activation, in either the first or second experiment. The test item was shown to be non-mutagenic to V79 cells at the HPRT locus under the conditions of the test.



  1. MNT (chromosome aberration)


Bohnenberger, 2013


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 Methyl Benzoate, dissolved in DMSO, was assessed for its potential to induce micronuclei in human lymphocytes in-vitro in two independent experiments.


 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 (1360.0 µg/mL of the test item, approx. 10 mM) was chosen about 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 and the occurrence of test item precipitation in accordance with OECD Guideline 487. The evaluated experimental points and the results are summarised in Table 2 (see Attached document). In both cytogenetic experiments, in the absence and presence of the S9 mix, no cytotoxicity was indicated as cytostasis was observed up to the highest applied concentration. In both independent experiments, neither a statistically significant nor a biologically relevant increase in the number of micronucleated cells was observed after treatment with the test item. Appropriate mutagens were used as positive controls. They induced statistically significant increases in cells with micronuclei. The test item did not induce micronuclei as determined by the in-vitro micronucleus test in human lymphocytes under the experimental l conditions reported. Therefore, Methyl Benzoate is considered to be non-clastogenic and non-aneugenic in this in-vitro micronucleus test, when tested up to the highest required concentration.


Conclusion


In conclusion, the test substance did not produce significant genetic toxicity in the reliable bacterial reverse mutation assays with S. Typhimurium and E. coli, in an in-vitro micronucleus assay with human lymphocytes and in an in-vitro Gene Mutation Assay in Chinese Hamster V79 Cells (V79/HPRT). It is therefore concluded that the substance is not genotoxic. Further testing on the genetic toxicity of Methyl benzoate is not required.

Justification for classification or non-classification

Based on the above-stated assessments of genotoxicity, the test substance Methyl benzoate is not considered genotoxic and does not need to be classified according to CLP (Regulation (EC) No 1272/2008).