Methyl benzoylformate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

All key studies in this section were carried out according to guideline and with GLP compliance.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

his, trp

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital i.p. and β-naphthoflavone induced liver S9 mix from rats

Test concentrations with justification for top dose:

33 μg - 5 200 μg/plate (SPT)
33 μg - 5 200 μg/plate (PIT)

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

with S9 mix

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

other: 4-nitro-o-phenylenediamine, N-methyl-N'-nitro-N-nitrosoguanidine

Remarks:

without S9 mix

Details on test system and experimental conditions:

DETAILS ON TEST SYSTEM
METHOD OF APPLICATION: in agar (plate incorporation); preincubation
DURATION
- Preincubation period: 20 min
- Exposure duration: 37°C for 48 - 72 hours
NUMBER OF REPLICATIONS: in triplicate
NUMBER OF CELLS EVALUATED: all revertants / colonies counted
DETERMINATION OF CYTOTOXICITY
Toxicity detected by a
• decrease in the number of revertants
• clearing or diminution of the background lawn (= reduced his- or trp- background growth)
• reduction in the titer is recorded for all test groups both with and without S9 mix in all experiments

Evaluation criteria:

The test substance was considered positive in this assay if the following criteria were met:
• A dose-related and reproducible increase in the number of revertant colonies, i.e. at least doubling (bacteria strains with high spontaneous mutation rate, like TA 98, TA 100 and E.coli WP2 uvrA) or tripling (bacteria strains with low spontaneous mutation rate, like TA 1535 and TA 1537) of the spontaneous mutation rate in at least one tester strain either without S9 mix or after adding a metabolizing system.

A test substance was generally considered non-mutagenic in this test if:
• The number of revertants for all tester strains were within the historical negative control data range under all experimental conditions in at least two experiments carried out independently of each other.

Species / strain:

S. typhimurium TA 1535

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

Species / strain:

S. typhimurium TA 1537

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

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

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

Species / strain:

E. coli WP2

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

Additional information on results:

SOLUBILITY: No precipitation of the test substance was found with and without S9 mix.

TOXICITY: A weak bacteriotoxic effect was occasionally observed depending on the strain and test conditions from about 2 600 μg/plate onward.

Conclusions:

Under the experimental conditions of this study, the test substance is considered not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay in the absence and the presence of metabolic activation.

Executive summary:

In this guideline (OECD 471) study conducted with GLP certification, the test material (EC 239-263-3) did not meet the criteria to be considered genotoxic in all tested strains. Testing was performed with and without metabolic acitivation and no precipitation was observed at any test concentration. The reverse mutagenicity test was performed upon strains of Salmonella typhimurium and Escherichia coli.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015

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:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

hprt locus

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

Culture medium: Ham's F12 medium
Treatment medium: Ham's F12 medium
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital and β-naphthoflavone induced rat liver S9 mix

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without S9 mix

Untreated negative controls:

yes

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
- Exposure duration: 4h
- Expression time (cells in growth medium): 7-9 days at 37°C
- Selection time (if incubation with a selection agent): 6-7 days in TG-medium
- Fixation: At the end of the selection period, the medium will be removed and the remaining colonies will be fixed with methanol, stained with Giemsa and counted

SELECTION AGENT (mutation assays): TG-medium

NUMBER OF REPLICATIONS: two independent experiments, every sample in triplicate

NUMBER OF CELLS EVALUATED: all colonies are counted

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

OTHER EXAMINATIONS:
- pH
- osmolarity
- solubility
- cell morphology

Evaluation criteria:

A finding is assessed as positive if the following criteria are met:
• Increase in the corrected mutation frequencies (MFcorr.) both above the concurrent negative control values and our historical negative control data range (see Appendix 6).
• Evidence of the reproducibility of any increase in mutant frequencies.
• A statistically significant increase in mutant frequencies and the evidence of a doseresponse relationship. Isolated increases of mutant frequencies above our historical negative control range (i.e. 15 mutants per 10^6 clonable cells) or isolated statistically significant increases without a dose-response relationship may indicate a biological effect but are not regarded as sufficient evidence of mutagenicity.

Statistics:

An appropriate statistical trend test (MS EXCEL function RGP) was performed to assess a dose-related increase of mutant frequencies. The number of mutant colonies obtained for the test substance treated groups was compared with that of the respective vehicle control groups. A trend is judged as statistically significant whenever the one-sided p-value (probability value) is below 0.05 and the slope is greater than 0. However, both, biological and statistical significance will be considered together.

Species / strain:

Chinese hamster Ovary (CHO)

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

Additional information on results:

In this study, in the 1st Experiment in the presence of metabolic activation the two highest applied concentrations were clearly cytotoxic. In the 1st Experiment in the absence of metabolic activation and in the 2nd Experiment in the absence and presence of metabolic activation no cytotoxicity was observed when tested up to clearly precipitating concentrations.

Conclusions:

Under the conditions of the test, the test material was considered to be non-genotoxic.

Executive summary:

In this guideline (OECD 476) study conducted with GLP certification, the test material was considered to be non-genotoxic. The HPRT test was conducted in Chinese Hamster Ovary (CHO) cells with and without metabolic activation. Cytotoxicity was clearly observed in the 1st Experiment in the presence of metabolic activation at the two highest concentrations tested. Test concentrations were tested up to levels of precipitation.

Reference 3

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Target gene:

not applicable

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: Minimum essential medium (MEM) supplemented with 10% fetal bovine serum
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes

Metabolic activation:

with and without

Metabolic activation system:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with penobarbital and b-naphthoflavone

Test concentrations with justification for top dose:

Experiment I and II:
54.7-1750 ug/ml

Vehicle / solvent:

DMSO 1 %

Untreated negative controls:

yes

Negative solvent / vehicle controls:

other: no solvent/vehicle used

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without metabolic activation

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 and 24 hours
- Expression time (cells in growth medium): 20 hours
- Fixation time (start of exposure up to fixation or harvest of cells): 20 hours

SPINDLE INHIBITOR (cytogenetic assays): Colcemid
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: at least 200

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index in 1000 cells

OTHER EXAMINATIONS:
- Determination of polyploidy: yes

Evaluation criteria:

A test substance is considered "positive" if the following criteria are met:
• A significant, dose-related and reproducible increase in the number of cells containing micronuclei was observed.
• The number of micronucleated cells exceeded both the value of the concurrent vehicle control and the range of our laboratory’s historical negative control data
A test substance generally is considered "negative" if the following criteria are met:
• The number of micronucleated cells in the test groups is not distinctly increased above the concurrent vehicle control and is within our laboratory’s historical negative control data range

Statistics:

The statistical evaluation of the data was carried out using the MUVIKE program system (BASF SE). The proportion of cells containing micronuclei was calculated for each group. A comparison of each dose group with the concurrent vehicle control group was carried out using Fisher's exact test for the hypothesis of equal proportions. This test is Bonferroni-Holm corrected versus the dose groups separately for each time and was performed one-sided. If the results of this test were statistically significant compared with the respective vehicle control, labels (* p ≤ 0.05, ** p ≤ 0.01) have been printed in the tables.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

In this study, cell morphology/attachment was not adversely influenced (grade > 2) at any concentration tested for micronuclei.

In the main experiments, no distinct cytotoxicity indicated by reduced cell count or proliferation index (CBPI) was observed up to the highest required test substance concentration.

Osmolarity and pH values were not influenced by test substance treatment.

Test substance precipitation in culture medium at the end of exposure period (macroscopical assessment) was observed in the 1st Experiment in the absence and presence of S9 mix from 875 μg/mL onward and in the 2nd Experiment in the absence and presence of S9 mix from 437.5 μg/mL onward.

Conclusions:

Under the conditions of the test, the test material was considered to be no-genotoxic.

Executive summary:

In this guideline (OECD 487) study conducted with GLP certification, the test material was considered to be non-genotoxic. The micronucleus test was conducted in Chinese Hamster Lung Fibroblasts (V79), with and without metabolic activation, at concentrations up to precipitation. No cytotoxicity was observed during the test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

The test substance was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of several bacterial strains, i.e. Salmonella typhimurium and Escherichia coli, at concentration of 33 - 5200 ug/plate in a reverse mutation assay.

No precipitation of the test substance was found with and without S9 mix. A weak bacteriotoxic effect was occasionally observed depending on the strain and test conditions from about 2 600 μg/plate onward. A relevant increase in the number of his+ or trp+ revertants was not observed in the standard plate test or in the preincubation test either without S9 mix or after the addition of a metabolizing system. Thus, under the experimental conditions of this study, the test substance is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay in the absence and the presence of metabolic activation.

The test substance was assessed for its potential to induce micronuclei in V79 cells in vitro. Two independent experiments were carried out at concentrations up to 1750 ug/ml, both with and without the addition of liver S9 mix from induced rats. No distinct cytotoxicity indicated by reduced cell count or proliferation index (CBPI) was observed up to the highest required test substance concentration. The test substance did not cause any biologically relevant increase in the number of cells containing micronuclei either without S9 mix or after adding a metabolizing system. Thus, under the experimental conditions described, the test item is considered not to have a chromosome-damaging (clastogenic) effect nor to induce numerical chromosomal aberrations (aneugenic activity) under in vitro conditions in V79 cells in the absence and the presence of metabolic activation.

At last, the test material was assessed for its potential to induce gene mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese hamster ovary (CHO) cells in vitro. Two independent experiments were carried out, both with and without the addition of liver S9 mix from induced. Based on the solubility properties of the test substance and according to an initial rangefinding cytotoxicity test doses from 53 - 1700 µg/ml were tested in presence and absence of a metabolic activation system. On the basis from the results of the present study, the test substance did not cause any relevant increase in the mutant frequencies both without S9 mix

Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. As a result the substance is not considered to be classified for genotoxicity under Regulation (EC) No. 1272/2008.