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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2008-06-18 to 2008-08-08
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD Guideline study report

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report Date:
2008

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
Name: Me-3K4A2
CAS no.: 132900-75-5
Chemical name: Benzoic acid, 4-[4-[(1-oxo-2-propenyl)oxy]butoxy]-, 2-methyl-1,4-phenylene ester
Puropse: industrial chemical
Colour: beige
Physical state: powder Storage: at room temperature, protected from light
Stability in solution: water: probably stable
Special procedures: prepare freshly

Method

Target gene:
not applicable
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
TA 98: his D 3052, rfa-; uvrB-; R-factor
TA 100: his G 46, rfa-; uvrB-; R-factor
TA 1535: his G 46, rfa-; uvrB-
TA 1537: his C 3076, rfa-; uvrB-
Species / strain / cell type:
S. typhimurium TA 102
Details on mammalian cell type (if applicable):
TA 102: his G 428 (pAQ1), rfa-; uvrB-; R-factor
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
Pretest: 3.16, 10, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate with/without activation
Main experiment 1 and 2: 31.6, 100, 316, 1000, 2500 and 5000 µg/plate with/without activation
Vehicle / solvent:
DMSO
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
For TA 100, TA 1535 without S9
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
other: 4-Nitro-o-phenylene-diamine
Remarks:
For TA 98, TA 1537 without S9
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene
Remarks:
all strains with S9
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
For TA 102 without S9
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation, 1st main experiment); preincubation (2nd main experiment)


DURATION
- Preincubation period: 60 min
- Exposure duration: 48 h


NUMBER OF REPLICATIONS: 3

Evaluation criteria:
The test is considered acceptable if the bacteria demonstrate their typical responses to ampicil-lin (TA 98, Ta 100, TA 102); the control plates with and without S9 are within the following ranges (mean values of the spontaneous reversion frequency are within the historical control data range: TA 98 18-54 (-S9), 18-71 (+S9); TA 100 75-167 (-S9), 81-168 (+S9); TA 102 165-391 (-S9), 163-594 (+S9); TA 1535 5-29 (-S9), 6-31 (+S9); TA 1537 5-30 (-S9), 6-36 (+S9)); corresponding background growth on negative control, solvent control and test plates is ob-served; the positive controls show a distinct enhancement of revertant rates over the control plate. A biologically relevant increase with or without metabolic activation is
- if in tester strains TA100 and TA 102 the number of reversions is at least twice as high
- if in the other tester strains the number of reversions is at least three times higher
than the reversion rate of the solvent control.
Statistics:
not reported

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
>= 2500 µg/plate, preincubation
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
>= 2500 µg/plate, preincubaition
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
5000 µg/plate, plate incorporation
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
5000 µg/plate, plate incorporation
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 102
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: none
- Effects of osmolality: none
- Evaporation from medium: none
- Water solubility: none
- Precipitation: yes, 2nd experiment > = 1000 µg/plate (without S9), 5000 µg/plate (with S9)


RANGE-FINDING/SCREENING STUDIES: yes


COMPARISON WITH HISTORICAL CONTROL DATA: yes


ADDITIONAL INFORMATION ON CYTOTOXICITY: not reported
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

The substance did not induce any mutagenic effect in the Ames test with and without metabolic activation under the conditions used.
Executive summary:

The test item Me3K4A2 was investigated for its potential to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using Salmonella typhimurium strains TA 98, TA 100, TA 102, TA 1535 and TA 1537.

In two independent experiments several concentrations of the test item were used. Each assay was conducted with and without metabolic activation. The concentrations, including the controls, were tested in triplicate. The concentrations of 31.6, 100, 316, 1000, 2500 and 5000 µg/plate were prepared in DMSO as a vehicle and used. Precipitation of the test item was observed in all tester strains used in experiment II (with and without metabolic activation). In experiment I no precipitation of the test item was found in any of the five tester strains used (with and without activation), in experiment II precipitation of the test item was found at doses of 1000 µg/plate and higher (without metabolic activation) and at a dose of 5000 µg/plate (with metabolic activation).

Toxic effects of the test item were noted in some tester strains evaluated in experiments I and II. In experiment I toxic effects of the test item were observed only in tester strain TA 1537 at a dose of 5000 µg/plate with metabolic activation. In experiment II toxic effects of the test item were noted in tester strain TA 98 at doses of 2500 µg/plate and higher with and without metabolic activation. The reduction in the number of revertants down to a mutation factor of 0.5 found in tester strain TA 1537 at a dose of 31.6 µg/plate (without metabolic activation) was regarded as not biologically relevant due to lack of the dose-response relationship.

No biologically relevant increases in revertant colony numbers of any of the five tester strains were observed following treatment with Me3K4A2 at any concentration level, neither in the presence nor absence of metabolic activation in experiments I and II.

The reference mutagens induced a distinct increase of revertant colonies indicating the validity of the experiments.

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, Me3K4A2 did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used. Therefore, Me3K4A2 is considered to be non-mutagenic in this bacterial reverse mutation assay according to OECD guideline 471.