2-({2-[4-(dimethylamino)benzoyloxy]ethyl}(methyl)amino)ethyl 4-(dimethylamino)benzoate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 April 2006 to 08 May 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2006

Report date:

2006

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

2lst July, 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

EEC Directive 2000/32, L 136, Annex 4D, 19 May 2000

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Version / remarks:

EPA 712-C-98-247, August 1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

466-080-8

EC Name:

-

Molecular formula:

C23H31N3O4

IUPAC Name:

466-080-8

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

Name: LFC 2098
Product/common name: LFC 2098
Purpose: Industrial chemical
Colour: white
Physical state: solid, powder
Purity: 99.69%
Storage: at room temperature, protected from light
Molecular formula: C23H3104N3
Molecular weight: 413
Safety precautions: Routine hygienic procedures were sufficient to assure personnel health and safety.

Method

Target gene:

The Salmonella Uphimurium histidine (his) reversion system measures his- → his+ reversions. The S. typhimurium strains are constructed to differentiate between base pair (TA 100, TA 1535, TA 102) and frameshift (TA 98, TA 1537) mutations

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 102

Metabolic activation:

with and without

Metabolic activation system:

S9 liver microsomal fraction prepared at BSL BIOSERVICE GmbH. Male Wistar rats were induced with Phenobarbital (80 mg/kg bw) and B-Naphtoflavone (100 mg/kg bw) for 3 consecutive days by oral route.

Test concentrations with justification for top dose:

31.6, 100, 316, 1000, 2500 and 5000 µg/plate
For soluble, non-toxic test compounds the recommended maximum test concentration is 5 mg/plate or 5 µL/plate

Vehicle / solvent:

Test substance dissolved in dimethyl sulfoxide (DMSO)

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Dimethyl sulfoxide

True negative controls:

no

Positive controls:

yes

Remarks:

Without metabolic activation

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine, 4-NOPD

Details on test system and experimental conditions:

For the plate incorporation method the following materials were mixed in a test tube and poured over the surface of a minimal agar plate:
100 µL Test solution at each dose level, solvent (negative control) or reference mutagen solution (positive control),
500 µL S9 mix (for testing with metabolic activation) or S9 mix substitution buffer (for testing without metabolic activation),
100 µL Bacteria suspension (cf. Preparation of Bacteria, pre-culture of the strain),
2000 µL Overlay agar.

For the pre-incubation method 100 µL of the test item preparation was pre—incubated with the tester strains (100 µL) and sterile buffer or the metabolic activation system (500 µL) for 60 minutes at 37 °C prior to adding the overlay agar (2000 µL) and pouring onto the surface of a minimal agar plate.
For each strain and dose level, including the controls, three plates were used.
After solidification the plates were inverted and incubated at 37 °C for at least 48 h in the dark.

Rationale for test conditions:

According to the direct plate incorporation or the pre-incubation method the bacteria are exposed to the test item with and without metabolic activation and plated on selective medium. After a suitable period of incubation, reveitant colonies are counted.
At least five different amounts of the test item are tested with approximately half log (i.e. √10) intervals between test points for an initial test. More narrow spacing between dose levels may be appropriate when a dose response is investigated. For soluble, non-toxic test compounds the recommended maximum test concentration will be 5 mg/plate or 5 µL/plate.

Evaluation criteria:

The Mutation Factor is calculated by dividing the mean value of the revertant counts through the mean values of the solvent control (the exact and not the rounded values are used for calculation).
A test is considered acceptable if for each strain:
- the bacteria demonstrate their typical responses to crystal violet and ampicillin
- the control plates without S9 mix are within the following ranges (mean values of the spontaneous reversion frequency are within the historical control data range):

TA 98: 18-63
TA 100: 79-197
TA 1535: 5-30
TA 1537: 4-32
TA 102: 173-396

- corresponding background growth on both negative control and test plates is observed.
- the positive controls show a distinct enhancement of revertant rates over the control plate.

A test item is considered as mutagenic if:
- a clear and dose-related increase in the number of revertants occurs and/or
- a biologically relevant positive response for at least one of the dose groups occurs in at least one tester strain with or without metabolic activation.

A biologically relevant increase is described as follows:
- if in tester strains TA 100 and TA 102 the number of reversions is at least twice as high
- if in tester strains TA 98, TA 1535 and TA 1537 the number of reversions is at least three times higher
as compared to the spontaneous reversion rate.

According to the OECD guidelines, the biological relevance of the results will be the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.
A test item producing neither a dose related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups is considered to be non-mutagenic in this system.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and 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:

Precipitation of the test item was observed in all tester strains used in experiment I and II (with and without metabolic activation). In experiment l and II precipitation of the test item was found at a dose of 316 µg/plate and higher (with and without metabolic activation).
No toxic effects of the test item were noted in any of the five tester strains used up to the highest dose group evaluated (with and without metabolic activation) in experiment I and II.
No biologically relevant increases in revertant colony numbers of any of the five tester strains were observed following treatment with LFC 2098 at any concentration level, neither in the presence nor absence of metabolic activation in experiment I and II.

Applicant's summary and conclusion

Conclusions:

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, LFC 2098 did not cause gene mutations by base pair changes or frameshifts in the genome of the
tester strains used.
Therefore, LFC 2098 is considered to be non-mutagenic in this bacterial reverse mutation assay.

Executive summary:

In order to investigate the potential of LFC 2098 for its ability to induce gene mutations the plate incorporation test (experiment I) and the pre-incubation test (experiment II) were performed with the Salmonella thphimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 102.

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 following concentrations of the test item were prepared and used in the experiments:

Experiment I and Experiment II:

31.6, 100, 316, 1000, 2500 and 5000 µg/plate

Precipitation of the test item was observed in all tester strains used in experiment I and II (with and without metabolic activation). In experiment l and II precipitation of the test item was found at a dose of 316 µg/plate and higher (with and without metabolic activation).

No toxic effects of the test item were noted in any of the five tester strains used up to the highest dose group evaluated (with and without metabolic activation) in experiment I and II.

No biologically relevant increases in revertant colony numbers of any of the five tester strains were observed following treatment with LFC 2098 at any concentration level, neither in the presence nor absence of metabolic activation in experiment I and II.

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