2-Propenoic acid, methyl ester, reaction products with 2-ethyl-1-hexanamine and sodium hydroxide

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2-Propenoic acid, methyl ester, reaction products with 2-ethyl-1-hexanamine and sodium hydroxide

EC Number:

271-865-1

EC Name:

2-Propenoic acid, methyl ester, reaction products with 2-ethyl-1-hexanamine and sodium hydroxide

Cas Number:

68610-44-6

Molecular formula:

not applicable, as UVCB substance

IUPAC Name:

2-Propenoic acid, methyl ester, reaction products with 2-ethyl-1-hexanamine and sodium hydroxide

Test material form:

solid

Remarks:

gummy solid at 25 °C

Details on test material:

- Color: Slight Pale Yellow Color
- Expiry date: August 2005
- Storage conditions: Room temperature
- Batch: R-0469-203-15
- Analytical purity: 82.9%
- Impurities: 17.1%

Method

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction was obtained from the liver of rats treated with Aroclor 1254 by the IP route. Each batch is validated by the Supplier for its ability to activate benzo(a)pyrene and 2-anthramine to mutagenic intermediates. It was preserved at -80°C.

Test concentrations with justification for top dose:

A preliminary test was conducted up to 5000 µg/plate (maximum dose level recommended by the OECD guideline).
No precipitate was observed. Moderate to marked cytotoxicity was noted without S9 mix at dose-levels ≥ 1000 µg/plate in the TA 98 and TA 102 strains and ≥ 2500 µg/plate in the TA 100 strain. No toxicity was noted with S9 mix.

On the basis of the results obtained in the preliminary test, the main experiments were conducted as following:
> Experiments without S9 mix:
156.3, 312.5, 625, 1250 and 2500 µg/plate, for TA 1535 and TA 100 strains in both experiments,
78.1, 156.3, 312.5, 625 and 1250 µg/plate, for the TA 1537, TA 98 and TA 102 strains in both experiments.
> Experiments with S9 mix:
312.5, 625, 1250, 2500 and 5000 µg/plate, for all the strains in both experiments.

Vehicle / solvent:

- Vehicle used: water
- Justification: freely soluble in the vehicle (water for injections) at 50 mg/mL

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

TA 1535 and TA 100, without S9 Mix

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

TA 1537, without S9 Mix

Controls 3

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

TA 98, without S9 Mix

Controls 4

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

TA 102, without S9 Mix

Controls 5

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Anthramine

Remarks:

All the strains, with S9 Mix

Details on test system and experimental conditions:

METHOD OF APPLICATION:
The preliminary test and the first main experiment were conducted using the plate incorporation method.
The second main experiment was conducted using the preincubation method, incubation for 60 minutes at 37°C, under shaking).

EXPERIMENTAL DESIGN:
> In the preliminary test, 6 dose-levels (one plate/dose-level) were tested in 3 strains (TA 98, TA 100 and TA102) with and without S9 Mix.
> The main experiments were conducted using 3 plates/dose levels in the 5 strains (TA 98, TA 100, TA 1535, TA 1537 and TA 102), with and without S9 Mix. At least 5 dose-levls were tested in each experiment, with vehicle controls and positive controls.

EVALUATION OF THE RESULTS:
The number of revertants and the aspect of the bacterial lawn were scored per plates.

Evaluation criteria:

> Acceptance criteria:
This study is considered valid if the number of revertants in the vehicle controls and in the positive controls are consistent with the historical data of the testing facility.
> Evaluation criteria
A reproducible 2-fold increase (for the TA 98, TA 100 and TA 102 strains) or 3-fold increase (for the TA 1535 and TA 1537 strains) in the number of revertants compared with the vehicle controls, in any strain at any dose-level and/or evidence of a dose-relationship was considered as a positive result. Reference to historical data, or other considerations of biological relevance may also be taken into account in the evaluation of the data obtained.

Statistics:

The individual results and the mean number of revertants, with the corresponding standard deviation and ratio (mutants obtained in the presence of the test item/mutants obtained in the presence of the vehicle), are presented in tabular form.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium, other: TA 98, TA 100, TA 1535, TA 1537 and TA 102

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Moderate to marked toxicity at ≥ 1250 µg/plate in the TA 1535, TA 1537, TA 98 and TA 102 strains and at 2500 µg/plate in the TA 100 strain.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium, other: TA 98, TA 100, TA 1535, TA 1537 and TA 102

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Moderate to marked toxicity in the second experiment (using preincubation method) at ≥ 2500 µg/plate in the TA 1535 and TA 1537 strains, and at 5000 µg/plate in the TA 98, TA 100 and TA 102 strains

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

The test item was tested according to the international guidelines (OECD 471) with Salmonella typhimurium strains, using both the plate incorporation and the preincubation methods, in the absence and in the presence of metabolic activation.
Under the experimental conditions, the test item did not show mutagenic activity.

Executive summary:

The objective of this study was to evaluate the potential of the test item to induce reverse mutation in Salmonella typhimurium. The study was performed according to the international guidelines (OECD 471, Commission Directive No. B13/14) and in compliance with the Principles of Good Laboratory Practice Regulations.

A preliminary toxicity test was performed to define the dose-levels to be used for the mutagenicity study.

Then, the test item was then tested in two independent main experiments, with and without a metabolic activation system, the S9 mix, prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254. Both experiments were performed according to the direct plate incorporation method except for the second test with S9 mix, which was performed according to the preincubation method (60 minutes, 37°C). Five strains of bacteria Salmonella typhimurium: TA 1535, TA 1537, TA 98, TA 100 and TA 102 were used. Each strain was exposed to five dose-levels of the test item (three plates/dose-level). After 48 to 72 hours of incubation at 37°C, the revertant colonies were scored.

The evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn.

The test item was dissolved in water for injections.

The test item was testd up to the maximum recommended dose level of 5000 µg/plate or up to the cytotoxicity, according to the strains and according to the presence or the absence of the metabolic activation.

The number of revertants for the vehicle and positive controls was in the acceptance criteria. The study was therefore considered valid.

No noteworthy increase in the number of revertants was noted in all the five strains.