2-Propenamide, N-(2-hydroxyethyl)-

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 May 2006 to 3 July 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 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: ICH (1996) Guideline S2A: Genotoxicity: Guidance on Specific Aspects of Regulatory Genotoxicity Tests for Pharmaceuticals. PAB/PAD Notification No. 444.

Deviations:

no

Test guideline 5

Qualifier:

according to guideline

Guideline:

other: ICH (1998) Guideline S2B: Genotoxicity: A Standard Battery for Genotoxicity Tests for Pharmaceuticals. PMSB/ELD Notification No. 544.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

N-(2-hydroxyethyl)prop-2-enamide

EC Number:

700-169-7

Cas Number:

7646-67-5

Molecular formula:

C5H9NO2

IUPAC Name:

N-(2-hydroxyethyl)prop-2-enamide

Specific details on test material used for the study:

Batch number: 050804
Purity: >99%

Method

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: R0 RPMI 1640, buffered with 2 mg/mL sodium bicarbonate, supplemented with 2.0 mM L-glutamine and 50 μg/mL gentamicin.
R10p R0, supplemented with 0.1% v/v Synperonic F68, 1.0 mM sodium pyruvate and HiDHS at 10% v/v.
R30p R0, supplemented with 0.02% v/v Synperonic F68, 1.0 mM sodium pyruvate and HiDHS at 30% v/v.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no
- Periodically "cleansed" against high spontaneous background: no

Metabolic activation:

with and without

Metabolic activation system:

S9 mix.

Test concentrations with justification for top dose:

39.06, 50, 78.13, 100, 156.25, 200, 300, 312.5, 400, 600, 625, 700, 800, 900, 1250 µg / ml.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: vehicle chosen is purified water

- Justification for choice of solvent/vehicle: Purified water was chosen as the vehicle because test substance is water soluble.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

3-methylcholanthrene

Remarks:

in presence of S9 mix

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

in absence of S9 mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period:
- Exposure duration: 3 hours
- Expression time (cells in growth medium): 24 and 48 hours

Evaluation criteria:

The following criteria were applied for assessment of individual assay results using data for MF where the RTG normally exceeded 10%:
Definitions:
GEF = Global Evaluation Factor. For microwell assays this is 126 x 10-6. The assay was considered valid in accordance with the assay acceptance criteria.
The test agent was regarded as negative if:
the mean concurrent solvent control mutant frequency and the GEF.
If the mutant frequency of any test concentrations exceeded the sum of the mean concurrent solvent control mutant frequency and the GEF, a linear trend test was applied:
If the linear trend test was negative, the result was regarded as negative.
If the linear trend test was positive, this indicated a positive, biologically relevant response.
Where appropriate, other factors were considered in the interpretation of the results, for example, the reproducibility within and between tests, the overall number of mutant colonies (as opposed to mutation frequency) and the nature of any concentration-related effect(s).
Results that only partially satisfied the assessment criteria described above were considered on a case-by-case basis. In cases where the results were inconclusive, further testing and/or a test modification may have been required to better define the assay response.

Results and discussion

Test results

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Main mutation test - 3 hour treatment in the absence of S9 mix:

In the 3 hour exposure, cultures were exposed to HEAA at concentrations from 39.06 to 1250 μg/mL. No precipitate was observed by eye at the end of treatment. Cultures exposed to HEAA at concentrations from 39.06 to 1250 μg/mL were assessed for determination of mutation frequency. Relative total growth (RTG) values from 104 to 86% were obtained relative to the solvent control. Data are presented for concentrations tested up to the maximum concentration, in accordance with current guidelines (approximately 10 mM). There were no clear increases in the mean mutant frequencies of any of the test concentrations assessed that exceeded the sum of the mean concurrent solvent control mutant frequency and the Global Evaluation Factor, (GEF), within acceptable levels of toxicity.

The positive control, methyl methanesulphonate, induced an acceptable increase in mutation frequency and an acceptable increase in the number of small colony mutants.

Main mutation test - 3 hour treatment in the presence of S9 mix:

In the 3 hour exposure, cultures were exposed to HEAA at concentrations from 50 to 1250 μg/mL. No precipitate was observed by eye at the end of treatment. Cultures exposed to HEAA at concentrations from 50 to 600 μg/mL were assessed for determination of mutation frequency. RTG values from 97 to 16% were obtained relative to the solvent control. There was a clear increase in the mean mutant frequency of the cells when exposed to HEAA at 600 μg/mL that exceeded the sum of the mean concurrent solvent control mutant frequency plus the GEF, within acceptable levels of toxicity. At 400 μg/mL the mean mutant frequency was marginally below the GEF. This dose-dependent increase in mutant frequency was accompanied by a decrease in toxicity which was reduced to an acceptable level of 16% RTG.

This dose-response showed a significant positive linear trend (p<0.001). The increases in mean mutant frequency were predominantly due to an increase in small colony formation, when compared to the concurrent solvent control. These increases fulfilled the criteria for a positive response, with a continuous exposure to the test substance not deemed to be necessary.

The positive control, 3-methylcholanthrene, induced an acceptable increase in mutation frequency and an acceptable increase in the number of small colony mutants.

Applicant's summary and conclusion

Conclusions:

It was concluded that HEAA did demonstrate mutagenic potential in this in vitro cell mutation assay, under the experimental conditions described.

Executive summary:

HEAA was tested for mutagenic potential in an in vitro mammalian cell mutation assay with mouse lymphoma L5178Y cells, according to OECD Guideline 476, under GLP.

It was concluded that HEAA did demonstrate mutagenic potential in this in vitro cell mutation assay, under the experimental conditions described.