N-ethyl-N-(3-methylphenyl)propionamide

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 September to 29 October

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1997

Report date:

1997

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

N-ethyl-N-(3-methylphenyl)propionamide

EC Number:

423-640-6

EC Name:

N-ethyl-N-(3-methylphenyl)propionamide

Cas Number:

179911-08-1

Molecular formula:

C12H17NO

IUPAC Name:

N-ethyl-N-(3-methylphenyl)propanamide

Test material form:

liquid

Method

Target gene:

Not applicable

Species / strain

Species / strain / cell type:

lymphocytes: human lymphocytes from whole blood culture

Details on mammalian cell type (if applicable):

Human blood samples were obtained by venepuncture in lithium heparin tubes on the days of culture initiation from healthy, non-smoking male donors

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

The metabolic activation system (S9-mix) used in this study was prepared as required (on each day of culture treatment) as a 1:1 mixture of S9 fraction and cofactor solution.
S9 was prepared from male Sprague Dawley rats, dosed once daily (by oral gavage) for 3 days with a combined phenobarbital (80mg/kg bodyweight) and beta-naphthoflavone (100mg/kg bodyweight) corn oil preparation. The treated animals were sacrificed on the day following the third dose. A 25% w/v homogenate (the S9 fraction) was prepared according to the method given in Callander et al (1995).
The cofactor solution was prepared as a single stock solution of Na2HPO., KCl, glucose-6- phosphate, NADP (Na salt) and MgCl2 (150: 49.5 : 7.5 : 6: 12mM) in sterile double deionised water and adjusted to a final pH of 7.4

Test concentrations with justification for top dose:

Donor 1: 500 µg/ml, 250 µg/ml, 50 µg/ml (with S9 mix), 68 hours harvest
Donor 1: 250 µg/ml, 150 µg/ml, 25 µg/ml (without S9 mix), 68 hours harvest
Donor 2: 500 µg/ml, 250 µg/ml, 50 µg/ml (with S9 mix), 68 hours harvest
Donor 2: 250 µg/ml, 150 µg/ml, 25 µg/ml (without S9 mixture), 68 hours harvest
Donor 2: 500 µg/ml (with S9 mix) 92 hours harvest
Donor 2: 100 µg/ml (without S9 mix) 92 hours harvest

Vehicle / solvent:

dimethylsulphoxide

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

Without S9-mix

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with S9-

Details on test system and experimental conditions:

EXPERIMENTAL DESIGN
Duplicate human peripheral blood cultures were exposed to the solvent, test substance or positive control substances at appropriate concentrations in the following experiments:
a) A cytogenetic test using blood from Donor I in the presence and absence of S9-mix with a standard sampling time of 68 hours after culture initiation. Solvent and positive control cultures were included.
b) A second independent cytogenetic test using blood from Donor 2 in the presence and absence of S9-mix with a standard sampling time of 68 hours after culture initiation and a later sampling time of92 hours after culture initiation. Solvent control cultures were included at both sampling times whereas the positive control cultures were only included at the 68 hour sampling time

In both experiments a range of concentrations of the test item was used in order to define suitable concentrations for chromosomal aberration analysis.
The standard sampling time of 68 hours after culture initiation used in this study was based on a measured mean cell cycle time for cultured human peripheral blood lymphocytes of 13.5 hours in this Laboratory (September 1992 and November 1993). The later sampling time was selected to be 24 hours after the standard sampling time.

CULTURE HARVESTING
Approximately 2 hours prior to harvesting, the cultures were treated with colcemid at a final concentration of 0.4 µg/ml. Sixty-eight hours or 92 hours after culture establishment the cultures were centrifuged, the supernatant was removed and the cells were re-suspended in approximately 10ml of 0.075M KC! at room temperature for approximately 10 minutes. The cultures were centrifuged, the supernatant was removed and the remaining cells were fixed in freshly prepared methanol/glacial acetic acid fixative (3:1 v/v) added dropwise and made up to a volume of approximately 10ml. The fixative was removed following centrifugation and replaced with freshly prepared fixative. This fixation process was repeated at least twice prior to slide preparation on clean, moist labelled microscope slides. The slides were air dried, stained in filtered Giemsa stain (10% Gurr's R66 in buffered [pH 6.8] double deionised water) for 7 minutes, rinsed in water, air-dried and mounted with coverslips in DPX

SLIDE ANALYSIS
Where appropriate, the mitotic index was determined by examining 1000 lymphocytes per culture and calculating the percentage of cells in metaphase. The number of polyploid metaphases, in 1000 cells, was determined during scoring of the mitotic index.
For each donor, both in the presence and absence of S9-mix, duplicate cultures treated with Agarbois at three concentrations were selected for chromosomal aberration analysis at the 68 hour sampling time along with the appropriate solvent and positive control cultures. In each case the highest concentration was selected on the basis of a significant reduction in mean mitotic activity (approximately 50%) and the suitability of the metaphase preparations for chromosomal aberration analysis. In addition, duplicate cultures from the Donor 2 treated with Agarbois at the highest concentration suitable for chromosomal aberration analysis (but not exceeding that selected at the 68 hour sampling time) in the presence and absence of S9- mix were selected for chromosomal aberration analysis at the 92 hour sampling time along with the appropriate solvent control cultures.

The slides were coded prior to analysis and one hundred cells in metaphase, where possible, were analysed blind using light microscopy from each selected culture for the incidence of structural chromosomal damage, according to the principles of the criteria recommended by Scott et al (1990).

Rationale for test conditions:

The human peripheral blood lymphocyte was used as it is a sensitive target cell for the induction of in vitro chromosomal damage when stimulated to provide large numbers of rapidly dividing cells in culture (Scott et al, 1990).

Evaluation criteria:

a) No statistically significant increase in the percentage of aberrant cells (at any concentration) above concurrent solvent control values - NEGATIVE.
b) A statistically significant increase in the percentage of aberrant cells above concurrent solvent control values, which falls within the laboratory solvent control range - NEGATIVE.
c) An increase in the percentage of aberrant cells, at least at one concentration, which is substantially greater than the laboratory historical solvent control values - POSITIVE.
d) A statistically significant increase in the percentage of aberrant cells which is above concurrent solvent values and which is above the historical solvent control range upper value but below that described in (c) may require further evaluation.

Results and discussion

Test results

Key result

Species / strain:

lymphocytes: human lymphocytes

Metabolic activation:

with and without

Genotoxicity:

ambiguous

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Small increases in the percentage of aberrant cells (up to 6%) were observed in cultures treated with the test item in the absence of S9-mix. Although these increases were small, they were statistically significant and were outside the laboratory historical control range of 5.5% aberrant cells. It is concluded that the test item has shown weak evidence of clastogenic activity in this test system.

Applicant's summary and conclusion

Conclusions:

Under the conditions of this assay, small increases in the percentage of aberrant cells (up to 6%) were observed in cultures treated with the test item in the absence of S9-mix at only the top concentration tested. Although these increases were small, they were statistically significant and were outside the laboratory historical control range. Therefore, under the conditions of this study, the test item showed evidence of weak mutagenic potential

Executive summary:

The test item was evaluated for its clastogenic potential in anin vitrocytogenetic assay using human lymphocytes from two male donors treated in the presence and absence of a rat liver­ derived metabolicactivationsystem(S9 -mix).Cultures from both donors were harvested at the standard time of 68 hours after culture initiation and additional cultures from Donor 2 were harvested at the later time of 92 hours after culture initiation. Donors I and 2 were assayedonseparateoccasions.

Cultures treated with the test item at the following concentrations were selected for chromosomal aberration analysis along with the appropriate solvent and positive control cultures.

Donor 1 68 Hours		Donor268 Hours			Donor 2 92 Hours
+S9-mix	-S9-mix	+S9-mix	-S9-mix		+S9-mix	-S9-mix
500µg/ml	250µg/ml	500µg/ml	250µg/ml		500µg/ml	100 µg/ml
250µg/ml	150µg/ml	250µg/ml	150µg/ml		-	-
50µg/ml	25µg/ml	50µg/ml	25µg/ml		-	-

Concentration related reductions in mitotic activity were observed in cultures from both donors, thus demonstrating that test item is biologically active in this test system.

 

No statistically or biologically significant increases in the percentage of aberrant cells, compared to the solvent control values, were observed at the 68 hour sampling time in either Donor I or Donor 2 cultures treated with test item in the presence of S9-mix, or at the 92 hour sampling time in Donor 2 cultures treated with test item in the presence of S9-mix.

Small, but statistically significant increases in the percentage of aberrant cells, compared to the solvent control values, were observed at the 68 hour sampling time in both Donor I and Donor 2 cultures treated with Agarbois in the absence of S9-rnix. A small increase was also observed at the 92 hour sampling time in Donor 2 cultures treated with Agarbois in the absence of   S9-mix.

The sensitivity of the test system, and the metabolic activity of the S9 -mix employed, were clearly demonstrated by the increases in the percentage of aberrant cells induced by the positive control agents, mitomycin C and cyclophosphamide.

Under the conditions of this assay, small increases in the percentage of aberrant cells (up to 6%) were observed in cultures treated with the test item in the absence of S9-mix at only the top concentration tested. Although these increases were small, they were statistically significant and were outside the laboratory historical control range. Therefore, under the conditions of this study, test item showed evidence of weak mutagenic potential.