N-(2-ethoxyphenyl)-N'-(2-ethylphenyl)oxamide

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2011-08-30 until 2011-10-25

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study conducted according to international guidelines. No relevant deviations have been reported so that the study is considered reliable.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

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:

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

Deviations:

no

Principles of method if other than guideline:

first experiment 4 hours treatment with and without metabolic activation
second experiment 24 hours treatment without metabolic activation, 4 hours treatment with metabolic activation

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-ethoxyphenyl)-N'-(2-ethylphenyl)oxamide

EC Number:

245-950-9

EC Name:

N-(2-ethoxyphenyl)-N'-(2-ethylphenyl)oxamide

Cas Number:

23949-66-8

Molecular formula:

C18H20N2O3

IUPAC Name:

N-(2-ethoxyphenyl)-N'-(2-ethylphenyl)ethanediamide

Details on test material:

- Molecular weight (if other than submission substance):312 g/mol
- Physical state: Solid, white
- Analytical purity: 99.23 g/mol
- Purity: 99.23 %
- Purity test date 2010-09-01:
- Lot/batch No.: CHA0054786
- Expiration date of the lot/batch: 2013-12-03
- Stability under test conditions: Not indicated by the sponsor
- Storage condition of test material: Room temperature

Method

Target gene:

HPRT

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: MEM
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/Beta-Naphtoflavone induced Rat liver S9

Test concentrations with justification for top dose:

Experiment I:
without metabolic activation: 6.3; 12.5; 25.0; 50.0; 100; 200; 400 µg/mL
with metabolic activation: 6.3; 12.5; 25.0; 50.0; 100; 3200 µg/mL
Experiment II:
without metabolic activation: 6.3; 12.5; 25.0; 50.0; 100; 200; 3200 µg/mL
with metabolic activation: 6.3; 12.5; 25.0; 50.0; 100; 200 µg/mL
In the first experiment the concentration of 6.3 µg/mL with metabolic activation was not continued since a minimum of only four analysable concentrations is required by the guidelines. The cultures at 100 µg/mL and above in experiment I without metabolic activa-tion were not continued to avoid analysis of too many precipitating concentrations. In the second experiment the concentration of 100 and 200 µg/mL without metabolic activation and at 100 µg/mL with metabolic activation were not continued for the same reason.

Vehicle / solvent:

DMSO

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium


DURATION
- Exposure duration: Experiment I: 4 hours with and without metabolic activation, Experiment II: 24 hours without metabolic activation, 4 hours with metabolic activation
- Expression time (cells in growth medium): 72 hours
- Selection time (if incubation with a selection agent): 10 days

SELECTION AGENT (mutation assays): 6-Thioguanine


NUMBER OF REPLICATIONS: 2


NUMBER OF CELLS EVALUATED: >1,5x10exp. 6


DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Evaluation criteria:

A test item producing neither a concentration-related increase of the mutant frequency nor a reproducible positive response at any of the test points is considered to be non-mutagenic in this system.
A mutagenic response is described as follows:
The test item is classified as mutagenic if it induces reproducibly with one of the concen¬trations a mutation frequency that is three times higher than the spontaneous mutation fre¬quency in the experiment.
The test item is classified as mutagenic if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be considered also in the case that a threefold increase of the mutant frequency is not observed.
In a case by case evaluation this decision depends on the level of the correspon¬ding solvent control data.

Statistics:

A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies using SYSTAT11 (SYSTAT Software, Inc., 501, Canal Boulevard, Suite C, Richmond, CA 94804, USA) statistics software. The number of mutant colonies obtained for the groups treated with the test item was compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological relevance and statistical significance were considered together.

Results and discussion

Test results

Test results 1

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with

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:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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: Not effected
- Effects of osmolality: Not increased
- Evaporation from medium: Not examined
- Water solubility: Not indicated by the sponsor
- Precipitation: Precipitation was observed in the first experiment: at 12.5 to 400 µg/mL without metabolic activation; at 50.0 to 3200 µg/mL with metabolic activation; in the second experiment: at 25.0 to 3200 µg/mL without metabolic activation; at 50.0 to 200 µg/mL with metabolic activation.
- Other confounding effects: None


RANGE-FINDING/SCREENING STUDIES:
In the range finding pre-experiment test item concentrations between 25 and 3200 µg/mL (≈10 mM) were used to evaluate toxicity in the presence (4 hours treatment) and absence (4 hours and 24 hours treatment) of metabolic activation. No relevant toxic effects oc-curred up to the maximum concentration with and without metabolic activation following 4 and 24 hours treatment.

The test medium was checked for precipitation or phase separation at the end of each treatment period (4 or 24 hours) prior to removal to the test item. Precipitation occurred at 50 µg/mL and above in the presence and absence of metabolic activation (4 and 24 hours treatment).
There was no relevant shift of pH and osmolarity of the medium even at the maximum concentration of the test item. The basic level of osmolarity however, was relatively high even at the solvent control based on a DMSO concentration of 1%. DMSO interferes with the freezing point depression technique used to measure osmolarity.
Based on the results of the pre-experiment, the individual concentrations of the main experiments were selected. A series of concentrations spaced by a factor of 2 was placed into the lower range. Larger spacing was used at high concentrations to avoid analysis of too many precipitating concentrations.

COMPARISON WITH HISTORICAL CONTROL DATA: Complies


ADDITIONAL INFORMATION ON CYTOTOXICITY:
Relevant cytotoxic effects indicated by a relative cloning efficiency I or a relative cell density below 50% of the corresponding solvent control occurred in the first experiment without metabolic activation at 50 µg/mL and above.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Summary Table

				relative	relative	relative	mutant		relative	relative	relative	mutant
	conc.	P	S9	cloning	cell	cloning	colonies/	induction	cloning	cell	cloning	colonies/	induction
	µg/mL		mix	efficiency I	density	efficiency II	10^6cells	factor	efficiency I	density	efficiency II	10^6cells	factor
				%	%	%			%	%	%
Column	1	2	3	4	5	6	7	8	9	10	11	12	13
Experiment I / 4 h treatment				culture I					culture II
Solvent control with DMSO			-	100.0	100.0	100.0	17.7	1.0	100.0	100.0	100.0	5.5	1.0
Positive control (EMS)	150.0		-	71.1	102.0	103.8	84.4	4.8	73.5	150.2	93.9	93.0	16.9
Test item	6.3		-	79.0	115.2	97.5	8.7	0.5	81.5	154.1	96.0	21.8	4.0
Test item	12.5	P	-	69.9	95.3	103.6	3.5	0.2	71.1	168.5	97.7	8.8	1.6
Test item	25.0	P	-	67.6	109.7	106.7	25.1	1.4	70.4	85.9	96.2	17.7	3.2
Test item	50.0	P	-	48.3	90.7	105.6	6.3	0.4	47.1	116.6	92.0	18.3	3.3
Test item	100.0	P	-	41.6	culture was not continued^#				31.1	culture was not continued^#
Test item	200.0	P	-	30.0	culture was not continued^#				31.7	culture was not continued^#
Test item	400.0	P	-	29.2	culture was not continued^#				40.4	culture was not continued^#
Solvent control with DMSO			+	100.0	100.0	100.0	21.0	1.0	100.0	100.0	100.0	14.3	1.0
Positive control (DMBA)	1.1		+	41.6	55.3	86.4	647.1	30.8	49.2	49.4	100.1	862.1	60.4
Test item	6.3		+	103.7	culture was not continued^##				94.8	culture was not continued^##
Test item	12.5		+	100.8	119.7	99.6	7.4	0.4	100.3	98.5	102.8	19.6	1.4
Test item	25.0		+	102.2	98.9	102.7	14.2	0.7	91.8	93.2	93.5	17.9	1.3
Test item	50.0	P	+	103.9	91.5	96.7	20.2	1.0	92.5	83.3	106.6	13.8	1.0
Test item	100.0	P	+	81.5	79.0	82.7	22.2	1.1	91.6	82.5	110.7	11.6	0.8
Test item	3200.0	P	+	101.2	95.2	91.5	14.9	0.7	86.5	97.3	112.5	14.0	1.0
Experiment II / 24 h treatment				culture I					culture II
Solvent control with DMSO			-	100.0	100.0	100.0	6.0	1.0	100.0	100.0	100.0	7.4	1.0
Positive control (EMS)	150.0		-	98.7	86.9	91.7	221.2	36.6	102.3	86.8	108.6	225.1	30.4
Test item	6.3		-	100.1	91.2	94.8	6.3	1.0	99.8	89.5	100.7	9.9	1.3
Test item	12.5		-	103.4	119.2	87.9	15.2	2.5	110.6	96.4	94.2	14.2	1.9
Test item	25.0	P	-	102.7	81.8	101.7	10.1	1.7	103.9	75.1	107.0	11.8	1.6
Test item	50.0	P	-	96.4	93.6	85.8	6.2	1.0	103.1	73.5	116.7	4.3	0.6
Test item	100.0	P	-	96.7	culture was not continued^#				102.0	culture was not continued^#
Test item	200.0	P	-	92.0	culture was not continued^#				104.0	culture was not continued^#
Test item	3200.0	P	-	96.9	69.8	88.9	11.8	2.0	102.6	80.1	120.0	10.0	1.3
Experiment II / 4 h treatment
Solvent control with DMSO			+	100.0	100.0	100.0	4.8	1.0	100.0	100.0	100.0	9.8	1.0
Positive control (DMBA)	1.1		+	47.1	36.1	67.8	352.6	73.1	74.1	44.3	87.3	589.4	60.4
Test item	6.3		+	86.2	70.6	74.9	7.0	1.4	99.4	116.6	78.2	13.5	1.4
Test item	12.5		+	94.6	81.3	81.7	12.6	2.6	94.8	102.3	76.7	7.1	0.7
Test item	25.0		+	91.0	92.8	80.5	9.6	2.0	93.9	115.8	88.8	13.0	1.3
Test item	50.0	P	+	91.0	75.3	66.4	10.5	2.2	91.6	106.6	70.5	10.2	1.0
Test item	100.0	P	+	86.4	culture was not continued^#				98.9	culture was not continued^#
Test item	200.0	P	+	84.3	107.9	73.1	5.0	1.0	105.8	135.9	96.2	6.4	0.7

#     culture was not continued to avoid analysis of too many precipitating concentrations

##  culture was not continued since a minimum of only four analysable concentrations is required

P    precipitation

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

Conclusion:
It can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells.

Executive summary:

The test item Hostavin VSU P was assessed for its potential to induce gene mutations at the HPRT locus using V79 cells of the Chinese hamster.

The study was performed in two independent experiments, using identical experimental procedures. In the first experiment the treatment period was 4 hours with and without metabolic activation. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation.

The maximum concentration of the test item equals a molar concentration of 10 mM, with respect to the purity of the test item (99.23 %).

Precipitation at the end of treatment, visible to the unaided eye occurred at 50.0 µg/mL and above with and without metabolic activation in the pre-experiment. In experiment I precipitation was noted at 12.5 µg/mL and above without metabolic activation and at 50.0 µg/mL and above with metabolic activation. In experiment II precipitation was observed at 25.0 µg/mL and above without metabolic activation and at 50.0 µg/mL and above with metabolic activation.

Relevant cytotoxic effects indicated by a relative cloning efficiency I or a relative cell density below 50% of the corresponding solvent control occurred in the first experiment without metabolic activation at 50 µg/mL and above.

No relevant and reproducible increase in mutant colony numbers/10⁶cells was observed in the main experiments up to the maximum concentration with and without metabolic activation. The induction factor reached the threshold of three times the corresponding solvent control in the second culture of experiment I without metabolic activation at 6.3, 50, and 100 µg/mL (4 hours treatment). This effect however, was judged as based upon the rather low solvent control of just 5.5 mutant colonies/10⁶cells. The absolute values of the mutation frequencies (21.8, 17.7, and 18.3 mutant colonies per 10⁶cells) remained well within the historical range of solvent controls.

A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of the mutation frequency. No significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was determined in any of the experimental groups.

In both experiments of this study (with and without S9 mix) the range of the solvent controls was from 4.8 up to 21.0 mutants per 10⁶cells; the range of the groups treated with the test item was from 3.5 up to 25.1 mutants per 10⁶cells.

(150 µg/mL) and DMBA (1.1 µg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies.