2-ethyl-N-(3-methoxyphenyl)-N-methylbutanamide

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 18 January 2017 and 24 May 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well conducted and well described study in accordance with GLP and OECD guideline 473 without any deviation

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

2-ethyl-N-(3-methoxyphenyl)-N-methylbutanamide

Cas Number:

2101947-22-0

Molecular formula:

C14H21NO2

IUPAC Name:

2-ethyl-N-(3-methoxyphenyl)-N-methylbutanamide

Test material form:

liquid

Specific details on test material used for the study:

Identification: FRET 15-0735
Chemical Name: 2-ETHYL-N-(3-METHOXYPHENYL)-N-METHYL-BUTANAMIDE
Physical state/Appearance: Extremely pale yellow liquid
Storage Conditions: Approximately 4 ºC in the dark

Method

Species / strain

Species / strain / cell type:

lymphocytes: cultures prepared from whole blood

Details on mammalian cell type (if applicable):

For each experiment, sufficient whole blood was drawn from the peripheral circulation of a non smoking volunteer (aged 18-35) who had been previously screened for suitability. The volunteer had not knowingly been exposed to high levels of radiation or hazardous chemicals and had not knowingly recently suffered from a viral infection. Based on over 20 years in house data for cell cycle times for lymphocytes using BrdU (bromodeoxyuridine) incorporation to assess the number of first, second and third division metaphase cells to calculate the average generation time (AGT) for human lymphocytes it is considered to be approximately 16 hours. Therefore using this average the in-house exposure time for the experiments for 1.5 x AGT is 24 hours.
The details of the donors used are:
Preliminary Toxicity Test: male, aged 25 years
Main Experiment (4(20)-hour without S9 and 24-hour): female, aged 33 years
Main Experiment (4(20)-hour with S9): female, aged 27 years

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

induced rat liver homogenate metabolizing system (S9), at a 2% final concentration

Test concentrations with justification for top dose:

Range-finder experiment: 7.81 to 2000 µg/mL (with and without S-9)
Main study:

4-hour exposure to the test item without S9-mix, followed by 20-hour culture in treatment-free media prior to cell harvest. The dose range of test item used was 31.25, 62.5, 125, 250, 312.5, 375 and 500 µg/mL.

4-hour exposure to the test item with S9-mix (2%), followed by 20-hour culture in treatment-free media prior to cell harvest. The dose range of test item used was 62.5, 125, 250, 312.5, 375, 437.5 µg/mL.

24-hour continuous exposure to the test item without S9-mix prior to cell harvest. The dose range of test item used was 7.81, 15.63, 31.25, 62.5, 125, 187.5 and 250 µg/mL.


The 4(20)-hour exposure group in the presence of S9 was initially performed with the dose range:- 31.25, 62.5, 125, 187.5, 250, 312.5 and 500 µg/mL. However, the toxicity curve was very steep and optimum toxicity was not achieved and therefore this exposure group was repeated with the revised dose range stated above.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

0.4 µg/mL for 4-hour exposure and 0.1 µg/mL for 24-hour exposure

Positive control substance:

mitomycin C

Remarks:

without metabolic activation

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

2 µg/mL for 4-hour exposure

Positive control substance:

cyclophosphamide

Remarks:

with metabolic activation

Details on test system and experimental conditions:

PREPARATION OF CULTURES: Cells (whole blood cultures) were grown in Eagle's minimal essential medium with HEPES buffer (MEM), supplemented “in-house” with L-glutamine, penicillin/streptomycin, amphotericin B and 10 % foetal bovine serum (FBS), at approximately 37 ºC with 5 % CO2 in humidified air. The lymphocytes of fresh heparinized whole blood were stimulated to divide by the addition of phytohaemagglutinin (PHA).

METHOD OF APPLICATION: In medium

DURATION
- Preincubation period: approximately 48 hours incubation at approximately 37 ºC with 5% CO2 in humidified air
- Exposure duration:
Three exposure groups were used:
i) 4-hour exposure to the test item without S9-mix, followed by a 20-hour recovery period in treatment-free media, 4(20)-hour exposure.
ii) 4-hour exposure to the test item with S9-mix (2%), followed by a 20-hour recovery period in treatment-free media, 4(20)-hour exposure.
iii) 24-hour continuous exposure to the test item without S9-mix.

- Fixation time (start of exposure up to fixation or harvest of cells): 24hrs

SPINDLE INHIBITOR (cytogenetic assays): Colcemid 0.1 µg/mL 2.5 hours before the required harvest time.

STAIN (for cytogenetic assays): 5% Giemsa for 5 minutes

NUMBER OF REPLICATIONS: duplicate

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
After incubation with demecolcine, the cells were centrifuged, the culture medium was drawn off and discarded, and the cells re-suspended in 0.075M hypotonic KCl. After approximately fourteen minutes (including centrifugation), most of the hypotonic solution was drawn off and discarded. The cells were re-suspended and then fixed by dropping the KCl cell suspension into fresh methanol/glacial acetic acid (3:1 v/v). The fixative was changed at least three times and the cells stored at approximately 4 ºC to ensure complete fixation prior to slide preparation. The lymphocytes were re-suspended in several mL of fresh fixative before centrifugation and re-suspension in a small amount of fixative. Several drops of this suspension were dropped onto clean, wet microscope slides and left to air dry. Each slide was permanently labeled with the appropriate identification data. When the slides were dry they were stained in 5% Giemsa for 5 minutes, rinsed, dried and a cover slip applied using mounting medium.


NUMBER OF CELLS EVALUATED: A total of 2000 lymphocyte cell nuclei were counted and the number of cells in metaphase recorded and expressed as the mitotic index and as a percentage of the vehicle control value.

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): Where possible, 300 consecutive well-spread metaphases from each concentration were counted (150 per duplicate), where there were at least 15 cells with aberrations (excluding gaps), slide evaluation was terminated. If the cell had 44-48 chromosomes, any gaps, breaks or rearrangements were noted according to the simplified system of Savage (1976) recommended in the 1983 UKEMS guidelines for mutagenicity testing and the ISCN (1985). Cells with chromosome aberrations were reviewed as necessary by a senior cytogeneticist prior to decoding the slides.


DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: cells with 69 chromosomes or more were scored as polyploid cells and the incidence of polyploid cells (%) (including the incidence of cells with endoreduplicated chromosomes) reported

Evaluation criteria:

The following criteria were used to determine a valid assay:
- The frequency of cells with structural chromosome aberrations (excluding gaps) in the vehicle control cultures was within the laboratory historical control data range.
- All the positive control chemicals induced a positive response (p≤0.01) and demonstrated the validity of the experiment and the integrity of the S9-mix.
- The study was performed using all three exposure conditions using a top concentration which meets the requirements of the current testing guideline.
- The required number of cells and concentrations were analyzed.

Criteria for determining the Study Conclusion
Providing that all of the acceptability criteria are fulfilled, a test item can be considered to be clearly negative if, in any of the experimental conditions examined:
1) The number of cells with structural aberrations in all evaluated dose groups should be within the range of the laboratory historical control data.
2) No toxicologically or statistically significant increase of the number of cells with structural chromosome aberrations is observed following statistical analysis.
3) There is no concentration-related increase at any dose level

A test item can be classified as genotoxic if:
1) The number of cells with structural chromosome aberrations is outside the range of the laboratory historical control data.
2) At least one concentration exhibits a statistically significant increase in the number of cells with structural chromosome aberrations compared to the concurrent negative control.
3) The observed increase in the frequency of cells with structural aberrations is considered to be dose-related
When all of the above criteria are met, the test item can be considered able to induce chromosomal aberrations in human lymphocytes.

Statistics:

The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test.
A toxicologically significant response is recorded when the p value calculated from the statistical analysis of the frequency of cells with aberrations excluding gaps is less than 0.05 when compared to its concurrent control and there is a dose-related increase in the frequency of cells with aberrations which is reproducible. Incidences where marked statistically significant increases are observed only with gap-type aberrations will be assessed on a case by case basis.

Results and discussion

Test results

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Preliminary Toxicity Test
The dose range for the Preliminary Toxicity Test was 7.81, 15.63, 31.25, 62.5, 125, 250, 500, 1000 and 2000 µg/mL. The maximum dose was the maximum recommended dose level.
A precipitate of the test item was observed in the parallel blood-free cultures at the end of the exposure, at 2000 µg/mL, in the 4(20)-hour exposure groups and at and above 500 µg/mL in the continuous exposure group.
Hemolysis was observed following exposure to the test item at and above 1000 µg/mL and 500 µg/mL in the 4(20)-hour exposure groups in the absence and presence of S9, respectively and at and above 250 µg/mL in the 24-hour exposure group. Hemolysis is an indication of a toxic response to the erythrocytes and not indicative of any genotoxic response to the lymphocytes.
Microscopic assessment of the slides prepared from the exposed cultures showed that metaphase cells were present up to 250 µg/mL in the 4(20)-hour exposures in the presence and absence of metabolic activation (S9). The maximum dose with metaphases present in the 24 hour continuous exposure was 125 µg/mL. The test item induced marked evidence of toxicity in all three of the exposure groups.
The selection of the maximum dose level for the Main Experiment was based on toxicity and was 500 µg/mL for the 4(20)-hour exposure groups and was 250 µg/mL for the continuous exposure group.

Chromosome Aberration Test – Main Experiment
The qualitative assessment of the slides determined that the toxicity was less than that observed in the Preliminary Toxicity Test in the 4(20)-hour exposure in the absence of S9 and there were metaphases suitable for scoring present up to 500 µg/mL. In the 4(20)-hour exposure in the presence of S9 and in the 24-hour exposure the toxicity was similar to that seen in the Preliminary Toxicity Test and there were metaphases suitable for scoring up to 437.5 µg/mL and 125 µg/mL, respectively.
Precipitate observations were made at the end of exposure and no precipitate was observed in any of the three exposure groups. Haemolysis was observed at the end of exposure at and above 375 µg/mL in the presence of S9 and at 250 µg/ml in the 24-hour exposure.group.
They confirm the qualitative observations in that a dose-related inhibition of mitotic index was observed. In the 4(20)-hour exposure group in the absence of S9, 56% mitotic inhibition was achieved at 500 µg/mL. In the presence of S9, the toxicity plateaued at 250, 312.5 and 375 µg/mL with 50%, 38% and 63% mitotic inhibition, respectively. The 24-hour exposure group achieved marginally greater than optimum toxicity with 67% mitotic inhibition at 62.5 µg/mL.
The maximum dose level selected for metaphase analysis was based on toxicity and was 500 µg/mL and 375 µg/mL for the 4(20)-hour exposures in the absence and presence of S9, respectively and was 62.5 µg/mL for the 24-hour exposure group.
The assay was considered valid as it met all of the following criteria:
The frequency of cells with chromosome aberrations (excluding gaps) in the vehicle control cultures were within the current historical control data range.
All the positive control chemicals induced a demonstrable positive response (p≤0.01) and confirmed the validity and sensitivity of the assay and the integrity of the S9-mix.
The study was performed using all three exposure conditions using a top concentration which meets the requirements of the current testing guideline.
The required number of cells and concentrations were analyzed.
The test item did not induce any statistically significant increases in the frequency of cells with aberrations either in the absence or presence of metabolic activation.
The test item did not induce a significant increase in the numbers of polyploid cells at any dose level in any of the three exposure groups.

Any other information on results incl. tables

The dose levels of the controls and the test item are given in the table below:

Exposure Group	Final concentration of FRET 15-0735 (µg/mL)
4(20)-hour without S9	0*, 31.25, 62.5, 125, 250*, 312.5*, 375*, 500*, MMC0.4*
4(20)-hour with S9 (2%)	0*, 62.5, 125*, 250*, 312.5*, 375, 437.5, 500, CP2*
24-hour without S9	0*, 7.81*, 15.63*, 31.25*, 62.5*, 125, 187.5, 250, MMC 0.1*

* = Dose levels selected for metaphase analysis

MMC = Mitomycin C

CP = Cyclophosphamide

Mitotic Index - Preliminary Toxicity Test

Dose Level (µg/mL)	4(20)-Hour Without S9		4(20)-Hour With S9		24-Hour Without S9
	Mitotic Index	% of Control	Mitotic Index	% of Control	Mitotic Index	% of Control
0	7.15	100	8.10	100	4.70	100
7.81	-	-	-	-	-	-
15.63	-	-	-	-	4.90	104
31.25	5.80	81	6.05	75	3.35	71
62.5	7.50	105	7.20	89	2.30	49
125	6.45	90	6.95	86	2.30	49
250	6.60	92	4.05	50	NM H	NM
500	NM	NM	NM	NM	NM H P	NM
1000	NM H	NM	NM H	NM	NM H P	NM
2000	NM H P	NM	NM H P	NM	NM H P	NM

-      = Not assessed for mitotic index

NM   = No metaphases or insufficient metaphases suitable for scoring

P      = Precipitate observed at end of exposure period in blood-free cultures

H     = Hemolysis observed at the end of exposure in blood cultures

Mitotic Index – Main Experiment (24-hour Exposure Group)

Dose Level (µg/mL)	24-Hour Without S9
	A	B	Mean	% of Control
0	6.60	6.40	6.50	100
7.81	5.45	5.60	5.53	85
15.63	4.60	3.95	4.28	66
31.25	6.50	4.30	5.40	83
62.5	2.45	1.80	2.13	33
125	1.30	1.50	1.40	22
187.5	NM	NM	NM	NM
250	NM H	NM H	NM	NM
MMC 0.1	3.10	2.50	2.80	43

MMC           =Mitomycin C

-                    =Not assessed for mitotic index

NM              =No metaphases suitable for scoring

H                  =Hemolysis

Applicant's summary and conclusion

Conclusions:

Interpretation of results:
negative

Under the test conditions, FRET 15-0735 is not considered as clastogenic in human lymphocytes according to the criteria of the Annex VI to the Directive 67/548/EEC and CLP Regulation (EC) N° (1272-2008).

Executive summary:

In an in vitro chromosome aberration test performed according to OECD guideline 473 and in compliance with GLP, human primary lymphocyte cultures were exposed to FRET 15-0735 in DMSO at concentration range of 7.81 to 2000 µg/mL., for 4 + 20 h (treatment + recovery) with metabolic activation (induced rat liver homogenate metabolizing system (S9), at a 2% final concentration), and for 4 + 20 h or 24 + 0 h (treatment + recovery) without metabolic activation for a preliminary cytotoxicity test (Lloyd 2010). In the main test, two experiments were performed at concentrations up to 500 µg/mL without S-9 and up to 437.5 µg/mL with S-9 and the following concentrations were selected for analysis:

4(20)-hour without S9: 0, 250, 312.5, 375 and 500 μg/mL

4(20)-hour with S9 (2%): 0, 125, 250 and 312.5 μg/mL

24-hour without S9: 0, 7.81, 15.63, 31.25 and 62.5 μg/mL

Proportion of cells with structural aberrations in negative control cultures fell within historical vehicle control ranges. Positive controls (Mitomycin C at 0.4 and 0.1 µg/mL without S-9 and cyclophosphamide at 2 µg/mL with S-9) induced the appropriate response. Treatment of cells with FRET 15-0735 in the presence or absence of S-9 in both experiments resulted in frequencies of cells with structural or numerical aberrations that were generally similar to those observed in concurrent vehicle controls for all concentrations analysed. Numbers of aberrant cells (excluding gaps) in treated cultures fell within the normal range.

Under the test conditions, FRET 15 -0735 is not considered as clastogenic in human lymphocytes.