3-methoxy-N,N-dimethylpropionamide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test (OECDTG 471): negative

In vitro chromosome aberration test (OECDTG 473): negative

Genemutation in mammalian cells (OECDTG 490): negative

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03 October 2006 - 25 October 2006

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

Duplicate plating instead of triplicate plating without scientifically justification.

Principles of method if other than guideline:

Standards testing facilities should possess' of Notice No. 76 of the Ministry of Labor (revised on Mar. 29, 2000, Notice No. 13 of the Ministry of Labor), dated Sep. 1, 1988, and 'Standards for mutagenicity study (only using microorganisms)' of Notice No. 77 of the Ministry of Labor, dated Sep. 1, 1988, its revision and Notice No. 67 of the Ministry of Labor, dated Jun. 2, 1997.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

- S. typhimurium: Histidine gene
- E. coli: Tryptophan gene

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9-mix induced by a combination of phenobarbital and 5,6-benzoflavone

Test concentrations with justification for top dose:

Range finding study (without and with S9), all five strains: 4.88, 19.5, 78.1, 313, 1250 and 5000 µg/plate
Main study (without and with S9), all five strains: 156, 313, 625, 1250, 2500 and 5000 µg/plate

Vehicle / solvent:

- Solvent used: Water
- Justification for choice of solvent: As a result of dissolving property test the test material was dissolved at the concentrations of 50 mg/mL in water. Therefore, solvent for the test material was decided to be water.

Untreated negative controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

furylfuramide

other: 2-aminoanthracene in DMSO for all tester strains

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 minutes
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS:
- Doses of the test substance were tested in duplicate in each strain. Two independent experiments were conducted.

NUMBER OF CELLS EVALUATED: 1.6 - 4.3x10E8 per plate

DETERMINATION OF CYTOTOXICITY
- Method: The reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies.

OTHER EXAMINATIONS:
- The presence of precipitation of the test compound on the plates was determined.

Evaluation criteria:

In principle the test material will be considered positive in case that the maximum value of reverse mutation colony number of the test material group increases 2 times or more of that of negative control group and further that properties of dose reaction and reproduction were confirmed.

Statistics:

Not performed.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

tested up to and including 5000 µg/plate

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

tested up to and including 5000 µg/plate

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

tested up to and including 5000 µg/plate

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

tested up to and including 5000

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

tested up to and including 5000

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation was observed up to and including the top dose of 5000 µg/plate.

RANGE-FINDING/SCREENING STUDIES:
- No toxicity or mutagenicity was observed up to and including the top dose of 5000 µg/plate.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data (April 2006 - July 2006):
TA98: -S9: 395 - 665; +S9: 383 - 714
TA100: -S9: 496 - 804; +S9: 839 - 1805
TA1535: -S9: 229 - 451; +S9: 123 - 435
TA1537: -S9: 128 - 496; +S9: 117 - 311
WP2uvrA: -S9: 165 - 307; +S9: 544 - 979
- Negative (solvent/vehicle) historical control data (April 2006 - July 2006):
TA98: -S9: 6 - 24; +S9: 10 - 36
TA100: -S9: 67 - 142; +S9: 77 - 155
TA1535: -S9: 4 - 14; +S9: 4 - 16
TA1537: -S9: 1 - 13; +S9: 4 - 20
WP2uvrA: -S9: 12 - 41; +S9: 14 - 46

COMPARISON WITH HISTORICAL CONTROL DATA:
- The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- No toxicity or mutagenicity was observed up to and including the top dose of 5000 µg/plate.

Conclusions:

In an AMES test, performed equivalent to OECD 471 guideline and GLP principles, 3-methoxy-N,N-dimethylpropanamide was found not to be mutagenic with or without metabolic activation.

Executive summary:

An AMES test was performed according to OECD 471 guideline and GLP principles. All bacterial strains (TA98, TA100, TA1535, TA1537 and WP2uvrA) showed negative responses up to and including 5000 µg/plate, i.e. no significant dose-related increase in the number of revertants with or without metabolic activation was seen in two independently repeated pre-incubation experiments. No cytotoxicity and/or precipitation of the test substance was observed. The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Based on the results of this study it is concluded that 3-methoxy-N,N-dimethylpropanamide is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay with or without metabolic activation.

Reference 2

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:

29 September 2006 - 26 December 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Principles of method if other than guideline:

Standards testing facilities should possess' of Notice No. 76 of the Ministry of Labor (revised on Mar. 29, 2000, Notice No. 13 of the Ministry of Labor), dated October 1, 1988, 'Standards for investigation by chromosome aberration test using mammalian cultured cell and revision of report form for result of chromosome aberration study using mammalian cultured cell' of Kihatsu No.652 of the Ministry of Labor, dated Sep. 29, 1997, and 'Method and result evaluation method of chromosome aberration study using mammalian cultured cell' of Affairs Correspondence by the director of Chemicals Investigation Section of Safety Health Division of Labors Standards Bureau of the Ministry of Labor, dated Sep. 16, 1999.

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

other: CHL/IU (Fibroblast from Chinese Hamster Lung)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Supplier: Health Science Research Resources Band of Japan Health Science Foundation.
- Suitability of cells: Data on many chemicals using CHL/IU cells are stocked in Japan.
- Cell doubling time: approximately 15 hours
- Number of passages: 5 passages at reception. 8 - 24 passages were used for the test.
- Modal number of chromosomes: 25

MEDIA USED
- Type and identity of media including CO2 concentration if applicable:
Culture medium: 10% of foetal bovine serum (made by JRH Biosciences, Inc., Lot No.: 4J0214) added to Eagle's MEM (made by SIGMA-ALDRICH).
Temperature, CO2 concentration: 37°C, 5%
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: not applicable.

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9-mix induced by a combination of phenobarbital and 5,6-Benzoflavone.

Test concentrations with justification for top dose:

Dose range finding test:
Without and with S9-mix, 6hr exposure; 24 hr fixation: 0.0409, 0.082, 0.164, 0.328, 0.655 and 1.31 mg/mL
Without S9-mix, 24/48hr exposure; 24/48 hr fixation: 0.0409, 0.082, 0.164, 0.328, 0.655 and 1.31 mg/mL

First cytogenetic test:
Without and with S9-mix, 6 hr exposure time, 24 hr fixation time: 0.164, 0.328, 0.655 and 1.31 mg/mL
Without and with S9-mix, 24/48 hr exposure, 24/48 hr fixation time: 0.164, 0.328, 0.655 and 1.31 mg/mL

Vehicle / solvent:

- Solvent used: Physiological saline
- Justification for choice of solvente: The test substance was dissolved in physiological saline.

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

mitomycin C

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 72 hr
- Exposure duration: 6 hr (with and without S9-mix), 24 and 48 hr (without S9-mix)
- Fixation time (start of exposure up to fixation or harvest of cells): 24 and 48 hr

SPINDLE INHIBITOR (cytogenetic assays): colcemid
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: duplicates in one experiment

NUMBER OF CELLS EVALUATED: 200 metaphase chromosome spreads per dose level

DETERMINATION OF CYTOTOXICITY
- Method: cell growth rate calculated by counting living cells stained with Trypan blue.

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: no

Evaluation criteria:

Frequency (%) of the cells with structural and numerical aberrations was judged according to the following criteria per dose level:
<5%: Negative
≥5% -<10%: Equivocal
≥10%: Positive
The test material is judged as positive in case the frequency (%) is increased compared to that of negative control, and dose level increase is confirmed. In case only one dose level is positive or in case equivocal, a confirmation test will be performed. If the reproductivity is confirmed, the test material is judged as positive.

Statistics:

Not performed.

Key result

Species / strain:

other: CHL/IU (Fibroblast from Chinese Hamster Lung)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

tested up to and including the highest dose level of 1.31 mg/mL (=10 mM).

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation was observed up to and including the top dose of 1.31 mg/mL (=10 mM).

RANGE-FINDING/SCREENING STUDIES:
No toxicity was observed up to and including the top dose of 1.31 mg/mL (=10 mM).

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data:
Without S9:
Mitomycin-C 0.07 µg/mL for a 6 h exposure period: 17 - 46
Mitomycin-C 0.04 µg/mL for a 24 h exposure period: 16 - 45
Mitomycin-C 0.02 µg/mL for a 48 h exposure period: 13 - 82
With S9:
Benzo(a)pyrene 0.01 mg/mL: 18 - 94
- Negative (solvent/vehicle) historical control data:
Without S9:
6 h and 48 h exposure period: 0 - 4
48 h exposure period: 0 - 3
With S9: 0 -4

COMPARISON WITH HISTORICAL CONTROL DATA:
- The number of cells with chromosome aberrations found in the solvent and positive control cultures was within the laboratory historical control data range. Positive control chemicals, mitomycin C and Benzo(a)pyrene induced appropriate responses.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
No toxicity was observed up to and including the top dose of 1.31 mg/mL (=10 mM).

Conclusions:

A chromosome aberration study with 3-methoxy-N,N-dimethylpropionamide was performed equivalent to OECD 473 guideline and GLP principles, in CHL/IU (Fibroblast from Chinese Hamster Lung) cells in one experiment. It is concluded that 3-methoxy-N,N-dimethylpropionamide is not clastogenic in CHL/IU (Fibroblast from Chinese Hamster Lung) cells.

Executive summary:

In a chromosome aberration study, cultured CHL/IU (Fibroblast from Chinese Hamster Lung) cells were exposed to different concentrations of 3-methoxy-N,N-dimethylpropionamide (dissolved in physiological saline), in the presence and absence of S9-mix equivalent to OECD 473 guideline and GLP principles. In the cytogenetic assay, 3-methoxy-N,N-dimethylpropionamide was tested up to and including the highest dose level of 1.31 mg/mL (=10 mM) for a 6 h exposure time with a 24 h fixation time in the absence and presence of S9-mix, and for a 24 h and 48 h continuous exposure time with a 24 h and 48 h fixation time in the absence of S9-mix. Reliable negative and positive controls were included. 3-methoxy-N,N-dimethylpropionamide did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and presence of S9-mix. No effects of the substance on the number of polyploid cells were observed both in the absence and presence of S9-mix. Therefore it can be concluded that 3-methoxy-N,N-dimethylpropionamide does not disturb mitotic processes and cell cycle progression and does not induce polyploidy.

It is concluded that 3-methoxy-N,N-dimethylpropionamide is not clastogenic in CHL/IU (Fibroblast from Chinese Hamster Lung) cells.

 

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 September 2017 - 23 October 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Version / remarks:

29 July 2016

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Target gene:

Thymidine kinase (TK) locus in L5178Y mouse lymphoma cells.

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media:
- Basic medium: RPMI 1640 Hepes buffered medium (Dutch modification) containing penicillin/streptomycin (50 U/mL and 50 μg/mL, respectively), 1 mM sodium pyruvate and 2 mM L-glutamin supplemented with 10% (v/v) heat-inactivated horse serum (=R10 medium). Exposure medium: medium
supplemented with 5% (v/v) heat-inactivated horse serum (R5-medium). Selective medium: basic medium supplemented with 20% (v/v) heat-inactivated horse serum (total amount of serum = 20%, R20) and 5 μg/ml trifluorothymidine (TFT).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone.

Test concentrations with justification for top dose:

Dose range finding test:
Without and with S9-mix, 3 hours treatment: 63, 125, 250, 500, 1000 and 1312 μg/mL
Without S9-mix, 24 hours treatment: 63, 125, 250, 500, 1000 and 1312 μg/mL
Experiment 1:
Without and with S9-mix, 3 hours treatment: 15.6, 31.3, 63, 125, 250, 500, 1000 and 1312 μg/mL
All dose levels were selected to measure mutation frequencies at the TK-locus.
Experiment 2
Without S9-mix, 24 hours treatment: 15.6, 31.3, 63, 125, 250, 500, 1000 and 1312 μg/mL
All dose levels were selected to measure mutation frequencies at the TK-locus.

Vehicle / solvent:

- Solvent used: RPMI 1640 (exposure medium (R5) Hepes buffered medium)
- Justification for choice of solvent: A solubility test was performed based on visual assessment. The test item was dissolved in RPMI 1640 (exposure medium (R5)).

Negative solvent / vehicle controls:

yes

Remarks:

Exposure medium

Positive controls:

yes

Positive control substance:

cyclophosphamide

methylmethanesulfonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration:
Short-term treatment
With and without S9-mix: 3 hours
Prolonged treatment period
Without S9-mix: 24 hours
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 11 to 12 days

SELECTION AGENT (mutation assays): 5 μg/mL trifluorothymidine (TFT)
NUMBER OF REPLICATIONS:
- Solvent controls: Duplicate cultures
- Treatment groups and positive control: Single cultures

NUMBER OF CELLS EVALUATED: 9.6 x 10E5 cells plated/concentration

DETERMINATION OF CYTOTOXICITY
- Method: relative suspension growth (dose range finding test) and relative total growth (mutation experiments)

Evaluation criteria:

ACCEPTABILITY OF THE ASSAY
A mutation assay was considered acceptable if it met the following criteria:
a) The absolute cloning efficiency of the solvent controls (CEday2) is between 65 and 120% in orderto have an acceptable number of surviving cells analysed for expression of the TK mutation.
b) The spontaneous mutation frequency in the solvent control is ≥ 50 per 10^6 survivors and ≤ 170 per 10^6 survivors.
c) The suspension growth (SG) over the 2-day expression period for the negative controls should be between 8 and 32 (3 hours treatment) and 32-180 (24 hours treatment).
d) The positive control should demonstrate an absolute increase in the total mutation frequency above the spontaneous background MF (an induced MF (IMF) of at least 300 x 10^-6). At least 40% of the IMF should be reflected in the small colony MF. Furthermore, the positive control should have an increase in the small colony MF of at least 150 x 10^-6 above that seen in the concurrent solvent control (a small colony IMF of at least 150 x 10^-6).

DATA EVALUATION
Any increase of the mutation frequency should be evaluated for its biological relevance including comparison of the results with the historical control data range.
A test item is considered positive (mutagenic) in the mutation assay if it induces a MF of more than MF(controls) + 126 in a dose-dependent manner. An observed increase should be biologically relevant and will be compared with the historical control data range.
A test item is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study.
A test item is considered negative (not mutagenic) in the mutation assay if: none of the tested concentrations reaches a mutation frequency of MF(controls) + 126.

Statistics:

The global evaluation factor (GEF) has been defined by the IWGT as the mean of the negative/solvent MF distribution plus one standard deviation. For the micro well version of the assay the GEF is 126.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Remarks:

up to and including the concentration of 1312 μg/mL (=0.01 M).

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH and osmolarity: The pH and osmolarity at a concentration of 1312 μg/mL were 7.36 and 0.306 Osm/kg respectively (compared to 7.37 and 0.294 Osm/kg in the solvent control).
- Precipitation: The substance was fully soluble in exposure medium up to and including the concentration of 1312 μg/mL (=0.01 M).

RANGE-FINDING/SCREENING STUDIES: No toxicity in the suspension growth was observed up to and including the highest test item concentration of 1312 μg/mL compared to the suspension growth of the solvent control both in the absence of S9-mix and presence of S9-mix after 3 hours of treatment. After 24 hours of treatment without S9-mix, no toxicity in the relative suspension growth was observed up and including the highest test item concentration of 1312 μg/mL compared to the solvent control.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data:
Mutation frequency per 10^6 survivors
- S9-mix + S9-mix
3 hour treatment 24 hour treatment 3 hour treatment
Mean 808 684 1669
SD 239 206 843
n 136 124 146
Upper control limit
(95% control limits) 1465 1222 4196
Lower control limit
(95% control limits) 152 146 -859

SD = Standard deviation
n = Number of observations
Distribution historical negative control data from experiments performed between November 2014 and November 2017.

- Negative (solvent/vehicle) historical control data:
Mutation frequency per 10^6 survivors
- S9-mix + S9-mix
3 hour treatment 24 hour treatment 3 hour treatment
Mean 96 92 96
SD 29 30 29
n 268 248 285
Upper control limit
(95% control limits) 160 152 160
Lower control limit
(95% control limits) 32 31 32

SD = Standard deviation
n = Number of observations
Distribution historical negative control data from experiments performed between November 2014 and November 2017.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
No significant toxicity was observed and all dose levels were evaluated in the absence and presence of S9-mix in both experiments.

Conclusions:

A mouse lymphoma assay with the substance was conducted according to OECD 490 guideline and GLP principles. It is concluded that the substance is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions of the study.

Executive summary:

The mouse lymphoma assay with the substance was conducted according to OECD 490 guideline and GLP principles.

Based on the results of the dose-range finding test, KJCMPA®-100 was tested in two mutation assays. The first experiment was performed in the absence and presence of S9-mix with a 3 hour treatment period. The second mutation experiment was performed in the absence of S9-mix with a 24 hour treatment period. The following dose-range was selected for the mutagenicity tests in the absence and presence of S9-mix: 15.6, 31.3, 63, 125, 250, 500, 1000 and 1312 (=0.01 M) μg/mL exposure medium. No significant toxicity was observed and all dose levels were evaluated in the absence and presence of S9-mix in both experiments. No significant increase in the mutation frequency at the TK locus was observed after treatment with KJCMPA®-100 either in the absence or in the presence of S9-mix. The numbers of small and large colonies in the KJCMPA®-100 treated cultures were comparable to the numbers of small and large colonies of the solvent controls. It is concluded that the substance is not mutagenic in the mouse lymphoma L5178Y test system.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

In vivo micronucleus (OECDTG 474): negative

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06 September 2011 - 13 October 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

21 July 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

30 May 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)

Version / remarks:

August 1998

Deviations:

no

Principles of method if other than guideline:

ICH Guidance S2A: Guidance on Specific Aspects of Regulatory Genotoxicity Tests for Pharmaceuticals, 1996.
ICH Guidance S2B: Genotoxicity: A Standard Battery for Genotoxicity Testing of Pharmaceuticals, 1997.

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Species:

mouse

Strain:

other: RjHan: NMRI

Details on species / strain selection:

The NMRI mouse is one of the standard animals used internationally in this type of mutagenicity testing.

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Elevage Janvier, Route des Chènes Secs B.P. 4105, 53940 LE GENEST-ST-ISLE, France
- Age at study initiation: approximately 7 weeks
- Weight at study initiation: 34.4 - 36.6g
- Assigned to test groups randomly: yes, based on body weights.
- Fasting period before study: no data
- Housing: Group caging of 5 animals/ cage (type II. polypropylene/polycarbonate cage).
- Diet: free access to Autoclavable Complete Feed for mice and rats - breeding and maintenance (SM R/M-Z+H from SNIFF Spezialdiäten GmbH, D-59494 Soest, Germany)
- Water: free access to tap water from the municipal supply
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS (set to maintain, maintest)
- Temperature (°C): 20.1 - 22.8
- Humidity (%): 48 - 61
- Air changes (per hr): 15 - 20
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

- Vehicle: physiol. saline
- Justification for choice of vehicle: Based on the result of a preliminary solubility test, the test item was dissolved in physiological saline.
- Concentration of test material in vehicle: 50, 100 and 200 mg/mL

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The necessary amount of the test item was weighed into a calibrated volumetric flask; the appropriate amount of vehicle was added and stirred to obtain homogenous formulations.
The concentration of the test item formulation was chosen to assure the same dosing volumes in mice for all dose levels (10 mL/kg bw).
The formulations were prepared just before the treatment.

Duration of treatment / exposure:

24 and 48 hours

Frequency of treatment:

Single treatment.

Dose / conc.:

500 mg/kg bw (total dose)

Dose / conc.:

1 000 mg/kg bw (total dose)

Dose / conc.:

2 000 mg/kg bw (total dose)

No. of animals per sex per dose:

5 males

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide
- Justification for choice of positive control: no data
- Route of administration: intraperitoneal injection
- Doses / concentrations: 60 mg/kg bw / 6.0 mg/mL (dissolved in sterile physiological saline)

Tissues and cell types examined:

Two thousand polychromatic erythrocytes (PCEs) were scored per animal to assess the incidence of the micronucleated cells.
The proportion of immature among total (immature +mature) erythrocytes was be determined for each animal by counting a total of at least 1000 cells (immature + erythrocytes, PCEs plus mature normochromatic erythrocytes, NCEs), in which the number of micronuclei were recorded in both types of erythrocytes.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
Based on the results of the preliminary toxicity test, 2000 mg/kg body weight (the maximum recommended dose) was selected for the highest dose level of the test item in the main micronucleus test. Two lower dose levels separated by a factor of two (1000 and 500 mg/kg body weight) were also included in the main test.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
In the low and mid dose groups, furthermore in the positive control group the sampling was made once at 24 hours after treatment. In the high dose group and vehicle control group, sampling was made 24 and 48 hours after treatment. Five male animals per dose group were used for sampling on each one occasion.

DETAILS OF SLIDE PREPARATION:
The bone marrow was flushed out of each pair of femurs with foetal bovine serum (5 mL) using a syringe and needle into a sterile centrifuge tube. After mixing, the cell suspension was concentrated by a gentle centrifugation and the supernatant was discarded. Smears of the cell pellet were made on standard microscope slides (2 slides / animal). Slides were air-dried at room temperature for approximately 24 hours.
Dried slides were fixed in methanol for a minimum of 5 minutes and allowed to air-dry. Two slides per animal were stained with 10 % Giemsa solution for 20 minutes then thoroughly rinsed with distilled water, and then air-dried at room temperature for at least 12 hours. After staining, coverslips were mounted on them.

METHOD OF ANALYSIS:
Microscope analysis.
Two thousand polychromatic erythrocytes (PCEs) were scored per animal to assess the incidence of the micronucleated cells. The frequency of micronucleated cells was expressed as percent of micronucleated cells based on the first 2000 PCEs counted in the optic field. Fewer than 2000 PCEs may be counted in the event of a clear positive effect.
The proportion of immature among total (immature +mature) erythrocytes was be determined for each animal by counting a total of at least 1000 cells (immature + erythrocytes, PCEs plus mature normochromatic erythrocytes, NCEs), in which the number of micronuclei were recorded in both types of erythrocytes.

Evaluation criteria:

Criteria for a positive response:
The test item is considered to have shown genotoxic activity if statistically significant increases in the frequency of micronucleated polychromatic erythrocytes are observed in treated animals compared to the corresponding negative controls, and the increases are dose-related. Historical control data are taken into consideration when evaluating the biological significance of small increases.
Criteria for a negative response:
The test item is concluded to have given a negative response if no reproducible, statistically significant increases are observed above the concurrent and historical control values.
Equivocal response:
Results which do not meet the criteria for a positive or negative response are considered to be equivocal. Further investigations or scoring of additional cells may be necessary in case of an equivocal result.

Statistics:

Kruskal-Wallis test.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 2000, 1000, 500 and 250 mg/kg body weight (2 animals/sex/group; 35.6 - 37.4g: males; 28.7 - 30.6g: females).
- Solubility: the test item was soluble in physiological saline at 200 mg/mL concentration.
- Clinical signs of toxicity in test animals: In the highest dose group (2000 mg/kg body weight) piloerection and hunched back were observed for one male animal on the first day; other male animals and all the female animals were free of clinical signs on this day. All animals were free of clinical signs 24 and 48 hours after the treatment.
Based on the results of the preliminary toxicity test, dose levels of 2000, 1000 and 500 mg/kg body weight were selected for the micronucleus test. As there were no differences between male and female animals in the preliminary experiment, the main experiment was performed using male mice only.

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): No indication of an increase in the number of micronucleated normochromatic erythrocytes was observed in the study.
- Ratio of PCE/NCE (for Micronucleus assay): The ratio in the treated animals was comparable to the ratio in the negative control group at all dose levels.
- Clinical signs of toxicity in test animals:
No mortality or signs of systemic toxicity were observed during the study. In the high dose group (2000 mg/kg body weight), hunched back and/or piloerection was observed for one or more animals on the first day. The animals in the mid and low dose groups, furthermore in the negative (vehicle) control and positive control groups were symptom-free during the whole observation period.
No marked effect of test item treatment on the body weight of the mice was observed in the main test.
- Statistical evaluation:
The average number of micronuclei in the treated animals at 24h was less than the negative control group at all dose levels, therefore statistical analysis was not necessary. Comparison of the vehicle control data and the high dose of the test agent (2000 mg/kg bw) at 48h using the Kruskal-Wallis test gave a value of H = 1.3816. This is non-significant, giving a negative response.
The positive and negative control results were also compared, and gave a value of H = 6.991 (p<0.01). The positive control treatment therefore caused a large, statistically significant increase, demonstrating the sensitivity of the test system.
The frequency of micronucleated polychromatic erythrocytes of the negative (solvent) control group was within the range of historical laboratory control data; the positive control item produced a biologically and statistically relevant increase in the number of micronucleated polychromatic erythrocytes.

Conclusions:

A Mammalian Erythrocyte Micronucleus Test in male mice with 3-methoxy-N,N-dimethylpropanamide was performed according to OECD 474 guideline and GLP principles.
It is concluded that 3-methoxy-N,N-dimethylpropanamide is not genotoxic.

Executive summary:

In a Mammalian Erythrocyte Micronucleus Test, male mice were exposed to different concentrations of 3-methoxy-N,N-dimethylpropanamide (dissolved in physiological saline), according to OECD 474 guideline and GLP principles.

Based on the results of the preliminary toxicity test, 2000 mg/kg body weight (the maximum recommended dose) was selected for the highest dose level of the test item in the main micronucleus test. Two lower dose levels separated by a factor of two (1000 and 500 mg/kg body weight) were also included in the main test. In the low and mid dose groups, furthermore in the positive control group the sampling was made once at 24 hours after treatment. In the high dose group and vehicle control group, sampling was made 24 and 48 hours after treatment. Five male animals per dose group were used for sampling on each one occasion. Reliable negative and positive controls were included,. No mortality or signs of systemic toxicity were observed during the study. In the high dose group (2000 mg/kg body weight), hunched back and/or piloerection was observed for one or more animals on the first day. The animals in the mid and low dose groups, furthermore in the negative (vehicle) control and positive control groups were symptom-free during the whole observation period. No marked effect of test item treatment on the body weight of the mice was observed in the main test.

No indication of an increase in the number of micronucleated normochromatic erythrocytes was observed in the study.

Based on the results, it is concluded that 3-methoxy-N,N-dimethylpropanamide is not genotoxic in the Mammalian Erythrocyte Micronucleus Test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Ames test:

An AMES test was performed according to OECD 471 guideline and GLP principles. All bacterial strains (TA98, TA100, TA1535, TA1537 and WP2uvrA) showed negative responses up to and including 5000 µg/plate, i.e. no significant dose-related increase in the number of revertants with or without metabolic activation was seen in two independently repeated pre-incubation experiments. No cytotoxicity and/or precipitation of the test substance was observed. The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Based on the results of this study it is concluded that 3-methoxy-N,N-dimethylpropanamide is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay with or without metabolic activation.

Chromosome aberration test:

In a chromosome aberration study, cultured CHL/IU (Fibroblast from Chinese Hamster Lung) cells were exposed to different concentrations of 3-methoxy-N,N-dimethylpropionamide (dissolved in physiological saline), in the presence and absence of S9-mix equivalent to OECD 473 guideline and GLP principles. In the cytogenetic assay, 3-methoxy-N,N-dimethylpropionamide was tested up to and including the highest dose level of 1.31 mg/mL (=10 mM) for a 6 h exposure time with a 24 h fixation time in the absence and presence of S9-mix, and for a 24 h and 48 h continuous exposure time with a 24 h and 48 h fixation time in the absence of S9-mix. Reliable negative and positive controls were included. 3-methoxy-N,N-dimethylpropionamide did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and presence of S9-mix. No effects of the substance on the number of polyploid cells were observed both in the absence and presence of S9-mix. Therefore it can be concluded that 3-methoxy-N,N-dimethylpropionamide does not disturb mitotic processes and cell cycle progression and does not induce polyploidy.

It is concluded that 3-methoxy-N,N-dimethylpropionamide is not clastogenic in CHL/IU (Fibroblast from Chinese Hamster Lung) cells.

Mouse lymphoma test:

The mouse lymphoma assay with the substance was conducted according to OECD 490 guideline and GLP principles.

Based on the results of the dose-range finding test, KJCMPA®-100 was tested in two mutation assays. The first experiment was performed in the absence and presence of S9-mix with a 3 hour treatment period. The second mutation experiment was performed in the absence of S9-mix with a 24 hour treatment period. The following dose-range was selected for the mutagenicity tests in the absence and presence of S9-mix: 15.6, 31.3, 63, 125, 250, 500, 1000 and 1312 (=0.01 M) μg/mL exposure medium. No significant toxicity was observed and all dose levels were evaluated in the absence and presence of S9-mix in both experiments. No significant increase in the mutation frequency at the TK locus was observed after treatment with KJCMPA®-100 either in the absence or in the presence of S9-mix. The numbers of small and large colonies in the KJCMPA®-100 treated cultures were comparable to the numbers of small and large colonies of the solvent controls. It is concluded that the substance is not mutagenic in the mouse lymphoma L5178Y test system.

In vivo micronucleus:

In a Mammalian Erythrocyte Micronucleus Test, male mice were exposed to different concentrations of 3-methoxy-N,N-dimethylpropanamide (dissolved in physiological saline), according to OECD 474 guideline and GLP principles.

Based on the results of the preliminary toxicity test, 2000 mg/kg body weight (the maximum recommended dose) was selected for the highest dose level of the test item in the main micronucleus test. Two lower dose levels separated by a factor of two (1000 and 500 mg/kg body weight) were also included in the main test. In the low and mid dose groups, furthermore in the positive control group the sampling was made once at 24 hours after treatment. In the high dose group and vehicle control group, sampling was made 24 and 48 hours after treatment. Five male animals per dose group were used for sampling on each one occasion. Reliable negative and positive controls were included,. No mortality or signs of systemic toxicity were observed during the study. In the high dose group (2000 mg/kg body weight), hunched back and/or piloerection was observed for one or more animals on the first day. The animals in the mid and low dose groups, furthermore in the negative (vehicle) control and positive control groups were symptom-free during the whole observation period. No marked effect of test item treatment on the body weight of the mice was observed in the main test.

No indication of an increase in the number of micronucleated normochromatic erythrocytes was observed in the study.

Based on the results, it is concluded that 3-methoxy-N,N-dimethylpropanamide is not genotoxic in the Mammalian Erythrocyte Micronucleus Test.

Justification for classification or non-classification

Based on the results of the Ames test, in vitro chromosome aberration and mouse lymphoma studies and in vivo micronucleus test, the substance does not have to be classified for genotoxicity in accordance with Regulation (EC) No 1272/2008 and its updates.