3-methoxy-N,N-dimethylpropionamide

Genetic toxicity: in vivo

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

21 July 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

30 May 2008

Deviations:

no

Test guideline 3

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

Test material

Test material information

Constituent 1

Reference substance name:

3-methoxy-N,N-dimethylpropionamide

EC Number:

258-420-7

EC Name:

3-methoxy-N,N-dimethylpropionamide

Cas Number:

53185-52-7

Molecular formula:

C6H13NO2

IUPAC Name:

3-methoxy-N,N-dimethylpropanamide

Test material form:

liquid

Details on test material:

- Appearance: Clear colourless liquid
- Storage condition of test material: Room temperature (15-25°C)

Test animals

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

Administration / exposure

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.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

500 mg/kg bw (total dose)

Doses / concentrations 2

Dose / conc.:

1 000 mg/kg bw (total dose)

Doses / concentrations 3

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)

Examinations

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.

Results and discussion

Test results

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.

Applicant's summary and conclusion

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.