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

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:

October 1997

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

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian erythrocyte micronucleus 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

Test animals

Species:

mouse

Strain:

CD-1

Details on species / strain selection:

Male and female CD-1 mice from Charles River Breeding Laboratories, Margate, UK

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Margate, UK
- Age at study initiation:
Phase I: 4-12 weeks
Phase II: 6-13 weeks
Phase III: 6-7 weeks
- Fasting period before study: No
- Housing: mobile mouse cage racks
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum


ENVIRONMENTAL CONDITIONS
- Temperature (°C): l 9-25°C
- Humidity (%): 40-70%.
- Air changes (per hr): 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours artificial light followed by 12 hours darkness

Administration / exposure

Route of administration:

oral: unspecified

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
An individual stock solution of the test substance was prepared in com oil for each group of animals.
The positive control substance was prepared as a solution in sterile double deionised water (CTL test substance reference number Y045 l 7/0 IO).
All test and positive control substance dosing preparations were prepared as close to the time of dosing as possible. The test substance, vehicle and positive control substance were dosed at a volume of 10 ml/kg bodyweight.

Duration of treatment / exposure:

Bone marrow was collected 24 and 48 hours after dose administration.

Frequency of treatment:

Single oral dose

Post exposure period:

24, 48 h

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

125 mg/kg bw (total dose)

Doses / concentrations 2

Dose / conc.:

250 mg/kg bw (total dose)

Doses / concentrations 3

Dose / conc.:

500 mg/kg bw (total dose)

No. of animals per sex per dose:

2 (two males and two females)
Phase II: 4 animals at 500 mg/kg (four males and four females)

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide at 65 mg/kg

Examinations

Tissues and cell types examined:

Bone marrow cells

Details of tissue and slide preparation:

SLIDE PREPARATION:
The animals were killed by asphyxiation in halothane Ph. Eur. (FLUOTHANE, Zeneca Pharmaceuticals) followed by cervical dislocation 24 and 48 hours after dosing. All animals treated with Agarbois were examined internally for signs of colouration. or abnormalities to organs/tissues.
Femurs were removed and stripped clean of muscle. The iliac end of the femur was removed and a fine paint brush was rinsed in saline, wiped to remove the excess and wetted with a solution of albumin (6% w/v in physiological saline). This was then dipped into the marrow canal and two smears were painted on an appropriately labelled clean, dry microscope slide. This procedure was repeated to give four smears of marrow per slide.
The slides were allowed to air dry and were stained with polychrome methylene blue and eosin using an automatic staining machine.

SLIDE ANALYSIS:
Slides were coded and scored blind. Two thousand (2 x 1000) polychromatic erythrocytes were examined for the presence of micronuclei for each animal. The slides were also examined for evidence of cytotoxicity, which may be manifest by alterations in the ratio of different cell types in the bone marrow. This was assessed by counting the ratio of polychromatic to normochromatic erythrocytes in a sample of 1000 erythrocytes.

Evaluation criteria:

a) No statistically significant increase in the incidence of micronucleated polychromatic erythrocytes above concurrent vehicle control incidences - NEGATIVE.
b) A statistically significant increase in the incidence of micronucleated polychromatic erythrocytes above the concurrent vehicle control incidences but which falls within the laboratory historical vehicle control range - NEGATIVE.
c) A statistically and biological significant increase in the incidence of micronucleated polychromatic erythrocytes which is in excess of a three-fold increase when compared with both historical and concurrent vehicle control incidences - POSITIVE.
d) An incidence of micronucleated polychromatic erythrocytes which is statistically significantly different from the concurrent vehicle control incidences, but less than 3-fold in excess of both historical and concurrent vehicle control incidences may require further evaluation.

Statistics:

Analyses were carried out using the GLM procedure in SAS (1989). Each treatment group mean was compared with the control group mean at the corresponding sampling time using a one-sided Student's t-test, based on the error mean square in the analysis.

Results and discussion

Test results

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

No statistically significant increases in the incidence of micronucleated polychromatic erythrocytes were observed in the repeat experiment. When the data from the original test and the repeat test were combined, again no statistically significant increases in micronucleated polychromatic erythrocytes were observed.

No statistically significant differences in the percentage of polychromatic erythrocytes, between the vehicle control and test item treated animals, were observed in either males or females at any dose level or either sampling time investigated.

The test system positive control, cyclophosphamide, induced statistically and biologically significant increases in the frequency of micronucleated polychromatic erythrocytes in both male and female mice at the 24 hour sampling time.

Any other information on results incl. tables

MEAN INCIDENCE OF MICRONUCLEATED POLYCHROMATIC ERYTHROCYTES/1000 POLYCHROMATIC ERYTHROCYTES ± STANDARD DEVIATION (SD) AT ONE SAMPLING TIME

GROUP MEAN ANIMAL DATA - MALES (PHASEIll)

Group	Treatment	Dose	Mean incidence of MPE/1000 PE± SD
			24 hours
16	Vehicle Control	10 ml/kg	0.8 ± 0.8 .
17	Cyclophosphamide	65mg/kg	22.4 ± 1.5**
18	Agarbois	125mg/kg	0.3 ± 0.5
19	Agarbois	250mg/kg	0.4 ± 0.4
20	Agarbois	500mg/kg	0.6 ± 0.7

 

PE = polychromatic erythrocytes

MPE    = micronucleated polychromatic erythrocytes.

SD      = Standard deviation

**       Statistically significant increase in micronucleated polychromatic erythrocytes at p<0.01 in the Student's t-test (one-sided) on transformed data.

 

All means based on 5 counts of 2000 PE per animal.

MEAN INCIDENCE OF MICRONUCLEATED POLYCHROMATIC ERYTHROCYTES/1000 POLYCHROMATIC ERYTHROCYTES±

STANDARD DEVIATION (SD) AT ONE SAMPLING TIME

GROUP MEAN ANIMAL DATA- FEMALES (PHASEIll)

Group	Treatment	Dose	Mean incidence of MPE/1000 PE±SD
			24	hours
16	Vehicle Control	lOml/kg	0.4±0.4
17	Cyclophosphamide	65mg/kg	25.1±7.2**
18	Agarbois	125mg/kg	0.5±0.6
19	Agarbois	250mg/kg	0.1±0.2
20	Agarbois	500mg/kg	0.2±0.3

PE = polychromatic erythrocytes.

MPE    = micronucleated polychromatic erythrocytes.

SD = standard deviation.

**       Statistically significant increase in micronucleated polychromatic erythrocytesatp<0.01 in the Student's t-test (one-sided) on transformed data.

 

All means based 5 counts of 2000 PE per animal.

Applicant's summary and conclusion

Conclusions:

Under the conditions of test, the test item is not clastogenic in the mouse bone marrow micronucleus test.

Executive summary:

Study design

Agarbois has been evaluated for its ability to induce micronucleated polychromatic erythrocytes in the bone marrow of CD-1 mice. A single oral dose was given to groups of male and female mice at dose levels of 125, 250 and 500 mg/kg.The highest dose level used represents the maximum tolerated dose (MTD) based on patterns of clinical signs and lethalities over a four day observation. Bone marrow samples were taken 24 hours after dosing for the vehicle control, positive control and each of the Agarbois dose levels. Bone marrow samples were taken at the 48 hour sampling time for the vehicle control and the highest dose level of the test item.

A repeat experiment was conducted in which groups of male and female mice were given a single oral dose of test item at doselevels of 125,250 and 500mg/kg.Bone marrow samples were taken 24 hours after dosing.

Results

In the first experiment, small statistically significant increases in the incidence of micronucleated polychromatic erythrocytes were observed in males treated with test item at dose levels of 250 and 500mg/kg at the 24 hour sampling time.

A small statistically significant increase was also observed in females treated with Agarbois at the lowest dose level of  l 25mg/kg.

 

Further testing was conducted with both males and females at dose levels of 125, 250 and 500mg/kg. Bone marrow smears were taken 24 hours after dosing.

In the repeat experiment, no statistically significant increases in the incidence of micronucleated polychromatic erythrocytes were observed in either males or females at any of the dose levels tested. When the data from the original test and the repeat test were combined, again no statistically significant increases in micronucleated polychromatic erythrocytes were observed.

Comparison of the percentage of polychromatic erythrocytes showed no statistically or biologically significant differences at any of the dose levels at either of tlie sampling times between the vehicle control animals and those treated with the test item.

The test system positive control, cyclophosphamide, induced statistically significant and biologically meaningful increases in micronucleated polychromatic erythrocytes,compared to the vehicle control values, thus demonstrating the sensitivity of the test system to a known clastogen.

Conclusion:

Under the conditions of test, Agarbois is not clastogenic in the mouse bone marrow micronucleus test.