Ethyl [2-(4-phenoxyphenoxy)ethyl]carbamate

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:

13 Sep 1996 to 29 Nov 1996

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1996

Report date:

1996

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

1983

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1992

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OTS 798.5395 (In Vivo Mammalian Cytogenics Tests: Erythrocyte Micronucleus Assay)

Version / remarks:

1987

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: MITI Japan

Version / remarks:

1987

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian erythrocyte micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

Ethyl [2-(4-phenoxyphenoxy)ethyl]carbamate

EC Number:

276-696-7

EC Name:

Ethyl [2-(4-phenoxyphenoxy)ethyl]carbamate

Cas Number:

72490-01-8

Molecular formula:

C17 H19 N O4

IUPAC Name:

ethyl [2-(4-phenoxyphenoxy)ethyl]carbamate

Test animals

Species:

mouse

Strain:

Tif:MAGf

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 7 to 8 weeks
- Weight at study initiation: 23-28 g for females and 27-38 g for males.
- Housing: The animals were housed 5/cage (reserve animals: 3/cage) and marked with color pens.
- Diet: Pelleted standard diet, ad libitum
- Water: Tap water, ad libitum
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.5
- Humidity (%): 41-48
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: 13 Sep 1996 to 29 Nov 1996

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle used: Arachis oil
- Amount of vehicle: 10 mL/kg body weight
- Justification for choice of vehicle: Another in vivo study performed with the test substance has revealed arachis oil to be the best suited vehicle, yielding an applicable suspension at the dose level of 5000 mg/kg

Duration of treatment / exposure:

Negative control + high dose: 16, 24 and 48 hours
Intermediate dose + low dose + positive control: 24 hours

Frequency of treatment:

Once

Post exposure period:

16, 24 or 48 hours

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

1 250 mg/kg bw/day (actual dose received)

Remarks:

Low dose

Doses / concentrations 2

Dose / conc.:

2 500 mg/kg bw/day (actual dose received)

Remarks:

Intermediate dose

Doses / concentrations 3

Dose / conc.:

5 000 mg/kg bw/day (actual dose received)

Remarks:

High dose

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide
- Route of administration: Oral by stomach tube, 10 mL/kg body weight
- Doses: 64 mg/kg dissolved in bidistilled water

Examinations

Tissues and cell types examined:

Subsequently femoral bone marrow cells were prepared and polychromatic erythrocytes were scored for micronuclei

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
A tolerability test was not performed, since LD50- data obtained in another in vivo study performed (p.o.) on mouse with the test substance indicated a maximum tolerated dose (MTD) > 5000 mg/kg. Signs of toxicity were recorded hourly for the first few hours after application and once the following days. Body weight were recorded daily.

TREATMENT AND SAMPLING TIMES: The animals were sacrificed by CO2 gas. Bone marrow was harvested in fetal calf serum from the shafts of both femurs, centrifuged and resuspended in fetal calf serum. Smears prepared from this suspension were stained with May-Grünwald/Giemsa solution and mounted. See table 1 in ''any other information on material and methods''.

DETAILS OF SLIDE PREPARATION:
Micronuclei are uniform, darkly stained, more or less round bodies in the cytoplasm of erythrocytes. Inclusions which are reflective, improperly shaped or stained, or which are not in the focal plain of the cell are judged to be artifacts and are not scored as micronuclei. Cells containing more than one micronucleus are only counted once. Prior to analysis the slides were coded. The slides of five animals/sex/dose, showing good between mature and polychromatic erythrocytes, were scored by a laboratory technician. The incidence of micronucleated polychromatic erythrocytes (MNPCE) among at least 2000 polychromatic erythrocytes (PCB), and the ratio of PCB to normochormatic erythrocytes (NCE) among a total of at least 1000 erythrocytes was determined for each slide.

OTHER:
Analytical chemistry: To confirm that the animals were actually exposed to the intended doses of the test substance and to confirm the stability of the in vehicle used substance in the vehicle used, determinations of the applied concentrations of the test substance in the vehicle used performed by the analytical unit. These determinations were performed with the 16 h and 24/48 h samples of the test substance prepared for application in the micronucleus test, representing the high dose (5000 mg/kg) and with the 24h sample prepared which represents the low dose (1250 mg/kg).

Evaluation criteria:

The results of the experiments were evaluated with respect to the mean number pf PCEs with micronuclei. The groups compared differed by treatment, sampling time and sec of the animals. The data from females and males were pooled for evaluation. In case of significant sex differences data have to be evaluated for each sex separately in addition.

Statistics:

The significance of differences was assessed by the Chi-Squared-Contingency-Test (F=1, p<0.05).

Results and discussion

Test results

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

At all sampling times (16, 24 and 48 hours) there was no statistically significant increase in the number of micronucleated polychromatic erythrocytes in the animals treated with the respective doses of the test substance as compared with the negative control animals. The percentages of micronucleated PCEs found in the negative controls were within the range of the negative control (0.05 ± 0.04). In the positive control (24 hours) the percentage of micronucleated cells within polychromatic erythrocytes was clearly increased. The mean percentage of micronucleated PCEs was 1.85. In comparison with the negative control (0. 06) this value15 highly significant (p<0.05) and within the range of the historical positive control (1.40± 0.80).

Any other information on results incl. tables

Table 1. Micronuclues test

Overall mean percentages of micronucleated PCEs
Treatment time	High dose (GD) 5000 mg/kg	Intermediate dose (ID) 2500 mg/kg	Low dose (LD) 1250 mg/kg	Positive control (CPA)	Negative control (vehicle
16 hours	0.07				0.08
24 hours	0.06	0.08	0.08	1.85	0.06
48 hours	0.09				0.06

Applicant's summary and conclusion

Conclusions:

It is concluded that under the given experimental conditions, no evidence for clastogenic or aneugenic effects was obtained in mice treated with the test substance

Executive summary:

 

The test substance was investigated for clastogenic (and/or aneugenic) on mouse bone marrow cells in vivo according to OECD TG 474 and GLP principles. The test substance was administered once by oral gavage to groups of 5 male and 5 female Tif: MAGf (SPF) mice at doses of 5000, 2500 and 1250 mg/kg. Additional groups of animals were treated with the vehicle alone (arachis oil, 10mL/kg body weight) or with the positive control cyclophosphamide (64 mg/kg body weight). From the high dose group and from the negative control group animals were sacrificed 16, 24 and 48 hours thereafter. From the intermediate and the low dose group and from the positive control group animals sacrificed 24 hours after application. Subsequently femoral bone marrow cells were prepared and polychromatic erythrocytes were scored for micronuclei.

Results showed that none of the animals treated with the test material showed symptoms of toxicity. In all dosage groups assessed at the different periods post treatment, no statistically significant increase in the number of micronucleated polychromatic erythrocytes was observed when compared with the respective negative control group. In the positive control group the percentage of micronucleated cells with polychromatic erythrocytes was clearly increased.

It is concluded that under the given experimental conditions, no evidence for clastogenic or aneugenic effects was obtained in mice treated with the test substance