1-[(4-chlorophenyl)methyl]-1-cyclopentyl-3-phenylurea

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:

1990-03-28 to 1990-11-14

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1990

Report date:

1990

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

not specified

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

not specified

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: New and Revised Health Effects Test Guidelines October 1984. (U.S.) Environmental Protection Agency Washington, DC (PB 84-233295). HG - Chromo - Micronuc, October 1983

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian erythrocyte micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

1-[(4-chlorophenyl)methyl]-1-cyclopentyl-3-phenylurea

EC Number:

266-096-3

EC Name:

1-[(4-chlorophenyl)methyl]-1-cyclopentyl-3-phenylurea

Cas Number:

66063-05-6

Molecular formula:

C19H21ClN2O

IUPAC Name:

1-[(4-chlorophenyl)methyl]-1-cyclopentyl-3-phenylurea

Specific details on test material used for the study:

white powder

Test animals

Species:

mouse

Strain:

NMRI

Remarks:

Bor: NMRI (SPF Han)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Specifics: young adult male and virgin female mice.
- Age at study initiation: approximately 8 to 12 weeks.
- Weight at study initiation: 28-42 g.
- Assigned to test groups randomly: yes.
- Housing: The females were kept in groups of a maximum of three mice in Makrolon type I cages. Males were kept singly in type I cages.
- Diet: ad libitum.
- Water: ad libitum.
- Acclimation period: at least one week.

ENVIRONMENTAL CONDITIONS
- Temperature: 21.5°C to 22.5°C room temperature
- Humidity: 36-50% mean relative humidity.
- Air changes: at least ten times per hour.
- Photoperiod: twelve hours of electrical lighting daily (6.00 hours to 18.00 hours).

Administration / exposure

Route of administration:

intraperitoneal

Vehicle:

- Vehicle(s)/solvent(s) used: 0.5 % Cremophor emulsion, the positive control material was dissolved in deionized water.
- Concentration of test material in vehicle: 250 mg/ml.

Details on exposure:

Pencycuron was suspended in 0.5 % aqueous Cremophor emulsion using sonication (30 minutes), stirred with a magnetic mixer until administration and injected intraperitoneally.
Cyclophosphamide was dissolved in deionized water and administered in the same way.

Duration of treatment / exposure:

Single dose

Frequency of treatment:

Single dose

Post exposure period:

Animals were sacrificed 24, 48 and 72 hours after the administration.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw (total dose)

Remarks:

Negative control group. Time of sacrifice: 24 hours

Doses / concentrations 2

Dose / conc.:

5 000 mg/kg bw (total dose)

Remarks:

Pencycuron 3 Groups. Time of sacrifice: 24, 48, and 72 hours

Doses / concentrations 3

Dose / conc.:

20 mg/kg bw (total dose)

Remarks:

positive control (cyclophosphamide) group. Time of sacrifice: 24 hours

No. of animals per sex per dose:

5/sex/group/time point

Control animals:

yes

Positive control(s):

Cyclophosphamide
- Route of administration: intraperitoneal.
- Doses / concentrations: 10 ml/kg (20 mg/ml).

Examinations

Tissues and cell types examined:

Bone marrow

Details of tissue and slide preparation:

Schmid's method was used to produce the smears.
At least one intact femur was prepared from each sacrificed animal (not pre-treated with a spindle inhibitor). A suitable instrument was used to sever the pelvic bones and lower leg.
The femur was separated from muscular tissue.
The lower-leg stump, including the knee and all attached soft parts, was separated in the distal epiphyseal cartilage by a gentle pull at the distal end.
The proximal end of the femur was opened at its extreme end with a suitable instrument, e.g., fine scissors, making visible a small opening in the bone-marrow channel.
A suitable tube was filled with sufficient fetal calf serum.
A small amount of serum was drawn from the tube into a suitable syringe with a thin cannula.
The cannula was pushed into the open end of the marrow cavity.
The femur was then completely immersed in the calf serum and pressed against the wall of the tube, to prevent it slipping off.
The contents were then flushed several times and the bone marrow was passed into the serum as a fine suspension.
Finally, the flushing might be repeated from the other end, after it had been opened.
The tube containing the serum and bone marrow was centrifuged in a suitable centrifuge at approximately 1000 rpm for five minutes.
The supernatant was removed with a suitable pipette (e.g., Pasteur pipette), leaving only a small remainder.
The sediment was mixed to produce a homogeneous suspension.
One drop of the viscous suspension was placed on a well-cleaned slide and spread with a suitable object, to allow proper evaluation of the smear.
The labelled slides were dried overnight. If fresh smears needed to be stained, they needed to be dried with heat for a short period.
The Staining of Smears:
The smears were stained automatically with an Ames Hema Tek Slide Stainer from the Miles Company. The slides were then "destained" with methanol, rinsed with deionized water, and left to dry.
The Covering of Smears:
Following this treatment, the smears were transferred to a holder. A cuvette was filled with xylene, into which the holder was immersed for approximately ten minutes. The slides were removed singly (e.g., with tweezers) to be covered.
A small amount of covering agent was taken from a bottle with a suitable object (e.g., glass rod) and applied to the coated side of the slide. A cover glass was then placed in position without trapping bubbles. The slides were not evaluated until the covering agent had dried.

Evaluation criteria:

Coded slides were evaluated using a light microscope at a magnification of about 1000. Micronuclei appear as stained chromatin particles in the anucleated erythrocytes. They can be distinguished from artifacts by varying the focus.
Normally, 1000 polychromatic erythrocytes were counted per animal. The incidence of cells with micronuclei was established by scanning the slides in a meandering pattern.
It is expedient to establish the ratio of polychromatic to normochromatic erythrocytes for two reasons:
1. Individual animals with pathological bone-marrow depressions may be identified and excluded from the evaluation.
2. An alteration of this ratio may show that the test compound actually reaches the target.
Therefore, the number of normochromatic erythrocytes per 1000 polychromatic ones was noted. If the ratio for a single animal amount to distinctly more than 3000 normochromatic erythrocytes per 1000 polychromatic ones, or if such a ratio seems likely without other animals in the group showing similar effects, then the case may be regarded as pathological and unrelated to treatment, and the animal may be omitted from the evaluation. A relevant, treatmentrelated alteration of the ratio polychromatic to normochromatic erythrocytes can only be concluded if it is clearly lower for a majority of the animals in the treated group than in the negative control.
In addition to the number of normochromatic erythrocytes per 1000 polychromatic ones, the number of normochromatic erythrocytes showing micronuclei was also established. This information is useful in two ways. Firstly, it permits the detection of individuals already subject to damage before the start of the test. Secondly, combined with the number of micronucleated polychromatic erythrocytes, it permits a representation of the time-effect curve for positive substances.

Statistics:

The Pencycuron group with the highest mean (provided this superceded the negative control mean) and the positive control were checked by Wilcoxon's non-parametric rank sum test with respect to the number of polychromatic erythrocytes having micronuclei and the number of normochromatic erythrocytes. A variation was considered statistically significant if its error probability was below 5% and the treatment group figure was higher than that of the negative control.

The rate of normochromatic erythrocytes containing micronuclei was examined if the micronuclear rate for polychromatic erythrocytes was already relevantly increased. In this case, the group with the highest mean was compared with the negative control using the one-sided chi2-test. A variation was considered statistically significant if the error probability was below 5% and the treatment group figure was higher than that of the negative control.

In addition, standard deviations (1s ranges) were calculated for all the means.

Results and discussion

Test results

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

Animals treated with 5000 mg/kg bw showed apathy, stretching of body, roughened fur, staggering gait and spasm.

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

After single intraperitoneal administration of 5000 mg/kg Pencycuron, treated animals showed the following compound-related symptoms until sacrifice: apathy, stretching of body, roughened fur, staggering gait, and spasm. Their feeding behavior was normal. There were no substance-induced mortalities.

Since there were no relevant variations in results between males and females, they were evaluated jointly.

The ratio of polychromatic to normochromatic erythrocytes was altered by the treatment with Pencycuron, being 1000: 856 (1s=347) in the negative control, 1000: 1571 (1s=545) in the 24 hours group, 1000: 2045 (1s=1046) in the 48 hours group and 1000: 1089 (1s=282) in the 72 hours group. Relevant variations were thus noted.

No biologically important or statistically significant variations existed between the negative control and the groups treated intraperitoneally with 5000 mg/kg Pencycuron, with respect to the incidence of micronucleated polychromatic erythrocytes. The incidence of these micronucleated cells was 2.0/1000 (1s=0.9) in the negative control, and 2.3/1000 (1s=1.6), 2.1/1000 (1s=1.5) and 1.5/1000 (1s=1.0) in the Pencycuron groups.

Similarly, there could be no biologically significant variation between the negative control and Pencycuron groups in the number of micronucleated normochromatic erythrocytes, since normochromatic erythrocytes originated from polychromatic ones. As expected, relevant variations were not observed.

The positive control, cyclophosphamide, caused a clear increase in the number of polychromatic erythrocytes with micronuclei. The incidence of micronucleated cells was 24.8/1000 (1s=14.4), which represents a biologically relevant increase in comparison to the negative control.

There could not have been a biologically relevant effect on the number of micronucleated normochromatic erythrocytes in the positive control since, in conjunction with the cell-cycle duration, normochromatic erythrocytes originated from polychromatic ones.

No further effect of cyclophosphamide was found concerning the ratio of polychromatic to normochromatic erythrocytes, since this ratio did not vary to a biologically relevant degree [1000: 776 (1s=342), as against 1000: 856 in the negative control]. This clearly demonstrates that an alteration of the ratio of polychromatic to normochromatic erythrocytes is not necessary for the induction of micronuclei.

Applicant's summary and conclusion

Conclusions:

Animals treated with 5000 mg/kg bw showed apathy, stretching of body, roughened fur, staggering gait and spasm. Pencycuron did not induce micronuclei in mouse bone marrow cells.

Executive summary:

The micronucleus test was employed to investigate Pencycuron in male and female mice for a possible clastogenic effect on the chromosomes of bone-marrow erythroblasts. The known clastogen and cytostatic agent, cyclophosphamide, served as positive control.  The treated animals received a single intraperitoneal administration of either Pencycuron or cyclophosphamide. The femoral marrow of groups treated with Pencycuron was prepared 24, 48 and 72 hours after administration. All negative and positive control animals were sacrificed after 24 hours. The doses of Pencycuron and the positive control, cyclophosphamide, were 5000 and 20 mg/kg body weight, respectively.  The animals treated with Pencycuron showed lasting symptoms of toxicity after administration. In addition, there was an altered ratio between polychromatic and normochromatic erythrocytes.
No indications of a clastogenic effect of Pencycuron were found after a single intraperitoneal treatment with 5000 mg/kg bw.  Cyclophosphamide, the positive control, had a clear clastogenic effect, as is shown by the biologically relevant increase in polychromatic erythrocytes with micronuclei. The ratio of polychromatic to normochromatic erythrocytes was not altered.