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

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1986-06-02

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1986

Report date:

1986

Materials and methods

Test guideline

Qualifier:

no guideline available

Principles of method if other than guideline:

The test was carried out following a publication of Moorhead (Exp. Cell. Res. 20 (1960), 613-616). The positive control gave the expected response. The method and results are not fully described. However, there are no doubts about the integrity of the study and the results are in line with other genotoxicity studies.

GLP compliance:

no

Remarks:

Older study, predates mandatory GLP

Type of assay:

in vitro mammalian chromosome aberration 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

Method

Species / strain

Species / strain / cell type:

lymphocytes: Human Lymphocytes

Details on mammalian cell type (if applicable):

Blood from healthy test persons was obtained on the test day and mixed with Liquemin (0.5 ml per 10 ml blood). Portions of 2 ml were drawn up into 2 ml disposable syringes, which were placed in stands with the point upwards for sedimentation of the blood. The transparent layer down to 0.6 ml was then discarded and the remainder, together with 0.2 ml of the following layer(s), was transferred to 9 ml chromosome medium B in culture flasks. The finished medium contains a phytohaemagglutinin additive prepared specifically for lymphocyte cultivation for the purpose of chromosome representation. The mixture was incubated at 37° C.

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: The livers of at least six adult male Sprague Dawley rats.

- method of preparation of S9 mix:
For enzyme induction the animals were given a single intraperitoneal injection of Aroclor 1254, at a dose of 500 mg/kg body weight dissolved in peanut oil, five days before preparation. The S-9 mix was prepared fresh (Ames et al., 1973), and only used on the same day.

- concentration or volume of S9 mix and S9 in the final culture medium:
Until use it, S9 mix was kept in a vessel placed in iced water. Its composition per 100 ml was as follows:

MgCl2 x 6 H2O: 271.0 mg
KCl: 410.0 mg
glucose-6-phosphate, disodium salt: 298.5 mg
NADP, disodium salt: 525.0 mg
phosphate buffer 100.0 mM: 50.0 ml

The S-9 mix contained 50 % S-9 fraction

Test concentrations with justification for top dose:

0, 25, 50 and 100 µg/ml.
The concentrations used were based on a pilot test.

Vehicle / solvent:

DMSO

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

The test compound, dissolved in DMSO at a volume of 0.1 ml, was added to the culture 48 hours after start of cultivation. Only the solvent was added to the negative control. With quantities under 0.1 ml, the short volume was made up by a suitable quantity of Hank's saline solution. The positive controls cyclophosphamide and mitomycin C were dissolved in Hank's saline solution and added at a volume of likewise 0.1 ml.

At the same time 0.1 ml of Hank's saline solution was added to the test group cultures without metabolic activation, to make up the volume. Simultaneously 0.1 ml S9 mix was added to the cultures in the groups with metabolic activation. To reduce the toxic side effects of the S9 mix, these cultures were washed two and a half hours after it had been added. The total volume was therefore 10 ml for all the cultures.

Twenty-one hours after the substance had been added, colchicine to a final concentration of 0.4 µg/ml was added to the cultures, to arrest mitosis in the metaphase stage. Three hours later the slides were produced. Two to three slides were made for each culture.

At the end of cultivation, the cultures were shaken gently, and each transferred to a graduated tube. They were then prepared as follows:

1. Centrifuged for five minutes at 1500 rpm.
2. Supernatant carefully discarded.
3. Sediment gently disturbed.
4. To each culture 1 to 2 ml of approx. 37° C warm KCl solution (0.56 %) was added. This hypotonic solution had to act for about seven minutes at room temperature. During this time the volume was topped up to a total of 6 ml with KCl solution
The suspension was mixed.
5. See 1.
6. See 2.
7. See 3.
8. A few drops of cold fixative (ethanol + glacial acetic acid =3+1 , approx. 4° C) were added and mixed. The solution was then topped up with fixative to 6 ml and again mixed. It then stood for twenty to thirty minutes at room temperature.
9. See 1.
10. The supernatant was discarded, the sediment agitated and resuspended in 6 ml fixative.
11. See 1.
12. The supernatant was discarded; the sediment was tapped up and carefully resuspended in a little fixative (0.5 to 1 ml depending on the suspension's density).
13. This suspension was dropped on to clean slides cooled in water and was then allowed to dry.
14. The slides were then stained in 5 % Giemsa solution for about five minutes, and then rinsed twice briefly in water.
After they had dried, they were covered.

The concentrations in the repeat test were in the range of 12 µg/ml to 48 µg/ml without S9 mix, due to the figures at the lowest concentration without S-9 mix, which did not correlate with dose but were significantly higher.

Evaluation criteria:

Approx. 200 metaphases per concentration, both with and without S-9 mix, were examined for structural changes in the chromosomes. This structural chromosome damage was assessed for the most part by using the terminology defined by Rieger and Michaelis as follows.

1. Gap: A gap is an achromatic gap within a chromatid arm, the end of which is not dislocated.
2. Break: This is a broken-off chromatid end which is clearly dislocated but does not yet have to be contained in the metaphase concerned.
Breaks are the prerequisite for all further chromosome damage.
3. Fragment: This is a centromere-less chromosome fragment, which has resulted from a break and is not in the same metaphase as the chromosome concerned.
4. Deletion: This is the loss of a piece of a chromatid as a result of a break, and the detached part is no longer present in the metaphase.
5. Exchange: This is an exchange mutation between several chromosomes (interchange) or within one chromosome (intrachange).
Since exchanges occur spontaneously, with a probability of about 1:10000, the occurrence of more than one exchange in a treatment group is sufficient to indicate a potential mutagenic effect, even if the incidence of all the other types of aberration is not increased.
6. Multiple aberration: If there are more than four cases of structural damage excluding gaps, this is assessed as a multiple aberration.

If almost no chromosomal structures are present in a metaphase, then chromosome breakdown is assumed. These metaphases are separately noted and listed, additionally to the hundred metaphases evaluated in each case.

A light microscope at approx. 1000 magnification with plan apochromatic lenses was used for the evaluation, each damaged metaphase being photographed for documentation.

Results and discussion

Test results

Species / strain:

lymphocytes: Human Lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Cytotoxicity observed at dose level: > 12 µg/ml (mitotic index decreased)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

Pencycuron did not induce chromosome aberrations in human blood lymphocytes under the conditions of the test.

Executive summary:

Pencycuron was evaluated with human lymphocyte cultures after in vitro treatment at concentrations up to 100 µg/ml, both with and without S9 mix, for effects on chromosomes.  The results of the determination of the mitotic index indicate a significant concentration-related cytotoxic effect for Pencycuron in the test groups, both with and without S9 mix.  There were no treatment-related variations between the negative control and the groups treated in vitro with concentrations up to 100 µg/ml Pencycuron, in respect to the parameters relevant to the assessment of a clastogenic effect (metaphases with aberrations including and excluding gaps, and metaphases with exchanges). The positive controls mitomycin C and cyclophosphamide had a clear clastogenic effect, and so demonstrated the system's sensitivity to agents harmful for chromosomes.