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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The genetic toxicity of pencycuron has been investigated in vitro in studies of bacterial reverse mutation, mammalian cell (forward) mutation and mammalian cell cytogenicity.  All studies report negative results, both in the absence and presence of exogenous metabolic activation.  Although a study in vitro specifically investigating numerical chromosomal aberrations is not available for pencycuron, this endpoint has been investigated in a higher tier study in vivo with evidence of target tissue exposure.

Test Species/Type	Results	Assessment	Reference
None - Ames test: negative result in four strains of S. typhimurium; up to 1000 ug/plate (+/-S9)	Pencycuron was not mutagenic under the conditions of this study.	Not specified. Insufficient strains tested, highest tested concentration insufficient.  DAR notes some rsults tables are not comprehensible	Inukai & Iyatomi (1978)
None - Ames test: negative result in four strains of S. typhimurium and one of E. coli.  Tested concentrations up to 5000 ug/plate	Pencycuron was not mutagenic under the conditions of this study.	Supporting study	Shirasu (1981)
OECD 471 (1997) - Ames test: negative result in five strains of S. typhimurium, up to 5000 ug/plate (some precipitation and toxicity)	Pencycuron was not mutagenic under the conditions of this study.	Key study	Herbold (2008)
None - Cytogenicity study in human lymphocytes; concentrations up to 100 ug/mL (cytototoxic)	Pencycuron was not clastogenic under the conditions of this study.	Supporting study	Herbold (1986)
None - Cytogenicity study in CHL cells (concentrations sufficient to cause cytotoxicity)	Pencycuron was not clastogenic under the conditions of this study.	Supporting study	Shirasu  (1987)
None stated, but comparable to OECD 476 - Mammalian cell HPRT assay; tested up to cytotoxic concentrations	Pencycuron was not mutagenic under the conditions of this study.	Key study	Brendler (1991)
None - Rec assay	Pencycuron did not cause DNA damage in this assay	- not a REACH information requirement	Inukai & Iyatomi (1978)
None - Yeast assay	Pencycuron was not mutagenic under the conditions of this study	- not a REACH information requirement	Jagannath (1981)
None - Rec assay	Pencycuron did not cause DNA damage in this assay	- not a REACH information requirement	Shirasu  (1981)
None - Yeast assay	Pencycuron was not mutagenic under the conditions of this study	- not a REACH information requirement	Brusick (1982)
None - UDS assay in vitro	Pencycuron did not induce unscheduled DNA synthesis under the conditions of this assay	- not a REACH Information requirement	Lehn (1990)
None - Mouse micronuleus assay	Pencycuron did not induce micronuclei formation under the conditions of this assay	- not acceptable due to methodological deficiencies	Herbold  (1981)
None - Mouse micronuleus assay: ip dosing up to 5000 mg/kg bw	Pencycuron did not induce micronuclei formation under the conditions of this assay.	Key study	Herbold  (1990)
None - Dominant lethal assay	Pencycuron did not cause a dominant lethal effect under the conditions of this study	- not a REACH Information requirement	Herbold  (1983)
None - Dominant lethal assay	Pencycuron did not cause a dominant lethal effect under the conditions of this study	- not a REACH Information requirement	Herbold  (1983)

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1986-05-15 to 1987-05-11

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Qualifier:

no guideline available

Principles of method if other than guideline:

There is not referred to a guideline or publication for the method followed; the method and results are not fully described. The positive control gave the expected response. 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. An Quality Assurance Authorization was included.

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

White crystalline

Species / strain / cell type:

Chinese hamster lung (CHL/IU)

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: 4 Sprague-Dawley male rats (7 weeks old; average body weight, 284g).

- method of preparation of S9 mix:
Animals were given a single intraperitoneal injection of a polychlorinated biphenyl mixture (Aroclor 1254) at a dosage of 500 mg/kg. The animals were fasted overnight on the fifth night after the injection. On the next day, the animals were killed by cervical dislocation and the livers were removed immediately.
The livers were perfused with chilled 0.15 M KCl solution and homogenized in three volumes of the same solution (3 ml/g wet liver). The homogenate was centrifuged for 10 min. at 9000 x g.
All the steps were performed below 5°C with cold and sterile solutions and glassware. The 9000 x g supernatant (S-9 fraction) was stored at -80°C.

- concentration or volume of S9 mix and S9 in the final culture medium:
The components of the activation system (S9 mix) were 30% S9 fraction, 8 mM MgCl2, 33 mM KC1, 5 mM glucose-6-phosphate, 4 mM NADPH, 4 mM NADH, and 100 mM Na-phosphate buffer (pH 7.2).
The final concentration of S-9 fraction in the medium was 5%.

Test concentrations with justification for top dose:

1x10^-3, 3.3x10^-4, 1x10^-4, 3.3x10^-5, 1x10^-5 M (+ S9).
1x10^-4, 3.3x10^-5, 1x10^-5, 3.3x10^-6 M (- S9).

The highest concentration of the test compound for the cytogenetics test was determined by the results from a preliminary growth test.

Vehicle / solvent:

DMSO at 0.5%

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO at 0.5%

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

mitomycin C

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of independent experiments: Two independent cultures were used for each experimental point.

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 1 x 10^6 cells/10cm dish.
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: without metabolic activation: 24 or 48 hours. With metabolic activation: 6 hours.

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Spindle inhibitor (cytogenetic assays): colchicine, 0.5 µg/ml, in c-metaphase, two hours prior to harvesting.
- Analysis:
Only good metaphases which satisfying the karyotype of CHL cell were analyzed and structural chromosome aberrations were recorded and classified. The number of chromosomes was not counted. The number of cells analyzed was 200 per experimental point. Metaphase containing at least one structural chromosome aberration was considered as an aberrant metaphase and recorded as one aberrant metaphase.

Mitotic indices were calculated as the number of metaphases per 1000 cells.

Evaluation criteria:

Judgement of results was as follows:

Aberrant metaphase frequency (%) Judgement
10% or more Positive
5 - 10% Inconclusive
less than 5% Negative

Species / strain:

Chinese hamster lung (CHL/IU)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Cytotoxicity observed at dose level: > 3.3x10^-4 M (+S9) and > 3.3x10^-5 M (-S9)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

1) Direct method:
The results of the preliminary growth test were as follows:
At the concentrations of 1.0 x 10^-3 M and 3.3 x 10^-4 M, cell growth was 23% and 20% of solvent control respectively, which revealed suppression of cell growth. At 1.0 x 10^-4 M cell growth was 40.5% of solvent control, which revealed 50% or more suppression of cell growth. On the other hand, at 3.3 x 10^-5 M cell growth was 76.5% of solvent control. Based on the above results, experiments were carried out at the following 5 concentrations including the concentrations at which suppression of cell growth was revealed remarkably: 3.3 x 10^-4 M, 1.0x10^-4 M, 3.3 x 10^-5 M, 1.0 x 10^-5 M, 3.3 x 10^-6 M.

At the concentration of 3.3 x 10^-4 M no mitotic preparation could be prepared due to death of cells. At 1.0 x 1010^-4 M suppression of cell division was remarkably observed. In one mitotic preparation which was prepared after the treatment for 24 hours, the number of available metaphases observed was less than 100. In the mitotic preparations which were prepared after the treatment for 48 hours no metaphase could be observed. The aberrant metaphase frequencies were less than 5% at any sampling time and concentration.

On the other hand, MMC used as a positive control induced marked increases in the incidence of aberrant metaphases.

2) Metabolic activation method:
The results of the preliminary growth test were as follows:
At the concentration of 1.0 x 10^-3 M cell growth was 37.0% of solvent control, which revealed $0% or more suppression of cell growth. At 3.3 x 10^-4 M cell growth was 67.5% of solvent control. Based on the above results, experiments were carried out at the following 5 concentrations: 1.0 x 10^-3 M, 3.3 x 10^-4 M, 1.0 x 10^-4 M, 3.3 x 10^-5 M, 1.0 x 10^-5 M.

At the concentration of 1.0 x 10^-3 M the number of metaphases observed was less than 100 due to suppression of cell division in both mitotic preparations which were prepared after the treatment for 12 hours.

On the other hand, BaP used as a positive control induced marked increases in the incidence of aberrant metaphases.

Conclusions:

The aberrant metaphase frequencies were less than 5/6 at any sampling time and concentration either in the absence or presence of the metabolic activation enzymes.
From these results, it is concluded that pencycuron did not induce chromosome aberrations in CHL cells, either in the absence or presence of the metabolic activation enzymes, under the conditions used in this test.

Executive summary:

The in vitro cytogenetics test was performed on pencycuron using cultured CHL Chinese hamster lung cells to evaluate the clastogenic potential. In a direct method, mitotic preparations were prepared after single treatment with pencycuron at 3.3 x 10^-4 - 3.3 x 10^-6 M for 24 and 48 hours. In a metabolic activation method, cells were treated with pencycuron at 1.0 x 10^-3 - 1.0 x 10^-5 M in the presence of S9 mix for 6 hours, and then the treatment medium was replaced by a fresh medium. Twelve and 18 hours after the medium change, mitotic preparations were prepared.  The aberrant metaphase frequencies were less than 5/5 at any sampling time and concentration either in the absence or presence of the metabolic activation enzymes.  It is concluded that pencycuron did not induce chromosome aberrations in CHL cells, either in the absence or presence of the metabolic activation enzymes, under the conditions used in this test.

Reference 2

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

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

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

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.

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

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. 

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1981-Unclear to 1981-05-08

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

no guideline followed

Principles of method if other than guideline:

The positive controls gave the expected response. The test was carried out following a publication of Ames et. al. The study does not contain a GLP statement. However, there are no doubts about the integrity of the study and the results are in line with the other genotoxicity studies

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Target gene:

Not specified.

Species / strain / cell type:

bacteria, other: Bacillus subtilis

Additional strain / cell type characteristics:

other: recombination-wild (H17) and -deficient (M.45) strains of B. subtilis

Species / strain / cell type:

S. typhimurium TA 1538

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

For the preparation of a liver metabolic activation system, Sprague-Dawley male rats (7 weeks old, average body weight 243 g) were given a single intraperitoneal injection of a polychlorinated biphenyl (PCB) mixture (Aroclor 1254) at a dosage of 500 mg/kg. In the evening of the fourth day after the injection, the food was removed. On the fifth day the animals were killed by cervical dislocation and immediately livers were removed. The livers were perfused with an ice-cold 0.15 M KCl solution and were homogenized in three volumes of the same solution (3 ml/g wet liver). The homogenate was centrifuged for 10 minutes at 9000 x g. The 9000 x g supernatant (S-9 fraction) was used in the experiment. All the steps were performed below 5°C with cold and sterile solutions and glassware. The components of 1 ml of the metabolic activation system (S-9 mix) were as follows:
0.1 ml S-9 fraction
8 mM MgCl2
33 mM KCl
5 m mM glucose-6-phosphate
4 mM NADPH
4 mM NADH
100 mM sodium phosphate (pH 7.4)

Vehicle / solvent:

DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

N-ethyl-N-nitro-N-nitrosoguanidine

mitomycin C

other: AF-2; 2-AA

Details on test system and experimental conditions:

1) Rec-assay
The rec-assay using the recombination-wild (H17) and -deficient (M.45) strains of B. subtilis was carried out to survey the DNA-damaging capability of pencycuron. Frozen cultures of the two strains stored at -80°C were thawed and streaked with the use of small pipettes onto the surface of a B-2 agar plate with care not to let them touch each other. A filter paper disk, 10 mm in diameter, was soaked with 0.02 ml of a solution of the compound and was placed so as to cover the starting parts of the bacterial streaks. The length of the inhibitory zone of each streak was measured after overnight incubation at 37°c. Kanamycin and Mitomycin C were used as a negative and a positive control, respectively. The results were judged as positive when the length of the inhibitory zone in M.45 strain exceeded 3 mm at the concentration of the compound that induced 0 - 1 mm of inhibitory zone in H17 strain.
2) Reverse mutation test with or without a liver metabolic activation system
Five strains of the Ames' TA series of S. typhimurium were used to investigate the mutagenic potential of pencycuron in reverse mutation tests. These strains were TA1535, TA1537, TA1538, TA98 and TA100. They are all histidine auxotrophs. In addition, E. coli WP2 her requiring tryptophan was also employed.
These six strains stored at -80°0 were inoculated on nutrient broth liquid medium and cultured by shaking at 37°C over night. For the S. typhimurium strains a sterile solution of 0.5 mM L-histidine-0.5 mM D-biotin was added to molten soft agar (0.6% agar and 0.5% NaCl) at the rate of 1/10 (v/v), and for the E. coli strain a 0.5 mM L-tryptophan solution was added at the same rate. The prepared soft agar was named "top agar".
To 2 ml of the molten top agar were added 0.1 ml of a bacterial suspension, 0. 1 ml of a solution of the compound, and 0. 5 ml of 100 mM sodium phosphate (pH 7.4) or the S-9 mix. The contents were mixed uniformly and poured onto the surface of a minimal agar plate (1.5% agar and 0.5% glucose) with modified Vogel-Bonner E medium). All plates were incubated at 37°C for 2 days, after which the number of revertant colonies was counted. The following compounds were used as positive controls: AF-2(2-(2-furyl)-3- (5-nitro-2-furyl) acrylamide; ENNG(N-ethyl-N'-nitro-N-nitrosoguanidine); 9-AA(9-aminoacridine); 2-NF(2-nitrofluorene); 2-AA(2-aminoanthracene). The results were induced based on reproducibility and relation between doses and reactions.

Key result

Species / strain:

bacteria, other: B. subtilis

Metabolic activation:

not applicable

Genotoxicity:

negative

True negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

other: S. typhimurium TA 1535, TA 1537, TA 1538, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

True negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

1) Rec-assay
Pencycuron did not induce any inhibitory zone in either strain in all the doses tested. On the other hand, the negative control, Kanamycin, induced similar lengths of inhibitory zones in the two strains, whereas the positive control, Mitomycin C, caused a marked difference in the length of inhibitory zones of the two strains.
2) Reverse mutation tests with or without a liver metabolic activation system
Pencycuron did not induce any significant increase in the number of revertant colonies of any strains compared with those of the corresponding control whether the S-9 mix was added or not. In contrast, AF-2, ENNG, 9-AA and 2-NF, used as positive controls, induced reverse mutations in the absence of the S-9 mix, and 2-AA was mutagenic for all the strains in the presence of the S-9 mix.

Conclusions:

Acceptability
The study is considered acceptable.
Conclusions
The test substance did not induce point mutations in S. typhimurium or in E coli WP2 hcr under the conditions of the test.

Executive summary:

The microbial mutagenicity testing was performed on pencycuron. This compound was negative in the repair test (rec-assay) with Bacillus subtilis H17 (rec+) and M45 (rec-) at the maximum dose tested (5,000 pg/disk) and in the reverse mutation tests with or without a liver metabolic activation system employing Escherichia coli WP2 her and Salmonella typhimurium TA series (TA1535, TA1537, TA1538, TA100, and TA98) as tester strains.

Reference 4

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2008-03-19 to 2008-07-11

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

2000

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Version / remarks:

August 1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

white powder

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: from the livers of at least six adult male Sprague Dawley rats of approximately 200 to 300 g in weight.

- method of preparation of S9 mix:
For enzyme induction, the animals received a single intraperitoneal injection of Aroclor 1254, dissolved in corn oil, at a dose of 500 mg/kg body weight, five days prior to sacrifice. The animals were prepared unfasted, following the directions of Ames et al. (1975) and Maron and Ames (1983).

The rats were terminated. Livers were removed under sterile conditions immediately after sacrifice and kept at 4°C until all animals had been prepared. All the remaining steps were carried out under sterile conditions at 4°C.

The livers were washed with cold (4°C), 0.15 M KCI solution (approximately 1 ml KCI per 1 g liver), and then homogenized in fresh, cold (4°C), 0.15 M KCI (approximately 3 ml KCI per 1 g liver). The homogenate was then centrifuged in a cooling centrifuge at 4°C and 9000 g for 10 minutes. The supernatant (the S9 fraction) was stored at -80°C in small portions.

- concentration or volume of S9 mix and S9 in the final culture medium:
Seventy ml of cofactor solution are composed as follows:

MgCI2 x 6H2O: 162.6 mg
KCI: 246.0 mg
Glucose-6-phosphate, disodium salt: 179.1 mg
NADP, disodium salt 315.0 mg
Phosphate buffer 100.0mM

S9 mix consists of this cofactor solution, S9 fraction and, if needed, 0.15 M KCI.

Prior to first use, each batch was checked for its metabolizing capacity by using reference mutagens; appropriate activity was demonstrated. At the beginning of each experiment 4 aliquots of the S9 mix were plated (0.5 ml per plate) in order to assess its sterility. This was repeated after completion of test tube plating. The sterility control plates were then incubated for 48 hours at 37°C. No indication of contamination of S9 mix was found.

Test concentrations with justification for top dose:

0, 16, 50, 158, 500, 1581, 5000 µg per plate.

The doses for the first trial were routinely determined based on a standard protocol: if not limited by solubility 5000 µg or 5 µl per plate were used as the highest dose. At least five additional doses were routinely used. If less than three doses were used for assessment, at least two repeats were performed.

The doses of the preincubation trial were determined based on the results of the plate incorporation assay. Doses are given as µg/tube for better separation of plate incorporation and preincubation trials, despite the fact that µg/plate and µg/tube could be used synonymously.

Vehicle / solvent:

- Mitomycin C was dissolved in deionized water. Pencycuron and the other positive controls were dissolved in DMSO. Pencycuron formed clear colourless solutions in DMSO.

- The solvent used was chosen out of the following solvents, in the order given: water, DMSO, methanol, ethanol, acetone, ethylene glycol dimethylether (EGDE), and DMF according to information given by the sponsor. The order of these solvents is based on their bacteriotoxic effects in preincubation experiments.

- The amount of solvent for the test substance and for the controls was 0.1 ml/plate.

Untreated negative controls:

no

Remarks:

No "untreated" negative control was set up for the used solvent, since sufficient evidence was available in the literature and from our own experience, indicating that this solvent had no influence on the spontaneous mutant counts of the used strains.

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

cumene hydroperoxide

mitomycin C

other: Nitrofurantoin, 4-Nitro-1,2-phenylene diamine, 2-Aminoanthracene,

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments: 2, The independent repeat was performed as preincubation in a water bath at 37°C for 20 minutes.

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium in the preincubation period, in agar (plate incorporation)

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period: 20 min
- Exposure duration/duration of treatment: 20 min + 48 hours.
- Harvest time after the end of treatment (sampling/recovery times):

FOR GENE MUTATION:
- Method used: agar
- The dilution of bacterial suspensions used for the determination of titers was 1:1,000,000.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- The toxicity of the substance was assessed in three ways. The first method was a gross appraisal of background growth on the plates for mutant determination. Secondly, a toxic effect of the substance was assumed when there was a marked and dose-dependent reduction in the mutant count per plate, compared to the negative controls. Thirdly, the titer was determined. Total bacterial counts were taken on two plates for each concentration studied with S9 mix. However, if an evaluation was performed only without S9 mix, the bacterial count was taken without S9 mix.

METHODS FOR MEASUREMENTS OF GENOTOXICIY
The count was made after the plates had been incubated for 48 hours at 37°C. If no immediate count was possible, plates were temporarily stored in a refrigerator. If not interfered e.g., by precipitation on the plates or coloration of the plates, colonies were counted automatically using the Petri Viewer Mk2.

Evaluation criteria:

The following criteria determined the acceptance of an assay:
1. The negative controls had to be within the expected range, as defined by published data (e.g., Maron and Ames, 1983) and/ or the laboratories' own historical data.
2. The positive controls had to show sufficient effects, as defined by the laboratories'' experience.
3. Titer determinations had to demonstrate sufficient bacterial density in the suspension.

Only trials which complied with all three of the above criteria were accepted for assessment. Even if the criteria for points (b) and (c) were not met, a trial was accepted if it showed mutagenic activity of the test compound. Furthermore, an unacceptable trial would have been repeated.

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Toxicity was observed at the test substance concentration of 5000 µg/plate in the presence of S9-mix only. Precipitation was observed at test substance concentrations of 1581 µg/plate and above.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Toxicity was observed at the test substance concentration of 5000 µg/plate in the presence of S9-mix only. Precipitation was observed at test substance concentrations of 1581 µg/plate and above.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Toxicity was observed at the test substance concentration of 5000 µg/plate in the presence of S9-mix only. Precipitation was observed at test substance concentrations of 1581 µg/plate and above.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Toxicity was observed at the test substance concentration of 5000 µg/plate in the presence of S9-mix only. Precipitation was observed at test substance concentrations of 1581 µg/plate and above

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Toxicity was observed at the test substance concentration of 5000 µg/plate in the presence of S9-mix only. Precipitation was observed at test substance concentrations of 1581 µg/plate and above.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

There was no indication of a bacteriotoxic effect of Pencycuron at doses of up to and including 1581 ug per plate. The total bacteria count consistently produced results comparable to the negative controls or differed only insignificantly. No inhibition of growth was noted as well. 5000 µg per tube had only a weak, strain-specific bacteriotoxic effect. Therefore, this dose could nevertheless be used for assessment purposes. At 1581 µg per plate, the substance started to precipitate.

None of the five strains concerned showed in the plate incorporation test a dose- related and biologically relevant increase in mutant counts over those of the negative controls. This applied both to the tests with and without S9 mix and was confirmed by the results of the preincubation trials.

The positive controls sodium azide, nitrofurantoin, 4-nitro-1,2-phenylene diamine, mitomycin C, cumene hydroperoxide and 2-aminoanthracene increased mutant counts to well over those of the negative controls, and thus demonstrated the system's sensitivity and the activity of the S9 mix.

Summary of the Results with Pencycuron in the Salmonella/Microsome Test

S9 mix	TA 1535	TA 100	TA 1537	TA 98	TA 102
without	-ve	-ve	-ve	-ve	-ve
with	-ve	-ve	-ve	-ve	-ve

-ve = negative

Assessment

The Salmonella/rnicrosome test, employing doses of up to 5000 µg per plate, showed Pencycuron to produce bacteriotoxic effects at 5000 µg per plate. Substance precipitation occurred at 1581 µg per plate and above.
Evaluation of individual dose groups, with respect to relevant assessment parameters (dose effect, reproducibility) revealed no biologically relevant variations from the respective negative controls.In spite of the low doses used, positive controls increased the mutant counts to well over those of the negative controls, and thus demonstrated the system's high sensitivity. Despite this sensitivity, no indications of mutagenic effects of Pencycuron could be found at assessable doses of up to 5000 µg per plate in any of the Salmonella typhimurium strains used.  Due to these results Pencycuron has to be regarded as non-mutagenic.

Conclusions:

Pencycuron is not mutagenic in the Salmonella typhimurium reverse mutation assay in the absence and presence of metabolic activation.

Executive summary:

Pencycuron was initially investigated using the Salmonella/microsome plate incorporation test for point mutagenic effects in doses of up to and including 5000 µg per plate on five Salmonella typhimurium LT2 mutants. These comprised the histidine-auxotrophic strains TA 1535, TA 100, TA 1537, TA98 and TA 102. The independent repeat was performed as preincubation for 20 minutes at 37°C. Other conditions remained unchanged.
Doses up to and including 1581 µg per plate did not cause any bacteriotoxic effects. Total bacteria counts remained unchanged, and no inhibition of growth was observed. At 5000 µg per plate, the substance had only a weak, strain-specific bacteriotoxic effect. Due to the weakness of this effect this dose could nevertheless be used for assessment purposes. Substance precipitation occurred at the dose 1581 µg per plate and above.  Evidence of mutagenic activity of Pencycuron TC was not seen. No biologically relevant increase in the mutant count, in comparison with the negative controls, was observed.  The positive controls sodium azide, nitrofurantoin, 4-nitro-1,2-phenylene diamine, mitomycin C, cumene hydroperoxide and 2-aminoanthracene had a marked mutagenic effect, as was seen by a biologically relevant increase in mutant colonies compared to the corresponding negative controls.  Therefore, Pencycuron TC was considered to be non-mutagenic without and with S9 mix in the plate incorporation as well as in the preincubation modification of the Salmonella/microsome test.

Reference 5

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1990-01-29 to 1991-11-13

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test using the Hprt and xprt genes)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Specific details on test material used for the study:

white powder

Target gene:

HGPRT

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: Wistar male rats, induced by Aroclor 1254, served as the source of the S-9 fraction.

- The S-9 fraction was kept frozen at -80°C. Immediately prior to use, the S-9 fraction was thawed at room temperature and mixed with the freshly prepared cofactor solution in a sodium phosphate buffer pH 7.4.

- concentration or volume of S9 mix and S9 in the final culture medium:
The mixture of S-9 fraction with Co-factor solution is designated S-9 mix. The S-9 mix contained the following:

8mM MgCl2
33mM KC1
5mM glucose-6-phosphate
1mM NADP
40% S-9 fraction

The S-9 reaction mixture was kept on ice until use.

- quality controls of S9: Prior to use in the HGPRT test, the S-9 fraction was tested for contamination and cytotoxicity.

Test concentrations with justification for top dose:

6.25, 12.5, 25.0, 50.0, 100.0 µg/ml.

The dose range was selected according to a preliminary cytotoxicity study. for the mutagenicity study was chosen ranging from approximately 0% to 20% reduction in colony forming ability. A 90% reduction of colony forming ability could not be reached because of the limited solubility of the test substance-vehicle solution in medium.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

- Justification for choice of solvent/vehicle:
Pencycuron was dissolved in DMSO. A clear solution was obtained up to a concentration of 250 mg/ml.
Pencycuron was checked analytically in advance and the batch used was shown to be stable for the treatment period. Stability in the vehicle in a range from 100 jug/ml to 10 mg/ml was also approved. Test substance solutions were prepared in vehicle immediately prior to cell treatment. To prevent any interference of the vehicle with cell growth or viability, the final concentration of the vehicle in the medium was 1% (v/v) or less.


- Justification for percentage of solvent in the final culture medium:
Justified by a lack of toxicity at 1% (v/v) or less.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

other: Dimethylbenzanthracene (DMBA)

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments: 2

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding:
Nonactivation Assay: 1.5x10^6 cells, At the end of the expression period, the cultures from each dose level were reseeded at 2x10^5 cells per dish in culture medium without hypo-xanthine but containing 10 µg/ml 6-TG.
In addition, three dishes were seeded in culture medium at 200 cells/dish to determine the absolute cloning efficiency for each dose level.

- Test substance added in medium.

TREATMENT AND HARVEST SCHEDULE:
- Exposure time: 5 hours.

FOR GENE MUTATION:
- Expression time: 6 days
- Selective agent is used: 6-thioguanine or trifluorothymidinendicate, concentration: 10 µg/ml, duration, and period of cell exposure: incubation for 7 days.
- Number of cells seeded and method to enumerate numbers of viable and mutant cells:
Three dishes were seeded in culture medium at 200 cells/ dish to determine the absolute cloning efficiency for each dose level. After incubation for 7 days at 37°C in a humidified atmosphere with about 5% CO2, the colonies were fixed, stained with Giemsa, and counted to determine the number of 6-TG resistant colonies in the mutation assay dishes and the number of colonies in the cloning efficiency dishes. Colonies with 50 cells or less were excluded.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
Cytotoxicity is determined after treatment with the test article by the assay parameter "Relative Survival to Treatment".

Relative Survival (or Relative CE) (%) = (Average no. of colonies per treated culture/Average no. of
colonies per vehicle control dish) x 100
The assay parameter "Relative Population Growth" shows the cumulative growth of the treated cell populations, relative to the vehicle control, over the expression period and prior to mutant selection. Significant decreases of this parameter indicate growth inhibition as result of test substance toxic.

Rel.Pop Growth (%) = (Treated culture population increase over the expression period/Vehicle control population increase over the expression period) x 100

METHODS FOR MEASUREMENTS OF GENOTOXICIY

The ability of cells to form colonies at the time of mutant selection is measured by the parameter "Absolute Cloning Efficiency" (CE). The absolute CE is expressed by the average number of viable colonies per dish (200 cells/dish seeded).

Absolute CE (%) = (Average no. of viable colonies per dish/200) x 100
The mutant frequency is the endpoint of the assay and is calculated for each treatment condition by dividing the total number of mutant colonies by the number of cells seeded (usually 8-10 plates at 2x10^5 cells/plate), corrected for the absolute CE. The mutant frequency is expressed as 6-TG resistant mutants per 10^6 clonable cells.

Evaluation criteria:

Normally, an assay is only considered acceptable for evaluation if the following criteria are satisfied. However, the study conclusions are to be based upon the Study Director's evaluation and interpretation of the data.

- The activation and nonactivation assays were repeated independently in a second assay.
- The average cloning efficiency of the negative controls should be at least 50%. Assays below 50% cloning efficiency will be unacceptable.
- The highest test article concentration should produce a low level of survival (0 - 30%) and the survival at the lowest concentration should approximate the negative control. Cytotoxicity is determined after treatment with the test article by the assay parameter "Relative Survival to Treatment".
- The background mutant frequency (average for negative controls) should not exceed 25x10^6 cells.
Assays with higher spontaneous mutant frequencies are not necessarily invalid, however, if all other criteria are fulfilled.
- An experimental mutant frequency is considered acceptable only if the absolute cloning efficiency is 10% or greater.
- Mutant frequencies for at least four treated cultures are routinely determined in each assay.
- Mutant frequencies are normally derived from sets of 8-10 dishes for each dose level. To allow for contamination losses, an acceptable mutant frequency can be calculated from a minimum of 5 dishes.
- The positive control must induce a mutant frequency of at least three times that of the negative control.

Statistics:

The number of mutations per one million cells will be governed by the POISSON distribution provided that the following assumptions are met, as in the case of mutation experiments such as the HGPRT-assay:

1. The mean number of spontaneous mutations must be small relative to the maximum possible number of events per sampling units.

2. An occurrence of the event must be independent from prior occurrences within the sampling unit.

As recommended by Armitage (1971) a POISSON heterogeneity test is used to determine whether or not there are statistically significant increases in mutant frequency.
If the test is first taken as a global test with subsequent comparisons among less than s means with s being the number of groups including the negative controls (provided the global test yielded a significant result), the type I error rate may be adjusted to account for the multiplicity of tests.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

The test substance induced significant concentration related cytotoxic effects (decrease in relative population growth and cloning efficiency) in the absence and presence of metabolic activation at = 50 µg/ml.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

1- Solubility and Dose Selection:
Pencycuron was dissolved in DMSO. A clear solution was obtained up to a concentration of 250 mg/ml.
However, there was precipitation of the test article after addition of the test article-DMSO solution to the medium. A preliminary cytotoxicity test was conducted with a series of 9 concentrations of Pencycuron ranging from 10 µg/ml to 100 µg/ml with and without metabolic activation. Precipitation started at 60 µg/ml. The results were used to select 5 concentrations of Pencycuron for the mutation assays ranging from 6.25 µg/ml to 100 µg/ml with and without metabolic activation. The evaluation was limited by solubility of the test compound under culture conditions.

2- Mutation Assay without Metabolic Activation
Under nonactivation conditions 2 trials were performed. There was test substance precipitation at 100 µg/ml under culture conditions. In the lower concentrations a dose-related decrease was observed both in relative survival and relative population growth. There was no statistically significant increase in mutant frequency over the concurrent vehicle controls in both trials. On the other hand, the positive control EMS revealed a clear mutagenic effect in all assays.
Therefore, the test article was evaluated as non-mutagenic in the nonactivation trial.

3- Mutation Assay with Metabolic Activation
Under metabolic activation conditions, 4 trials were performed. The first two trials could not be evaluated due to not sufficiently high responses of the positive control and a too high spontaneous mutation frequency in the negative controls.

In the third assay nearly, no cytotoxicity was induced up to the limit of solubility of the test substance. In the fourth one the treated cultures showed dose-related decreases in both relative survival to treatment and relative population growth.

For one duplicate at 12.5 µg/ml in both trials a statistically significant increase in mutant frequency over the concurrent vehicle controls could be observed.
Only in the last trial also one duplicate showed a statistically significant elevated mutant frequency at 6.25 µg/ml and 100 µg/ml. However, these increases were neither dose-related nor confirmed by the duplicate treatment. Furthermore, precipitation already occurred at 100 µg/ml. In addition, these mutant frequencies did not exceed the range which is typical of vehicle control variation between trials.

In contrast the positive control article DMBA revealed a clear mutagenic effect in both trials.
Therefore, Pencycuron was evaluated as non-mutagenic with metabolic activation in this test system.

Conclusions:

Test substance induced significant concentration related cytotoxic effects (decrease in relative population growth and cloning efficiency) in the absence and presence of metabolic activation at 50 µg/ml. The test substance did not induce gene mutations in CHO cells.

Executive summary:

Pencycuron was evaluated for mutagenic effects at the HGPRT locus (forward mutation assay) in CHO cell cultures after in vitro treatment at concentrations up to 100.0 µg/ml, both with and without S-9 mix.  Under nonactivation conditions, Pencycuron induced cytotoxic effects as seen by decreases in relative population growth and cloning efficiency. These results revealed a significant concentration-related cytotoxicity of Pencycuron without metabolic activation. Under activation conditions, a significant dose-related cytotoxicity was also observed in one trial. In the second trial such a strong cytotoxic effect could not be seen; however, Pencycuron was tested up to its limit of solubility under culture conditions.  There was no significant dose-related or reproducible increase in mutant frequency above that of the negative controls. In contrast, the positive controls ethylmethanesulfonate (without S-9 mix) and dimethylbenzanthracene (with S-9 mix) produced a clearly mutagenic effect in the assay.  Based on these results, the test substance Pencycuron is considered to be non-mutagenic in the CHO-HGPRT Forward Mutation Assay, both with and without metabolic activation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

A study of bone marrow micronucleus formation in the mouse is available for pencycuron.  This study shows evidence of target tissue exposure and reports a clear negative result.  Based on the negative responses reported in the available studies in vitro and in vivo, further testing of the genotoxicity of pencycuron in vivo is not required.

Link to relevant study records

Reference

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

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

Deviations:

not specified

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

Specific details on test material used for the study:

white powder

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).

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.

Dose / conc.:

0 mg/kg bw (total dose)

Remarks:

Negative control group. Time of sacrifice: 24 hours

Dose / conc.:

5 000 mg/kg bw (total dose)

Remarks:

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

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).

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.

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.

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

The genetic toxicity of pencycuron has been investigated in vitro in studies of bacterial reverse mutation, mammalian cell (forward) mutation and mammalian cell cytogenicity.  All studies report negative results, both in the absence and presence of exogenous metabolic activation.  Although a study in vitro specifically investigating numerical chromosomal aberrations is not available for pencycuron, this endpoint has been investigated in a higher tier study in vivo with evidence of target tissue exposure.

Studies in vitro and in vivo with pencycuron covering all endpoints (gene mutation, structural and numerical chromosomal aberration) do not show any evidence of genotoxicity.  Consequently, classification of pencycuron under CLH for germ cell mutation in any category is not required.