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Toxicological information

Genetic toxicity: in vivo

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Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 17 January 2005 to 13 June 2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2005
Report date:
2005

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
Adopted : 21st July 1997
Deviations:
yes
Remarks:
The Major Computer System used to randomize animais was Cyscore version 2. The current applicable Covance SOP for randomizing animals (NA-GT 303) specifies the use of CDMS (Cytogenetics Data Management System) .
GLP compliance:
yes
Type of assay:
other: In vivo rats micronucleus assay

Test material

Constituent 1
Chemical structure
Reference substance name:
3-methyl-1-phenyl-5-pyrazolone
EC Number:
201-891-0
EC Name:
3-methyl-1-phenyl-5-pyrazolone
Cas Number:
89-25-8
Molecular formula:
C10H10N2O
IUPAC Name:
5-methyl-2-phenyl-2,3-dihydro-1H-pyrazol-3-one
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: No. 62
- Expiration date of the lot/batch: 09/2005
- Purity test date: 31 August 2004

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Approximately +4°C, protected from light and under nitrogen gas
- Stability under test conditions: Stability and homogeneity of the formulated test article were determined in CIT study No. 26977 AHS
- Solubility and stability of the test substance in the solvent/vehicle: formulations of Phenylmethyl pyrazolone (A039) were
stable in 0.5% aqueous carboxymethylcellulose for 6 hours in the range of 2-200 mg/mL (room temperature) protected from light in a nitrogen atmosphere. Such dosage formulations were also stable and homogeneous for 9 days when stored at +4°C, away from light and under inert gas .

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The top stock of the test article was ground to a fine powder using a mortar and pestle, mixed with the required quantity of vehicle, and
homogenized thereafter using a magnetic stirrer
- Final dilution of a dissolved solid, stock liquid or gel: 50, 100 and 200 mg/ml
- Final preparation of a solid: test item was ground with mortar and pestle

Test animals

Species:
rat
Strain:
other: Crl:CD(SD)BR
Details on species / strain selection:
This is an outbred strain that maximizes genetic heterogeneity and therefore tends to eliminate strain-specific response to test articles . The rat has been routinely utilized as an animal model of choice for the
mammalian bone marrow erythrocyte micronucleus assay .
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories,North Carolina
- Age at study initiation: Dose Rangefinding Assay : 9 weeks old
Micronucleus assay : 8 weeks old
- Weight at study initiation: Dose Rangefinding assay : male : 293 to 309 g / female 202 to 235g
Micronucleus assay : male : 255 to 303g / female : 194 to 228 g
- Assigned to test groups randomly: Cyscore version 2 was used to randomize animals
- Fasting period before study: not specified
- Housing: The animais were housed in sanitary, stainless-steel, hanging, wire cages . The animais were housed, separated by gender, up to two animais per cage during acclimation and individually alter randomization .
- Diet (e.g. ad libitum): PMI Certified Rodent Dieto #5002 was available ad libitum, unless otherwise noted . The manufacturer analyzed the diet for nutritional components and environmental contaminants.
- Water (e.g. ad libitum): Tap water, by an automatic watering system, was available ad libitum unless otherwise noted . Water samples are routinely analyzed for specified microorganisms and environmental contaminants. No contaminants were known to be present in the diet or water at levels which might interfère with this study .
- Acclimation period: For at least 5 days before initiation of study

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17.7°C to 26°C
- Humidity (%): relative humidity 30 to 70%
- Air changes (per hr): 10 or greater air changes/hour
- Photoperiod (hrs dark / hrs light): 12-hours light/12-hours dark

IN-LIFE DATE: Dose rangefinding assay : From 27 January 2005 To 2 February 2005
Micronucleus assay : From 10 February 2005 To 17 February 2005

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: CMC (carboxymethyl cellulose)
- Justification for choice of solvent/vehicle: Stability and homogeneity of the formulated test article were determined in CIT study No. 26977 AHS . In that study, formulations of Phenylmethyl pyrazolone (A039) were stable in 0.5% aqueous carboxymethylcellulose for 6 hours in the range of 2-200 mg/mL (room temperature) protected from light in a nitrogen atmosphere
- Concentration of test material in vehicle: 50,100 and 200 mg/ml
- Amount of vehicle (if gavage or dermal): 0.5% of carboxymethyl cellulose
- Type and concentration of dispersant aid (if powder): magnetix stirrer
- Lot/batch no. (if required): 034KO120
- Purity: not specified
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: All mixing containers were purged with nitrogen gas prior to use . Before preparation, the vehicle was degassed by sonication for at least 15 minutes, then saturated with nitrogen gas. The vehicle was kept under nitrogen atmosphere for at least 15 minutes prior to dosing and during the dosing procedure. The test article was kept under a nitrogen atmosphere during preparation .The top stock of the test article, Phenylmethyl pyrazolone (A039), was ground to a fine powder using a mortar and pestle, mixed with the required quantity of vehicle, and homogenized thereafter using a magnetic stirrer . The test article was administered as a suspension in the vehicle . Lower concentrations were obtained by dilution with the vehicle. The formulations were held at room temperature, protected from light in a nitrogen atmosphere prior to dosing . Dosing was performed within 6 hours of test article preparation. The treatment regimen was single oral gavage dose administration. The oral route of administration was selected because this is a relevant route of administration for this test article to utilize in this assay .
Dosing volume : 10 ml/kg bodyweight
Duration of treatment / exposure:
24 and 48 hours
Frequency of treatment:
single oral gavage dose administration
Post exposure period:
24 and 48 hours
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Vehicle control
Dose / conc.:
500 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Dose / conc.:
2 000 mg/kg bw/day (actual dose received)
Remarks:
limit test dose
No. of animals per sex per dose:
In Dose rangefinding assay, 3 males and 3 females were used in each dose group. 5 males and females per treatments groups per sex for 24 hours and for 48 hours for micronucleus assay.
Control animals:
yes, concurrent vehicle
Positive control(s):
- Positive control : cyclophosphamide
- Justification for choice of positive control(s): not specified
- Route of administration: oral gavage
- Doses / concentrations: 60 mg/kg

Examinations

Tissues and cell types examined:
Bone marrow
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: Based on the results of the dose rangefinding assay, the top dose selected for the micronucleus assay was 2000 mg/kg, the limit dose for this assay based on OECD guidelines.

Extraction of Bone Marrow
The hind limb bones (tibias) were removed for marrow extraction from five surviving
animais in each treatment and control group . For each animal, the marrow from the bone
was flushed into an individual centrifuge tube containing 3 to 5 mL fetal bovine serum
(one tube per animal) .

Preparation of Slides
Following centrifugation to pellet the marrow, the supernatant was removed by aspiration
and portions of the pellet were spread on slides and air-dried. The slides were fixed in
methanol, stained in acridine orange, and analyzed under fluorescent microscopy . For
control of bias, ail slides were coded prior to analysis .

Slide Analysis
Slides prepared from the bone marrow collected from five animals per group at the
designated harvest timepoints were scored for micronuclei and the PCE to NCE cell ratio .
The micronucleus frequency (expressed as percent micronucleated cells) was determined
by analyzing the number of micronucleated PCEs from at least 2000 PCEs per animal .
The PCE:NCE ratio was determined by scoring the number of PCEs and NCEs observed
while scoring at least 500 erythrocytes per animal .
Evaluation criteria:
The criteria for the identification of micronuclei are those of Schmid (1976) . Micronuclei were darkly stained and generally round, although almond and ring-shaped micronuclei occasionally occurred. Micronuclei were sharp bordered and were generally between 1/20 and 1/5 the size of the PCE. The unit of scoring is the micronucleated cell, not the micronucleus ; thus, the occasional cell with more than one micronucleus was counted as one micronucleated PCE, not two (or more) micronuclei. The staining procedure permits the differentiation by color of polychromatic and normochromatic erythrocytes (bright orange and ghost-like, dark green, respeclively). The criteria for a positive response is the detection of a statistically significant
positive response for at least one dose level and a statistically significant dose-related response . A test article that does not induce both of these responses will be considered negative . Equivocal results may require further investigation (as additional cost). Statistical significance will not be the only determinant of a positive response.
Statistics:
Assay data analysis will be performed using an analysis of variance (Winer, 1971) on untransformed proportions of cells with micronuclei per animal when the variances are homogeneous . Ranked proportions will be used for heterogeneous
variances . If the analysis of variance is significant (p < 0 .05), a Dunnett's t-test (Dunnett, 1955; 1964) will be used to determine which dose groups, if any, are significantly different from the vehicle control . Analyses wil be performed
separately for each sampling time (and sex combination, if more tan one sex). Additionally, parametric or nonparametric tests for trend may be employed to identify any dose-related response .

Results and discussion

Test results
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
Squinted eyes, oral discharge, slight hypoactivity, upright posture, eye discharge and/or irregular respiration were noted for test article treated groups only at the observation period approximately 1 hour after dosing .
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
No deaths were observed up to 2000 mg/kg. Observations of clinical signs at all doses (500,1000 and 2000 mg/kg bodyweight) included squinted eyes, oral discharge, slight hypoactivity, upright posture, ocular discharge and/or irregular respiration .

RESULTS OF DEFINITIVE STUDY
No deaths were observed up to 2000 mg/kg. All animals in the vehicle and positive control groups appeared normal after dosing and remained healthy until the appropriate harvest timepoint . Squinted eyes, oral discharge, slight hypoactivity, upright posture, eye discharge and/or irregular respiration were noted or test article treated groups only at the observation period approxirnately 1 hour after dosing .

Phenylmethyl pyrazolone (A039) did not induce statistically significant increases in micronucleated PCEs or statistically significant decreases in the PCE :NCE ratios at any test article dose examined (500, 1000 and 2000 mg/kg) . Although there were no indications of bone marrow toxicity in the present study, the oral bioavailability of phenylmethyl pyrazolone (A039) was evidenced by the clinical signs observed. Moreover, in a contemporary 13-week toxicity study (CIT Study No. 26900 TCR), systemic exposure to phenylmethyl pyrazolone (A039) was observed in rats given a single oral dose at lower dose levels (Area Under the Curve values of approxirnately 5, 80 and 300 μg .h/mL at 20, 100 and 500 mg/kg, respectively) .
The vehicle control group had less than the published value (0 .0 to 0.4%, Salamone and Mavournin, 1994) of micronucleated PCEs . The vehicle control group mean in the definitive micronucleus assay was within the Covance-Vienna 2003 historical vehicle control group mean of 0.085 ± 0 .009% and the historical vehicle control individual mean of 0 .085 ± 0 .007% for males at the 24-hour bone marrow sampling time . Additionally,
the vehicle control group mean in the definitive micronucleus assay was within the Covance-Vienna 2003 historical vehicle control group mean of 0 .073 ± 0 .008% and the historical vehicle control individual mean of 0 .073 ± 0 .007% for males at the 48-hour bone marrow sampling time . The vehicle control group mean in the definitive micronucleus assay was within the Covance-Vienna 2003 historical vehicle control group mean of 0.068 ± 0 .0014% and the historical vehicle control individual mean of 0 .068 ± 0 .011 % for females at the 24-hour bone marrow sampling time . Additionally,
the vehicle control group mean in the definitive micronucleus assay was similar to the Covance-Vienna 2003 historical vehicle control group mean of 0 .040 ± 0 .010% and the historical vehicle control individual mean of 0 .040 ± 0 .000% for females at the 48-hour bone marrow sampling time . However, this mean is based only on 5 animais (1 trial) . The positive control, cyclophosphamide, induced a statistically significant increase in micronucleated PCEs as compared to that of the vehicle control, with a mean and standard error of 4 .21 ± 0.25% for the males and 1 .83 ± 0.20% for the females .

Any other information on results incl. tables

Clinical Observations -

DoseRangefindingAssay

Target Dose Level (mg/kg)

Sex

Animal ID

Time after dosing

IPD

Approximately20 minutes

Approximately1hour

1day

2days

500

M

8884

0

1,2,3

0

0

0

8888

0

2,3

0

0

0

8891

0

2,3

0

0

0

F

8896

0

1,3,4

1

0

0

8898

0

1,3,4,6

1

0

0

8900

0

1,3,4,6

1

0

0

1000

M

8883

0

3,4,5

0

0

0

8885

0

3,4,6

0

0

0

8889

0

3,4

0

0

0

F

892

0

2,3

1,3

0

0

8893

0

2,3,4

0

0

0

8897

0

3,4

1,3

0

0

2000

M

8886

0

3

0

0

0

8887

0

2,3

0

0

0

8890

0

1,2,3

1,3

0

0

F

8894

0

1,2,3

3

0

0

8895

0

2,3

1,3

0

0

8899

0

2,3

0

0

0

 

 

 

Key: 0 = Normal; 1 = squinted eyes, 2 = oral discharge, 3 ° slightly hypoactive, 4 = upright

posture, 5 = ocular discharge, 6 = irregular respiration

IPD =Immediately post dose

Micronucleus Assay - Summary Table

Treatment

Dose

Harvest Time

Micronucleated PCEs Mean of 2000 per Animal ±SE

Ratio PCE:NCE Mean ± S.E.

Males

Females

Males

Females

Controls

 

 

 

 

 

 

Vehicle

0.5% MC 10mL/kg

24hr

0.06±0.02

0.07±0.03

0.80±0.05

0.86±0,05

48hr

0.10±0.02

0.18±0.01

1.29±0.01

1.20±0.10

Positive

CP 60 mg/kg

24hr

4.21±0.25

1.83±0.01

0.77±0.04

0.71±0.04**

Test article

500 mg/kg

24hr

0.08±0.03

0.07±0.02

0.94±0.05

0.98±0.06

1000 mg/kg

24hr

0.08±0.02

0.14±0.04

0.90±0.04

0.78±0.10

2000 mg/kg

24hr

0.18±0.05

0.12±0.05

0.97±0.03

0.81±0.04

 

 

48hr

0.14±0.02

0.16±0.05

1.05±0.06

0.89±0.13

 

* Significantly greater than the corresponding vehicle control, p <_ 0 .01 .

** Significantly less than the corresponding vehicle control, p<_ 0 .05 .

0.5% MC = 0 .5%aqueous methylcellulose

CP =Cyclophosphamide

PCE =Polychromatic erythrocyte

NCE =Normochromatic erythrocyte

 

Applicant's summary and conclusion

Conclusions:
The test article, Phenylmethyl pyrazolone (A039), was evaluated as negative in the rat bone marrow micronucleus assay under the conditions of this assay, when tested up to the testing limit of 2000 mg/kg . Although there were no indications of bone marrow toxicity in the present study, the oral bioavailability of phenylmethyl pyrazolone (A039) was evidenced by the clinical signs observed.
Executive summary:

This GLP-compliant study was assessed to determine damage induced by the test item Phenylmethyl Pyrazolone (A039) to the chromosomes or the mitotic apparatus of erythocytes sampled from bone-marrow of rats. The study method was perform according to OECD 474 method for Mammalian Erythrocyte Micronucleus Test.

Under the experimental conditions of this study, the test item phenylmethyl pyrazolone did not induce damage to chromosomes or mitotic apparatus leading to micronucleus formations on erythrocytes from treated rats bone marrow at the limited dose 2000mg/kg bodyweight. Some clinical signs were observed, as systemic exposure.