Registration Dossier

Administrative data

Endpoint:
basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 4 February 2004 to 18 May 2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Toxicokinetics study included in 90-days repeated toxicity study (CIT/Study No. 26900 TCR)

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
2005
Report date:
2005
Reference Type:
secondary source
Title:
Blood volume in the rat
Author:
H.B. Lee and M.D. Blaufox
Year:
1985
Bibliographic source:
J Nucl Med 25;72-76

Materials and methods

Objective of study:
toxicokinetics
Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
other: OECD 408 Guideline (90 Days sub-chronic oral toxicity - Rodent) dated : 21st September 1981
Version / remarks:
Toxicokinetics study included in 90-days repeated toxicity study (CIT/Study No. 26900 TCR
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 417 (Toxicokinetics)
Version / remarks:
Adopted 22 July 2010
Deviations:
no
Principles of method if other than guideline:
The plasma samples were deproteinized by addition of methanol. The clear supernatant (containing Phenylmethyl pyrazolone (A039) and the Internal Standard, 2-ACETAMIDOPHENOL) was evaporated and the dry residue was reconstituted with solvent D
(degassed). Then, the samples were analyzed by reversed phase chromatography (HPLC) with ultra-violet detection at 241 nm. The plasma concentrations were determined by interpolation with the calibration curve and the precision and accuracy of the method were checked by analysis of control quality plasma samples (QC) containing Phenylmethyl pyrazolone (A039) at three concentration levels: 2.5, 10 and 40 μg/mL. The following parameters were determined:
. Cmax, maximal plasma concentration measured,
. tmax, time of occurrence of this maximal plasma concentration,
. AUC0-t, area under the concentration-time curve (calculated according to the linear trapezoidal rule) from time 0 to the last quantifiable data-point.
GLP compliance:
yes

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: batch No. 62
- Expiration date of the lot/batch: September 2005
- Purity test date: 31 August 2004

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: in glass containers at +4°C, protected from the light and under nitrogen gas
The analysis of homogeneity, stability and concentration of dosage forms were performed in CIT/Study No. 26977 AHS at concentrations spanning those used in the present study.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item was ground to fine powder using a mortar and pestle, then suspended in the vehicle and homogenized using a magnetic stirrer.
The test item dosage forms were prepared under nitrogen atmosphere with a frequency
depending on their stability (up to 7 days) and were stored prior to use, at +4°C, under nitrogen atmosphere and protected from light with an aluminum foil.
- Final dilution of a dissolved solid, stock liquid or gel: 4, 20 and 100 mg/mL
- Final preparation of a solid: The test item was ground to fine powder using a mortar and pestle
Radiolabelling:
no

Test animals

Species:
rat
Strain:
Sprague-Dawley
Remarks:
Crl CD (SD) IGS BR
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, France
- Age at study initiation: 6 weeks old
- Weight at study initiation: male : mean 221g, female : mean 164g
- Fasting period before study: not specified
- Housing: The animals were housed in suspended wire-mesh cages (43.0 x 21.5 x 18.0 cm) and
each cage contained two rats of the same sex and group. A metal tray, containing autoclaved sawd
ust (SICSA, France), was placed under each cage.
- Diet and Water (e.g. ad libitum): The animals had free access to A04 C pelleted maintenance diet,
batch Nos. 31222, 40123 and 40227 (SAFE, France) distributed weekly. The animals had free acce
ss to bottles containing tap water (filtered
with a 0.22 μm filter). During periods of fasting, food, but not water, was removed.
- Acclimation period: a 7-day acclimation period to the conditions of the study preceded the beginning
of the treatment period
DETAILS OF FOOD AND WATER QUALITY:
The batches of diet and sawdust were analyzed by the suppliers for composition and contaminant
levels. Bacterial and chemical analyses of water are performed regularly by external laboratories.
These analyses include the detection of possible contaminants (pesticides, heavy metals and nitro
samines). No contaminants were present in the diet, drinking water or sawdust at levels, which may
be expected to interfere with or prejudice the outcome of the study.
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 50 ± 20%
- Air changes (per hr): approximately 12 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12h/12h (7:00 - 19:00)
IN-LIFE DATES: From: 2 March 2004 To: 1 July 2004

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
methylcellulose
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The test item was ground to fine powder using a mortar and pestle, then suspended in the vehicle and homogenized using a magnetic stirrer.
The test item dosage forms were prepared under nitrogen atmosphere with a frequency depending on their stability (up to 7 days) and were stored prior to use, at +4°C, under nitrogen
atmosphere and protected from light with an aluminum foil
Duration and frequency of treatment / exposure:
Once a day, during 13 weeks
Doses / concentrationsopen allclose all
Dose / conc.:
20 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
500 mg/kg bw/day (nominal)
Dose / conc.:
0 mg/kg bw/day (nominal)
No. of animals per sex per dose / concentration:
6 animals per sex per dose
Control animals:
no
Details on study design:
- Dose selection rationale:
The selection of dose-levels was based on the results of a 4-week oral toxicity study performed in rats at 40, 200 and 1000 mg/kg/day (CIT/Study No. 6482 TSR, 1990). In this study, adverse effects were observed at 1000 mg/kg/day and mainly consisted of decreased body weight gain and food intake together with marked clinical signs displayed throughout the study (decreased activity and half-closed eyes). Moreover, adverse effects on the spleen were observed at 1000 mg/kg/day (increased weight, congestion, coloration and presence of hemosiderin-laden macrophages). Based on these effects at 1000 mg/kg/day, the high-dose level for the present study was set at 500 mg/kg/day. The low and intermediate dose-levels were set at 20 and 100 mg/kg/day to assess the dose-relationship of any adverse effects.

The plasma concentrations were determined by interpolation with the calibration curve and the precision and accuracy of the method were checked by analysis of control quality plasma samples (QC) containing Phenylmethyl pyrazolone (A039) at three concentration levels: 2.5, 10 and 40 μg/mL.
The typical analytical sequence for the assay was as follows:
. calibration samples: one plasma blank and six concentrations levels (1 to 50 μg/mL) as
above-defined,
. investigated samples and QC samples distributed regularly along the run,
. at least one QC at the end of the sequence.
In each analytical sequence, at least six QC samples (each level in duplicate) were analyzed together with the investigated samples.
Each plasma sample was extracted once and one injection was performed for each extract obtained
Details on dosing and sampling:
TOXICOKINETIC (Absorption only)
- Tissues and body fluids sampled (delete / add / specify): plasma
- Time and frequency of sampling: Blood samples were taken on day 1 and in week 13 at the following time-points:
0, 0.5, 1, 2, 4, 8 and 24 hours post-dosing.
For each time-point, three animals/sex/group were sampled (groups 2, 3 and 4), and each animal was sampled on three occasions.

Results and discussion

Main ADME results
Type:
absorption
Results:
The maximum mean plasma level was observed 0.5 hour post-dosing.The systemic exposure, Cmax, and systemic exposure (AUC(0-24-h)) increased with dose-level.

Toxicokinetic / pharmacokinetic studies

Details on absorption:
On both occasions, the maximum mean plasma level was observed 0.5 hour post-dosing (first sampling time-point) in all groups. Thereafter, blood levels decreased and the test item was not quantifiable (limit of quantification = 1 μg/mL) 2 hours after dosing for animals given 20 mg/kg/day or 24 hours after dosing for animals given 100 or 500 mg/kg/day. The systemic exposure, as measured by Cmax, increased with dose-level but less than dose-proportionally. However, in term of AUC(0-24-h), systemic exposure generally increased proportionally with dose-level.

Amount of test substance was calculated in rat plasma with informations of rats weights of the main study, plasma volume determination by secondary source (H.B Lee and M.D. Blaufox, Blood volume in Rats, J Nucl Med, 1985), dose levels and blood plasma concentration quantified (HPLC/UV approach). The maximum amount of test substance in blood was 6.05% of the total admisitered substance observed in female of the 20 mg/kg bw/day. For 100 mg/kg bw/day group, the mean value was 2.29% and 0.89% for the highest dose group at 0.5 hours (Tmax).

Any other information on results incl. tables

Table 1 Test item concentration in blood plasma (µg/mL)

MALE

20

100

500

Samplingtime

Week1

Week13

Week1

Week13

Week1

Week13

0,5

10,6

17,3

39,9

47,3

107

76,1

1

2,28

nc

22,9

26,9

68,7

53,6

2

nc

nc

10,8

10,1

54,1

37,3

4

nc

nc

8,18

7,79

26,5

21,9

8

nc

nc

1,24

2,98

12,5

13,6

FEMALE

0,5

7,85

29,1

38,5

62,4

102

137

1

2,84

2,31

27,4

29,3

89,5

68,1

2

nc

nc

8,42

11

54,9

89

4

nc

nc

6,94

8,57

28,8

29

8

nc

nc

1,4

6,51

15,9

22,9

 

Table 2 Total amount of substance in rat plasma (mg)

MALE

20

100

500

Samplingtime

Week1

Week13

Week1

Week13

Week1

Week13

0,5

0,09975236

0,40372318

0,36874782

1,05391968

0,993388

1,37127634

1

0,02145617

nc

0,21163722

0,59937504

0,6378108

0,96583984

2

nc

nc

0,09981144

0,22504416

0,5022644

0,67212362

4

nc

nc

0,07559792

0,17357366

0,246026

0,39462486

8

nc

nc

0,01145983

0,06639917

0,11605

0,24506384

FEMALE

0,5

0,10482576

0,38858976

0,2674672

0,8176896

0,6916416

1,4988896

1

0,01546022

0,03084682

0,19035328

0,3839472

0,6068816

0,74506848

2

nc

nc

0,05849542

0,144144

0,37226592

0,9737312

4

nc

nc

0,04821357

0,11230128

0,19528704

0,3172832

8

nc

nc

0,00972608

0,08530704

0,10781472

0,25054432

 

Table 3 Percent of eaten substance in plasma (%)

MALE

20 mg/kg/day

100 mg/kg/day

500 mg/kg/day

Samplingtime

Week1

Week13

Week1

Week13

Week1

Week13

0,5

2,2366

3,6503

1,68378

1,99606

0,90308

0,642284

1

0,48108

nc

0,96638

1,13518

0,579828

0,452384

2

nc

nc

0,45576

0,42622

0,456604

0,314812

4

nc

nc

0,345196

0,328738

0,22366

0,184836

8

nc

nc

0,052328

0,125756

0,1055

0,114784

FEMALE

0,5

3,21551411

6,0528

1,6016

2,59584

0,84864

1,13984

1

0,47424

0,48048

1,13984

1,21888

0,74464

0,566592

2

nc

nc

0,350272

0,4576

0,456768

0,74048

4

nc

nc

0,288704

0,356512

0,239616

0,24128

8

nc

nc

0,05824

0,270816

0,132288

0,190528

 

Table 3 : Determination of Cmax, Tmax and AUC

Sampling period   Kinetic parameters               Dose level

                                              20mg/kg/day       100 mg/kg/day     500 mg/kg/day

Male

Female

Male

Female

Male

Female

Cmax(µg/mL)

10.6

7.9

39.9

38.5

107.0

102.0

tmax(h)

0.5

0.5

0.5

0.5

0.5

0.5

Day1     AUC(0-24h)g.h/mL)

5.9

4.6

80.3

76.1

290.0

319.0

Dose-ratio

1

1

5

5

25

25

AUC/dose

0.30

0.23

0.80

0.76

0.58

0.64

Cmax/dose

0.53

0.39

0.40

0.39

0.21

0.20

Cmax(µg/mL)

17.3

29.1

47.3

62.4

76.1

137.0

tmax(h)

0.5

0.5

0.5

0.5

0.5

0.5

Week13   AUC(0-24h)g.h/mL)

4.3

15.1

88.2

108.0

227.0

377.0

Dose-ratio

1

1

5

5

25

25

AUC/dose

0.22

0.76

0.88

1.08

0.45

0.75

Cmax/dose

0.87

1.46

0.47

0.62

0.15

0.27

 

AUC(0-24h), corresponds to area under curve (trapezoidal rule) from time 0 to last data point.

 

 

 

 

 

 

 

 

Applicant's summary and conclusion

Conclusions:
Phenylmethyl pyrazolone (A039) was detected in the plasma at all dose levels. Plasma levels were comparable after single dosing and at the end of the dosing period. The Cmax was measured 0.5 h after dosing, and systemic exposure increased with dose level.
Executive summary:

In a 90 -Days repeated dose toxicity study (CIT/Study No. 26900 TCR), the test substance was administered at 0, 20, 100 and 500 mg/kg/day by gavage on rats. This study was performed according to the OECD Guideline 408 (21st september 1998) and completed by toxicokinetics follow-up with three additional groups of 12 animals (6 males and 6 females). Plasma samples were collected and analysed by HPLC/UV method. Blood samples were taken on day 1 and in week 13 at the following time-points: 0.5, 1, 2, 4, 8 and 24 hours post-dosing.

The toxicokinetic parameters were calculated according to standard non-compartmental

methods. The following parameters were determined:

. Cmax, maximal plasma concentration measured,

. tmax, time of occurrence of this maximal plasma concentration,

. AUC0-t, area under the concentration-time curve (calculated according to the linear trapezoidal rule) from time 0 to the last quantifiable data-point.

Results

On day 1 and in week 13, the maximum mean plasma level (Cmax) of unchanged Phenylmethyl pyrazolone (A039) was observed 0.5 hour post-dosing (first sampling time-point) in all groups. Thereafter, blood levels decreased rapidly and the test item was not quantifiable 2 hours after dosing for animals given 20 mg/kg/day or 24 hours after dosing for animals given 100 or 500 mg/kg/day. The systemic exposure, as measured by Cmax, increased with dose-level but less than dose-proportionally. However, in term of AUC(0-24h), systemic exposure generally increased proportionally with dose-level. There were no apparent sex-related differences and no evidence of a time-effect since the values for the calculated parameters were globally within the same range in week 13 and on day 1.

Amount of test substance was calculated in rat plasma with informations of rats weights of the main study, plasma volume determination by secondary source (H.B Lee and M.D. Blaufox, Blood volume in Rats, J Nucl Med, 1985), dose levels and blood plasma concentration quantified (HPLC/UV approach).The maximum amount of test substance in blood was 6.05% of the total administered substance observed in female of the 20 mg/kg bw/day. For 100 mg/kg bw/day group, the mean value was 2.29% and 0.89% for the highest dose group at 0.5 hours (Tmax).

According this resutls, absorption rate was calculated according Cmax, high dose level (500mg/kg/day), rats weights at 13 weeks, and rat blood volume (64 ml/kg). The maximum amount of test item was 6.05% of total administered item observed in female 20 mg/kg bw/day group. This mean value decreased until 8 hours and in higher dose group.