Registration Dossier

Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 15 May 1990 to 3 December 1990
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1990
Report Date:
1990

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Version / remarks:
Adopted : 12 may 1981
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
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: lot 500 - ref. 8060110
- Expiration date of the lot/batch: not specified
- Purity test date: 12 december 1989

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: White powder, stored at room temperature and protected from the light

The chemical analysis of the preparations was performed by the sponsor. Sampling were taken as follows :
- Stability under test conditions: At the beginning of the study, for the highest concentration, the exact measurement of 2 x 10ml was taken just after preparation and frozen, then 2 x 10 ml, the exact measurement, was taken 4 hours after preparation and frozen immediately
- Solubility and stability of the test substance in the solvent/vehicle: At the beginning of the study , the exact measurement of 2 x 10 ml, was taken at three different levels of the suspension of the highest concentration frozen.
The resutls of these analyses are not included in this report.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test substance was suspended in the vehicle and homogenized by a magnetic stirrer
- Final dilution of a dissolved solid, stock liquid or gel: Chemical analysis was performed by the sponsor and results were not available during the study.

Test animals

Species:
rat
Strain:
Sprague-Dawley
Remarks:
Crl CD (SD) BR
Details on species / strain selection:
The rat was chosen as the rodent species recommended by the various National and International Regulations for this type of study.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River U.K. Limited (England)
- Females (if applicable) nulliparous and non-pregnant: not specified
- Age at study initiation: 6 weeks old
- Weight at study initiation: male : 201g and females : 154g
- Fasting period before study: not specified
- Housing: Housed in protected zone .Animals were housed in suspended wire-mesh cages (43.0 x 21.5 x 18 cm), each cage containing 2 rats of the same sex and group.
Diet (e.g. ad libitum): free access to A04 C pellet diet (U.A.R., France) distributed weekly. The batch was analized (composition, contaminants) by the supplier.
- Water (e.g. ad libitum): free access to bottles containing filtered tap water with a 0.22 micron filter (Millipore s.a., France). Routine analysis (nitrosamines, pesticides, heavy metals, bacterias) were made periodically.
- Acclimation period: a 8-day acclimatation period preceded the treatment period.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2°C
- Humidity (%): 50 ± 20%
- Air changes (per hr): about 13 cycles per hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12 hours of light and 12 hours of dark

IN-LIFE DATES: From: 15 may 1990 To: 22 June 1990

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
The oral route was chosen in order to attain higher resorption rates. The test substance was administered by gavage using a glass syringe fitted with a metal prob. Each an
imal was given the same volume : 5ml/kg/day.
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
0.5% of CMC in water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test substance was suspended in aqueous hydrogel of 0.5% carboxymethyl cellulose and homogeneized by a magnetic stirrer. The preparations were performed daily and delivered protected from light by aluminium foil.

VEHICLE
- Justification for use and choice of vehicle (if other than water): not specified
- Concentration in vehicle: not specified
- Amount of vehicle (if gavage): not specified
- Lot/batch no. (if required): water : batch no. 1298 (Biosédra), Carboxymethyl cellulose batch No. 88314 (Prolabo)
- Purity: not specified
Analytical verification of doses or concentrations:
yes
Remarks:
The results of these analyses were not included in this report
Details on analytical verification of doses or concentrations:
The chemical analysis of concentration was performed by the sponsor. Samplings of suspensions were taken as follows : The exact measurement of 2 x 10 ml of each preparation, control group included, was taken on weeks 1 and 4 and frozen. For the validation of analytical assay, 100 ml if the vehicle, 5g of the test substance and 100 ml if the lowest concentration were taken and frozen. All the samples were sent frozen to the sponsor.
Duration of treatment / exposure:
28 days
Frequency of treatment:
The test substance was administered once a day, at the same approximate daily time, in the morning, 7 days per week for 29 and 30 days, according to the necropsy dates.
Doses / concentrationsopen allclose all
Dose / conc.:
40 mg/kg bw/day (actual dose received)
Dose / conc.:
200 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Dose / conc.:
0 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
Three groups of 10 males and 10 females and one control group of 10 males and 10 females
Control animals:
yes
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were determined in agreement with the sponsor, following the results of a previously conducted study No. 5956 TSR
- Rationale for animal assignment (if not random): The assignement to the treatment groups were randomly selected by a computer

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: No

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once a day, at approximately the same daily time

BODY WEIGHT: Yes
- Time schedule for examinations: once before allocation of the animals into groups, on the first day of treatment, then once a week until the end of the study

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes / No / Not specified
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes / No / Not specified

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes / No / Not specified

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No
Food comsumption was recorded once a week over a period of 7 days until the end of the study. Food
intake per animal per day, was calculated using amount of food given and left in each cage.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Examinations were performed before the beginning of the treatment and in all the animals of the control gorup and the high dose level group at week 4.
- Dose groups that were examined: all groups at the beginning of the treatment, and control and high dose level groups at the week 4.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood samples were taken approximately 24 hours after treatment from the orbital sinus in animals while they were under light ether anaesthesia.
- Anaesthetic used for blood collection: Yes (ether)
- Animals fasted: Yes
- How many animals: 80 animals
- Parameters checked in table were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood samples were taken approximately 24 hours after treatment from the orbital sinus in animals while they were under light ether anaesthesia.
- Animals fasted: Yes
- How many animals: 80
- Parameters checked in table were examined.

URINALYSIS: Yes
--Time schedule for collection of urine: Animals were placed into metabolic cages and fasted overnight
for about 18 hours for an urine collection
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked in table were examined.

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: Yes
- Time schedule for examinations: : Blood samples were taken approximately 24 hours after treatment from the orbital sinus in animals while they were under light ether anaesthesia.
- How many animals: 80
- Dose groups that were examined: all groups
- Parameters checked in table were examined.

Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table)

HISTOPATHOLOGY: Yes (see table)
Statistics:
The sequence was used for the statistical tests of clinical parameters (bodyweight and food consumption), and for haematological and biochemical parameters, and for organ weight :

- The normal distribution of values in the samples was checked by Kolmogorov-Smirnov's test.

-If no significant heterogeneity of the variance is established the comparison between the control and treated groups was performed by Dunett's test.

-In the case of an abnormal distribution, this test was performed after the logarithmic transformation of the values. If a significant heterogeneity persisted after the transformation, a comparison between the treated groups and control group in order to prove a treatment-related difference was made by using Mann-Whitney's test (2 groups) or Dunn's test (more than 2 groups).

-In the case of the normal distribution of values according to the normal law an analysis of the variance was made by the Bartlett's test (more than 2 samples) or Fisher's test (2 samples).

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
No clinical signs were observed in the animals of the 40 and the 200 mg/kg/day groups. At the dose level of 1000 mg/kg/day, clinical signs observed were : hypersalivation (9/10 males and 7/10 females), decrease in activity in all the animals, eyes half-closed in 9/10 males and all females, lacrimation (7/10 males and 2/10 females), dyspnea in 1/10 males. This clinical signs could be due, in part, to the slight irritant properties of the test substance.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One male of the 200 mg/kg/day dose level was found dead on day 28, after blood sampling. No clinical signs preceded the death. The probable cause was not established.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The bodyweight gain in the males and the females of the 40 and 200 mg/kg/day groups was comparable to that of control groups. A slight decrease in the bodyweight gain was observed from the first week of treatment, in the males and the females of the 1000 mg/kg/day group. The difference in the mean bodyweight values, statistically significant from week 3 in the males and 4 in the females, was in mean 10% and 7% respctively.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
A very slight decrease in the food consumption was recorded in all animals of the high dose group (1000 mg/kg/day) throughout the study, in mean 8% (male) and 9% (female).
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
The exmainations revealed only minor changes : corneal vacuolisation and persistence of the hyaloid vessel) commonly observed in the laboratory rats.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Moderate prolongation in the Quicktime (QT) together with moderate increase in Activated Partial Thromboplastin Time (APTT), were observed in the males of the 1000 mg/kg/day group. The values for these two parameters in the females of the same group were comparable to those of the controls.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Slight to moderate increase in triglycerides values was found among the male rats receiving 1000 mg/kg/day, when compared with the controls. A tendancy towards such an effect was found among the females rats of the same group. The relationship of the increase in triglycerides values to the treatment cannot be ruled out. The variations observed in the other blood biochemical parameters, namely inorganic phosphorus, total bilirubin, glucose level, proteingram and aminotransferas activities, were considered to be of no toxicological significance, although they were statistically significant. This was because they were minor and not dose related.
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Immunological findings:
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
The statistical analysis of organ weights revealed the following changes in the 1000 mg/kg/day group versus control: a marked increase in absolute and relative spleen weights in the males (60% and 86%) and in the females (36% and 53%), a slight decrease in relative kidneys weight in the males (24%) and in females (9%), a moderate increase in relative liver weight in the males (15%) and females (8%), a slight increase in testes weight (24%) in the males, a slight decrease in absolute adrenals weight (16%) in the females.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
A blackish colouration of the spleen was observed in 9/10 males and 8/10 females of the 1000 mg/kg/day group, 2 females of the 200 mg/kg/day group and on male of the 40 mg/kg/day group. Moreover, enlargement of the spleen was seen in 5/10 males and 4/10 females of the 1000 mg/kg/day group.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
In spleen, Haemosiderin-laden macrophages were seen in 9/10 males and 10/10 females of the high dose level group vs 3/10 females of control group. The increased incidence and severity of haemosiderin pigment accumulation was also considered to be treatment-related.
Histopathological findings: neoplastic:
no effects observed

Effect levels

Key result
Dose descriptor:
NOEL
Effect level:
200 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
body weight and weight gain
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic

Target system / organ toxicity

Critical effects observed:
no

Any other information on results incl. tables

Bodyweight

Dose (mg/kg/day)

 

0

40

200

1000

0

40

200

1000

Week

 

Male

Female

-1

Mean

136

136

136

136

113

113

113

113

 

StandartDeviation

9.5

10.5

10.7

11.1

7.3

7.5

7.5

9.1

 

number

10

10

10

10

10

10

10

10

1

Mean

201

203

203

198

154

155

153

155

 

StandartDeviation

11.1

15.0

14.6

13.6

11.5

6.9

9.4

7.9

 

number

10

10

10

10

10

10

10

10

2

Mean

273

274

271

255

183

179

183

177

 

StandartDeviation

16.6

19.3

18.8

17.2

12.5

8.1

9.0

11.6

 

number

10

10

10

10

10

10

10

10

3

Mean

325

324

321

300*

207

203

205

198

 

StandartDeviation

22.2

25.6

20.5

18.6

18.4

8.1

12.3

13.4

 

number

10

10

10

10

10

10

10

10

4

Mean

367

362

353

326**

224

220

221

207*

 

StandartDeviation

28.3

26.3

24.8

19.9

21.0

7.8

15.8

13.6

 

number

10

10

10

10

10

10

10

10

5

Mean

381

376

378

327**

234

226

227

206**

 

StandartDeviation

28.1

26.1

34.4

19.1

21.0

7.1

15.5

13.7

 

number

10

10

9

10

10

10

10

10

Foodconsumption

 

Male

Female

1

Mean

30.2

30.7

30.0

27.6

20.4

19.3

20.1

18.8*

 

StandartDeviation

1.45

2.61

2.11

2.30

0.91

0.98

1.14

0.63

 

number

5

5

5

5

5

5

5

5

2

Mean

32.5

32.6

31.2

28.7*

21.3

20.2

20.4

19.2**

 

StandartDeviation

1.58

2.95

1.73

2.04

0.52

0.75

1.58

0.74

 

number

5

5

5

5

5

5

5

5

3

Mean

31.4

30.9

30.5

29.8

20.7

20.1

20.3

18.9*

 

StandartDeviation

2.26

2.62

2.62

3.20

1.22

0.52

1.28

0.75

 

number

5

5

5

5

5

5

5

5

4

Mean

28.4

28.6

28.6

26.6

20.5

19.3

20.1

18.5

 

StandartDeviation

1.86

2.92

4.36

3.38

1.73

0.88

1.38

0.87

 

number

5

5

5

5

5

5

5

5

 

 

Male

Female

Organ

Dose (mg/kg/day)

0

40

200

1000

0

40

200

1000

Liver

Mean

10.80

11.25

10.72

10.65

6.95

6.64

6.76

6.72

StandartDeviation

1.61

1.94

1.98

0.975

0.511

0.326

0.822

0.563

Means% Body

3.09

3.25

3.10

3.56**

3.29

3.24

3.27

3.56*

StandartDeviation

0.294

0.391

0.307

0.231

0.178

0.153

0.325

0.221

Kidney

Mean

2.91

3.05

2.95

3.11

1.88

1.78

1.92

1.83

StandartDeviation

0.222

0.296

0.425

0.396

1.148

0.114

0.162

0.161

Means% Body

0.837

0.884

0.858

1.04##

0.891

0.871

0.930

0.970*

StandartDeviation

0.031

0.037

0.081

0.089

0.063

0.056

0.076

0.066

Spleen

Mean

0.801

0.772

0.712

1.28##

0.604

0.577

0.561

0.825**

StandartDeviation

0.081

0.132

0.127

0.232

0.075

0.083

0.114

0.095

Means% Body

0.231

0.223

0.207

0.429##

0.285

0.281

0.271

0.437**

StandartDeviation

0.022

0.031

0.030

0.080

0.018

0.037

0.050

0.045

Testes

Mean

3.23

3.22

3.47

3.46

 

StandartDeviation

0.264

0.223

0.321

0.313

Means% Body

0.934

0.939

1.01

1.16**

StandartDeviation

0.109

0.066

0.091

0.120

Adrenal

Mean

0.071

0.062

0.065

0.061

0.075

0.067

0.079

0.063**

StandartDeviation

0.015

0.012

0.012

0.011

0.010

0.007

0.010

0.007

Means% Body

0.020

0.018

0.019

0.020

0.036

0.033

0.038

0.033

StandartDeviation

0.004

0.003

0.004

0.004

0.005

0.004

0.004

0.004

 

Dose

Control

40 mg/kg/day

200 mg/kg/day

1000 mg/kg/day

Organ

Sex

M

F

M

F

M

F

M

F

Numberofanimals

10

10

10

10

10

10

10

10

MacroscopicPathology

Spleen

Enlarged

 

 

 

 

 

 

5

4

Irregularsurface

 

 

 

 

 

 

1

 

Blackishcolor

 

 

1

 

 

 

9

8

MicroscopicPathology

Spleen

CapsularThickening

1

 

1

 

 

1

5

3

Sub-capsul. CHR.Infl.

2

 

1

 

 

1

4

1

Haemosid. Lad.Acrop.

 

3

 

 

 

 

9

10

Congestion

2

2

1

 

1

2

10

10

 

BloodBiochemistryatweek4

Dose (mg/kg/day)

 

0

40

200

1000

0

40

200

1000

Sexe

Male

Female

InorganicPhosphorus(I.PHOS)

Mean

3.51

3.44

3.31

2.85**

2.58

2.54

2.53

2.41

mmol/l

StandartDeviation

0.235

0.308

0.332

0.299

0.204

0.265

0.184

0.157

number

10

10

10

10

10

10

10

10

Glucose (GLUC)

Mean

6.45

6.90

6.46

6.09

7.15

6.43

6.98

6.22*

mmol/l

StandartDeviation

0.754

0.966

0.789

0.768

0.867

0.665

0.337

0.710

number

10

10

10

10

10

10

10

10

Totalbilirubin(TOT.BIL)

Mean

1

1

1

3

1

2

2

3**

µmol/l

StandartDeviation

0.0

0.7

0.5

1.2

0.5

0.5

0.5

0.4

number

10

10

10

10

10

10

10

10

Totalprotein(PROT)

Mean

69

68

67

67

69

68

68

71

g/l

StandartDeviation

2.5

2.8

2.7

2.9

2.8

4.1

1.7

2.6

number

10

10

10

10

10

10

10

10

Triglycerides

Mean

0.65

0.56

0.71

1.68**

0.38

0.34

0.33

0.56**

mmol/l

StandartDeviation

0.256

0.196

0.275

0.857

0.161

0.073

0.058

0.108

number

10

10

10

10

10

10

10

10

Aspartateaminotransferase(ASAT)

Mean

70

76

69

72

53

54

57

61

UI/l

StandartDeviation

11.2

9.6

8.8

8.5

5.8

7.5

9.6

28.5

number

10

10

10

10

10

10

10

10

Alanineaminotransferase(ALAT)

Mean

24

18*

21

20

15

16

24

33

UI/l

StandartDeviation

6.0

4.0

3.3

4.5

2.6

4.5

5.0

39.8

number

10

10

10

10

10

10

10

10

BloodHaematologyatweek4

Dose (mg/kg/day)

 

0

40

200

1000

0

40

200

1000

Sexe

Male

Female

Quick Time

Mean

13.9

13.9

13.7

17.8**

13.6

14.1

13.5

13.7

StandartDeviation

0.89

0.75

0.97

2.58

0.40

1.03

0.56

0.61

number

10

10

10

10

10

10

10

10

ActivatedPartialThromboplastinTime

Mean

24.5

21.9

23.8

39.1**

18.9

20.9

19.1

19.5

StandartDeviation

2.71

2.48

3.83

12.69

3.10

5.31

3.18

2.01

number

10

10

10

10

10

10

10

10

* P<0,05

**P<0,01

##Dunn’stest at 1%Level

 

Applicant's summary and conclusion

Conclusions:
Under experimental conditions of this study, the daily administration (gavage) of the test item 1-phenyl-3-methyl-5-pyrazolone to Sprague-Dawley rats for 28 days at doses of 0, 40, 200 and 1000 mg/kg/day induced clinical signs as hypersalivation, decrease in activity, eyes half-closed, lacrimation and a slight decrease in the body weight gain in line with a very slight decrease in food consumption in all the animals of 1000 mg/kg/day group. At the same dose, a moderate prolongation of the quicktime with a moderate increase in the activated partial thromboplastin time, and moderate increase in the triglyceride level were observed in males.On microscopic examination, a marked congestion of the spleen with haemosiderin-laden macrophages in line with blackish colouration were observed in animals of both sexes at the high dose level. Consequently, according to REACh regulation, the No Observed Adverse Effect Level (NOAEL) could be defined at the dose level of 200 mg/kg/day.
Executive summary:

The GLP-compliant study was performed to assess the potential toxicity of 1 -phenyl-3 -methyl-5 -pyrazolone when administered daily to Sprague-Dawley rats for 4 weeks (28 days) according to OECD guideline 407 (Repeated Dose oral toxicity - Rodent 28-day or 14-day study) (dated 12th May 1981).

Materials and Method

Groups of 20 animals (10 males and 10 females) each received daily the test substance at doses of 0 (control group), 40 , 200 or 1000 mg/kg/day by oral route (gavage) for 4 weeks (28 days).

Clinical signs was recorded once daily, twice for mortality. Food consumption and body weight were recorded on a weekly basis. Haematological and blood biochemical investigations were performed on

week 4.

At the end of the treatment period, all animals were sacrificed. Designated organs were weighed and all tissues were examined before and after dissection.

Results

No clinical signs were observed in the animals of the 40 and the 200 mg/kg/day groups. At the dose level of 1000 mg/kg/day, clinical signs observed were : hypersalivation (9/10 males and 7/10 females), decrease in activity in all the animals, eyes half-closed in 9/10 males and all females, lacrimation (7/10 males and 2/10 females), dyspnea in 1/10 males. This clinical signs could be due, in part, to the slight irritant properties of the test substance. The bodyweight gain in the males and the females of the 40 and 200 mg/kg/day groups was comparable to that of control groups. A slight decrease in the bodyweight gain was observed from the first week of treatment, in the males and the females of the 1000 mg/kg/day group. The difference in the mean bodyweight values, statistically significant from week 3 in the males and 4 in the females, was in mean 10% and 7% respctively. A very slight decrease in the food consumption was recorded in all animals of the high dose group (1000 mg/kg/day) throughout the study, in mean 8% (male) and 9% (female). Moderate prolongation in the Quicktime (QT) together with moderate increase in Activated Partial Thromboplastin Time (APTT), were observed in the males of the 1000 mg/kg/day group. The values  for these two parameters in the females of the same group were comparable to those of the controls.  

The statistical analysis of organ weights revealed the following changes in the 1000 mg/kg/day group versus control: a marked increase in absolute and relative spleen weights in the males (60% and 86%)  and in the females (36% and 53%), a slight decrease in relative kidneys weight in the males (24%) and in females (9%), a moderate increase in relative liver weight in the males (15%) and females (8%), a slight increase in testes weight (24%) in the males, a slight decrease in absolute adrenals weight (16%) in the females. A blackish colouration of the spleen was observed in 9/10 males and 8/10 females of the 1000 mg/kg/day group, 2 females of the 200 mg/kg/day group and on male of the 40 mg/kg/day group. Moreover, enlargement of the spleen was seen in 5/10 males and 4/10 females of the 1000 mg/kg/day group. In spleen, Haemosiderin-laden macrophages were seen in 9/10 males and 10/10 females of the high dose level group vs 3/10 females of control group. The increased incidence and severity of haemosiderin pigment accumulation was also considered to be treatment-related.

Conclusion

Under experimental conditions of this study, the daily administration (gavage) of the test item 1-phenyl-3-methyl-5-pyrazolone to Sprague-Dawley rats for 28 days at doses of 0, 40, 200 and 1000 mg/kg/day induced clinical signs as hypersalivation, decrease in activity, eyes half-closed, lacrimation and a slight decrease in the body weight gain in line with a very slight decrease in food consumption in all the animals of 1000 mg/kg/day group. At the same dose, a moderate prolongation of the quicktime with a moderate increase in the activated partial thromboplastin time, and moderate increase in the triglyceride level were observed in males.On microscopic examination, a marked congestion of the spleen with haemosiderin-laden macrophages in line with blackish colouration were observed in animals of both sexes at the high dose level. Consequently, according to REACh regulation, the No Observed Adverse Effect Level (NOAEL) could be defined at the dose level of 200 mg/kg/day.