3-methyl-1-phenyl-5-pyrazolone

Repeated dose toxicity: oral

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

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Version / remarks:

Adopted : 12 may 1981

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

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-2,4-dihydro-3H-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: 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 or test system 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 / concentrations

Doses / concentrations 1

Dose / conc.:

40 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

200 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Doses / concentrations 4

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:

body weight and weight gain

clinical signs

gross pathology

histopathology: non-neoplastic

organ weights and organ / body weight ratios

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.