4-[4-(diethylamino)benzylidene]-5-ethoxy-2-phenyl-2,4-dihydro-3H-pyrazol-3-one

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:

08 October 2003 to 05 November 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3050 Repeated dose 28-day oral toxicity study in rodents 2000.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Details on test material:

Identification: HSY-2701
Description: Red powder
Batch: 1004-1
Purity: 99.8 %
Test substance storage: At room temperature in the dark
Stability under storage conditions: Stable
Expiry date: 12 february 2004
Stability in vehicle (propylene glycol): At least 4 hours

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfield, Germany
- Age at study initiation: Approximately 6 weeks
- Weight at study initiation (Mean, ST. Dev): MALES; Control/Group 1 206 g ± 5.1, Group 2 206 ± 10.0, Group 3 200 ± 5.2, Group 4 202 ± 9.6. FEMALES; Control/Group 1 159 g ± 7.0, Group 2 154 ± 6.8, Group 3 163 ± 5.3, Group 4 162 ± 4.8
- Housing: Group housing of 5 animals per sex per cage in stainless steel suspended cages with wire mesh floors (55 x 34 x 21.5 cm height). During activity monitoring animals were individually housed overnight in Macrolon plastic cages (type III, height 15 cm) with sterilised sawdust (SAWI, Jelu Werk, Rosenberg, Germany) provided as bedding.
- Diet (e.g. ad libitum): Free access to standard pelleted laboratory animal diet (Altromin (code VRF-1, Lage germany)).
- Water (e.g. ad libitum): Free access to tap water
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.0 ± 3.0 °C (actual range 18.3 - 22.9 °C)
- Humidity (%): 30-70 % (actual range 41-75 %)
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 hours artificial fluorescent light and 12 hours darkness per day.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: Formulations were prepared daily within 4 hours prior to dosing and were homogenised to a visually acceptable level.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on previously performed trial formulations
- Amount of vehicle (if gavage): 5 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of formulations prepared prior to the main study were analysed to check homogeneity (highest and lowest concentration) and accuracy of preparation (all concentration). Stability in vehicle over 4 horus was also determined (highest and lowest concentration). In addition, from all formulations prepared on a single day in week 1 of the main study, accuracy of preparation was determined. The analytical method used was based on the results of a separate project for the development and validation of the analytical method.

Duration of treatment / exposure:

28 days

Frequency of treatment:

Daily

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 150, 500 and 100 mg/kg bw/day
Basis:

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based on the results of a 5 day dose range finding study

Positive control:

Not performed

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least twice daily
Once daily, detailed clinical observations were made in all animals.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once prior to the start of treatment and on days 8, 22 and 28 clinical observations were performed outside the home cage in a standard arena. The symptoms were graded according to fixed scales and the time of onset, degree and duration were recorded:
Maximum grade 1: grade 0 = absent, grade 1 = present
Maximum grade 3 or 4: grade 1 = slight, grade 2 = moderate, grade 3 = severe, grade 4 = very severe.

BODY WEIGHT: Yes
- Time schedule for examinations: On days 1, 8, 15, 22 and 28

FOOD CONSUMPTION : Yes
- Time schedule: Weekly


WATER CONSUMPTION : Yes
- Time schedule for examinations: Subjective appraisal was maintained through the study, but no quantitative investigation introduced as no effect was suspected.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Prior to scheduled post mortem
- Anaesthetic used for blood collection: Yes iso-flurane
- Animals fasted: Yes
- How many animals: All rats/sex/group
- Parameters: Erythrocytes, Haemoglobin, Haematocrit, Mean corpuscular volume, Mean corpuscular haemoglobin, Mean corpuscular haemoglobin concentration, Platelet count, Red cell distribution width, Total leucocytes count, Differential leucocyte count (neutrophils, lymphocytes, monocytes, eosinophils and basophils), prothrombin time, partial thromboplastin time


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Prior to scheduled post mortem
- Anaesthetic used for blood collection: Yes iso-flurane
- Animals fasted: Yes
- How many animals: All rats/sex/group
- Parameters: Alanine aminotransferase, Alkaline phosphatase, Aspartate aminotransferase, Bilirubin total, Chloride, Cholesterol, Creatinine, Glucose, Phosphorus, Protein total, Protein albumin, Urea, Calcium, Potassium and Sodium

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: During week 4
- Dose groups that were examined: All animals
- Battery of functions tested: hearing ability, pupillary reflex, static righting reflex and grip strength (Score 0 = normal/present, score 1 = abnormal/absent).
Motor activity test (recording period: 12 hours during overnight for individual animals, using a computerised monitoring system, Pearson Technical Services, Debenham, Stowmarket, England).

Sacrifice and pathology:

GROSS PATHOLOGY: Yes. All surviving animals were necropsied and descriptions of all macroscopic abnormalities recorded.
Adrenal glands, Aorta, Brain (cerebellum, mid-brain, cortex), Caecum, (Cervic), (Clitoral gland), Colon, Duodenum, Epididymides, (Eyes with optic nerve and Harderian gland), (Female mammary gland area), Femur including joint), Heart, Ileum, Jejunum, Kidneys, (Larynx), (Lacrimal gland, exorbital), Liver, Lung infused with formulin, Lymph nodes - mandibular and mesenteric, (Nasopharynx), Oesophagus, Ovaries, Pancreas, Peyer's Patches (jejenum, ileum) if detectable, Pituitary gland, (Preputial gland), Prostate gland, Rectum, (Salivary glands - mandibular, sublingual), Sciatic nerve, (Seminal vesicles), (Skeletal muscle), (Skin), Spinal cord - cervical - midthoracic - lumbar, Spleen, Sternum with bone marrow, Stomach, Testes, Thymus, Thyroid including parathyroid, (Tongue), Trachea, Urinary bladder, Uterus, (Vagina), All gross lesions
HISTOPATHOLOGY: Yes. All organ and tissue samples were processed embedded and cut at a thickness of 2-4 µm and stained with haemotoxylin and eosin. The following were examined by a pathologist:
- all tissues and organs collected at the scheculed sacrifice from all animals of the control and the highest dose group.
- all gross lesions of all animals. Tissues mentioned within brackets were not examined as there were no signs of toxicity of target organ involvement.

Other examinations:

The following organ weights were recorded from the animals on the scheduled necropsy:
Adrenal glands
Brain
Epididymides
Heart
Kidneys
Liver
Spleen
Testes
Thymus

Statistics:

The following statistical methods were used to analyse the data:
If the variable could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steet-test (many-to-one rank test) was applied when the data could not be assumed to follow a normal distribution
- The exact Fisher-test was applied to frequency data.
All tests were two sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded. Therefore two groups may display the same prented means for a different parameter yet display different test statistic values.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not specified

Details on results:

CLINICAL SIGNS AND MORTALITY
There were no clinical signs or behavioural changes over the 28 day observation period that were considered to be of toxicological significance.
Red faeces noted among all males and females at 500 and 1000 mg/kg/day from weeks 1 onwards was considered due to the staining properties of the substance. Salivation noted among all high dose males between days 14 and 18 was temporary. Incidental findings included alopecia, chromodacryorrhoea, broken upper incisors and scabs. These were not considered to be toxicologically significant.

BODY WEIGHT AND WEIGHT GAIN
Increased body weight gains were seen in males dosed at 150 mg/kg/day (statistically significat from week 1 or 3 onwards for relative and absolute bodyweights respectively). However this was considered to have occurred by chance or may represent a weak low-dosage growth stimulation effect, and was not considered to be toxicologically significant. Weight loss as noted in one high dose male between days 22 and 28 was not considered to be significant when compared with the rest of the group. The statistically significant lower weight gain of the high dose females in week 3 was not consistently noted during treatment and remained within the normal range for rats of this age and strain.

FOOD CONSUMPTION
The higher food intake of males at 150 and 500 mg/kg/day noted during the treatment was considered to be a chance observation or may represent a weak low-dosage growth stimulation effect and was considered of no toxicological significance. Food intake values of high dose males remained within the range expected for rats of this age and strain. Food consumption corrected for body weight of control females was considered to be slightly high in week 1. In addition, no dose-related changes in food consumption were noted among females treated with the test substance.

HAEMATOLOGY
Values acheiving statistical significance were not related to the dose and occurred within the normal variation. (lower partial thromboplastin time of males at 500 mg/kg/day and lower monocyt counts of females at 150 and 1000 mg/kg/day).

CLINICAL CHEMISTRY
Statistical significance for the lower creatinine level in high dose males was caused by a slightly low value for one male. As this was not observed in other animals it was not considered to be toxicologically important. No dose related response was noted for the higher cholesterol levels of females at 150 mg/kg/day and for the higher creatinine and chloride levels of females at 500 mg/kg/day.

NEUROBEHAVIOUR
No changes were observed in hearing ability, pupillary reflex, static righting reflex and grip strength. The vcariation in motor activity did not indicate a relation with treatment. The statistical significant increase of the high sensor counts in group 3 males was considered to have occurred by chance.

ORGAN WEIGHTS
An increased thymus weight was noted for two males at 150 mg/kg/day and to a lesser extent for two males at 1000 mg/kg/day. Statistical significance was achieved for the mean group responsed at 150 mg/kg/day. No further changes were noted.

GROSS PATHOLOGY
Incidental findings among control or treated animals included a liver grown together with the stomach, pelvic dilation of the kidneys, adrenal glands grown together with kidneys, enlargement of the thymus, watery -clear cysts on the kidneys, fluid in the uterus, reduced size of the thymus and scab formation on the skin. These were not considered toxicologically significant.

HISTOPATHOLOGY: NON-NEOPLASTIC
There were no microscopic findings recorded which could be attributed to treatment with the test substance.

Effect levels

1

Dose descriptor:

NOEL

Effect level:

> 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other:

Remarks on result:

not determinable due to absence of adverse toxic effects

2

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other:

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity

Critical effects observed:

no

Any other information on results incl. tables

In the attached document, the data is summarised and tabulated in Appendix I, graphical representations are also included in the figures detailed in Appendix I.

Applicant's summary and conclusion

Conclusions:

There were no changes at determination of clinical appearance, performance of functional observations, body weight and food consumption measurements, or alterations during clinical laboratory investigations, macroscopic examination, organ weight determination and microscopic examination that were considered to be of toxicological significance.
From the results a definitive NOAEL of 1000 mg/kg/day was established. This can also be considered a NOEL following HSY-2701 adminsitration.

Executive summary:

In a GLP compliant study conducted in accordance with the OECD guideline 407, HSY-2701 was found not to exhibit any toxicologically significant effects. The NOEL and NOAEL for HSY-2701 was established to be 1000 mg/kg bw/day.