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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
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1993
Report Date:
1993

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): RPA 405217
- Supplier: Rhone-Poulenc Secteur Agro
- Substance type: white and yellowish powder
- Lot/batch No.: DA 696
- Storage condition of test material: room temperature or at 4 °C

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:

TEST ANIMALS

- Source: Charles River France (76410 Saint-Aubin-Elbeuf, France).
- Age at study initiation: approx. 6 weeks
- Weight at study initiation: approx. 200 g for the males and 175 g for the females
- Housing: Upon their arrival at C.I.T., the animals were housed in a protected zone. The animal room was disinfected before the arrival of the animals and cleaned regularly thereafter.
The animals were housed in suspended wire-mesh cages (43.0 x 21.5 x 18.0 cm) and each cage contained 2 rats of thesame sex and group. A metallic tray was placed under each cage and contained autoclaved sawdust (Socité SICSA, 94142 Alfortville, France). Bacteriological analyses and detection of possible contaminants (pesticides, heavy metals) of the sawdust were performed periodically (Laboratoire D’partemental d'Analyses, 27000 Evreux, France; Laboratoire Municipal et Régional dc Rouen, 76000 Rouen, France).
Bottles and sawdust were changed once a week.
Cages were not randomized in theroom but were placed in numerical order, vertically on die
racks. Every fortnight, the racks were moved around clockwise, rack by rack.
- Diet: All animals had free access to A04 C pelleted diet (U.A.R., 91360 Villemoisson-sur-Orge, France). Batch numbers 20605, 20305 and 20701 were used. The batches were analysed (composition, contaminants) by the supplier.
- Water: The animals had free access to bottles containing potable water filtered with a 0.22 micron filter (Millipore S.A, 78140 V6hizy, France). Bacteriological and chemical analyses of the water and detection of possible contaminants (pesticides, heavy metals and nitrosamines) are made periodically (Laborazoire D’parzemenzal d'Analyses, 27000 Evreux, France; Laborazoire Municipal et Regional dc Rouen, 76000 Rouen, France; Centre dc Nutrition Humaine, 54000 Nancy, France).
There were no known contaminants in the diet, water or sawdust at levels likely to have influenced th eoutcome of the study.
- Acclimation period: 7 days

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


Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Remarks:
provided by C.P.F. (77000 Melun, France), batch No. A248999 1552.
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:

- Vehicle
The vehicle was corn oil provided by C.P.F. (77000 Melun, France), batch No. A 248999 1552.

- Preparation
The test substance was suspended in die vehicle and homogenized using a magnetic stirrer, before and during the treatment. The preparations were made by die C.I.T. Pharmacy for up to seven days of treatment, according to known stability.

The test substance was administered by gavage using a glass syringe fitted with a metal probe. A constant dose volume of 5ml/kg/day was used.
Each animal was given the test substance once a day, at the same approximate daily time, 7 days a week over a period of 4 weeks.
The quantity of the test substance or vehicle administered to individual animals was adjusted according to the most recently recorded body weight.
Control animals received the vehicle alone, under the same conditions.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Before the beginning of the study, the homogeneity of the test substance dissolved in corn oil was checked at concentrations of 10 and 200 mg/ml. From each preparation, samples were taken at 3 different levels (top, middle, and bottom) and analysed in duplicate. Before the beginning of the study, the chemical stability of the test substance preparations at the same concentrations was checked. Each preparation was sampled the day of preparation and after 4 and 7 days storage at +4 °C. Each sample was analysed in duplicate.
On weeks 1 and 4 of the study, each preparation (control group included) was checked for the achieved concentration of the test substance.
Duration of treatment / exposure:
- 4 weeks
Frequency of treatment:
- each animal was given die test substance once a day, at the same approximate daily time, 7 days a week over a period of 4 weeks.
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 50, 220 or 1000 mg/kg bw/d
Basis:
actual ingested
No. of animals per sex per dose:
- 6 males and 6 females
Control animals:
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 5- day preliminary toxicity study by oral route in rats (CIT/Study No. 9110 TSR). In the preliminary study, the test substance was administered at doses of 0, 250, 500, 1000 or 2000 mg/kg bw/day. A slightly (non dose-related) lower body weight gain was observed in the male treated groups, when compared to respective controls. Consequently, 1000 mg/kg/day were selected as the high dose for the 4-week study, and 50 mg/kg/day as the low dose. 220 mg/kg bw/day was selected as the intermediate dose, being the geometric mean between 50 and 1000 mg/kg bw/day.

- Rationale for animal assignment:
Groups were formed in order to obtain approximately the same mean body weight and standard deviation between groups. The stratified body weight procedure and assignment to the treatment groups were performed using a computer-derived randomisation.

The oral route was selected since it is a possible route of exposure in humans

Examinations

Observations and examinations performed and frequency:

- Clinical signs
Clinical signs were observed for each animal, at least once a day, at the same approximate daily time.
- Morbidity and mortality
The animals were checked at least twice each day for mortality or signs of morbidity, including weekends.
- Food consumption
The quantity of food consumption by the animals of each cage was recorded once a week over a 7-day period during the treatment period. Food intake per animal and per day, was calculated using the amount of food given and left in each cage.
- Body weight
Body weight was recorded for each animal, once before allocation of the animals into groups, on the first day of treatment, then once a week until the end of the study.

- Dose groups that were examined: all


LABORATORY INVESTIGATIONS

Approximately 24 hours after lasttreatment, blood samples were taken from the orbital Sinus of the overnight fasted animals under light ether anaesthesia. The samples were collected into tubes containing the appropriate anticoagulant. For urine collection, the animals were deprived of food and placed in metabolism cages. The urine was collected into a tube containing thymol crystals during an overnight period of about 18 hours.

Haematology

The following parameters were determined in all animals on week 4.

- Blood collected on EDTA:
Leucocytes, Haematology, Erythrocytes, Haemoglobin, Packed Cell Volume, Mean Cell Volume, Mean Cell Haemoglobin,Mean Cell Concentration (MCHC) Thrombocytes (eosinophils, basophils, lymphocytes, monocytes)

- Blood collected on sodium citrate:
Prothrombin Time, Thromboplastin Activated Partial Time, Fibrinogen Thromboplastin

Blood smears were prepared for all sampled animals. In the absence of abnormalities, a differential white ceIl count was only determined in animals of the control and high dose groups.

The following parameters were determined in all animals on week 4.
- Blood collected on lithium heparinate:
Sodium, Potassium, Chloride, Calcium, Ortho-cresol-phthalein, Inorganic phosphorus, Phosphomolybdic reaction,Glucose, Urea, Creatinine, Total bilirubin,Total proteins, Biuret, Albumin, Albumin/globulin ratio, Cholesterol, Triglycerides, Alkaline Phosphatase, Aspartate aminotransferase, Alanine aminotransferase


- Urinalysis
The following parameters were determined in all surviving animals on week 4.

Quantitative parameters
Volume, pH, Specific gravity,

Semi-quantitative Parameters
Proteins, Glucose, Ketones, Bilirubin, Nitrites, Blood, Urobilinogen


Cytology of sediment Microscopic
leucocytes, erythrocytes, cylinders, magnesium ammonium phosphate crystals, calcium phosphate crystals, calcium oxalate crystals, cells

Qualitative Parameters
Appearance, Colour
Sacrifice and pathology:
- Sacrifice
On completion of the treatment period, after about 18 hours fasting, all animals were asphyxiated by carbon dioxide and sacrificed by exsanguination.
- Organ weights
The animals were weighed before possible necropsy and the following organs weighed wet as soon as after dissection:
adrenals, heart, kidneys, liver, ovaries, spleen, testes, thymus.
Paired organs were weighed separately.
- Macroscopic examination
A complete macroscopic examination was performed on all animals.
All gross observations were recorded individually.
Other examinations:
- Preservation of tissues
In all animals, all the macroscopic lesions and the following tissues were preserved in 10 % buffered formalin (except for the eyes and pituitary gland which were fixed in formolsublimate):
adrenals, lungs with bronchi, spleen,
aorta, Lymph nodes, sternum (with bone brain including medulla/ mandibular and mesenteric marrow) , pons, cerebellar and marnmary glands, stomach , cerebral cortex oesophagus, salivary glands, caecum, ovaries, pancreas, duodenum, pituitary gland, testes and epididymides, eyes, prostate, thymus, rectum, thyroids with parathyroids, femoral bone with sciatic nerve, trachea, articulation, seminal vesicle, tongue, heart, skeletal muscle, urinary bladder, ileum, skin, uterus, jejunum, spinal cord, horns and cervix, kidneys, vagina, liver

- Microscopic examination
All tissues required for microscopic examination were embedded in paraffin wax, sectioned at approximately 4 microns in thickness and stained with hematoxylin-eosin.
Microscopic examination was performed on:
all macroscopic lesions and tissues listed above in animals of the high dose and control groups. liver and all macroscopic lesions of the animals of the low and intermediate dose groups.
Statistics:
The normality of the distribution of the values in each group was checked by Kolmogorov- Smirnov's test (1948). If the distribution was normal, the homogeneity of variances between the groups was assessed by Bardett's test (1937) (more than 2 groups) or Fisher's test (1934) (2 groups). If no significant heterogeneity of the variances was established, the comparison between treated and control groups was performed by Dunnett's test (1955). If the variances were heterogeneous, the comparison between treated and control groups was performed by Dunn's test (1964) (more than 2 groups) or by Mann Whitney's test (1947) (2 groups). If the distribution of values in the groups was not normal, the analysis was repeated after logarithmic transformation of the values (except for organ weights). If this logarithmic transformation failed to normalise the distribution of the values, comparison of treated and control groups was performed by Dunn's test using original values.

Results and discussion

Results of examinations

Details on results:
CLINICAL EXAMINATIONS

- Clinical signs
Ptyalism was observed in all treated groups and was clearly treatment-related. The incidence was dose-related.

- Mortality
No mortalities were observed during the course of the study.

- Food consumption
The mean food consumption of males and females in treated groups was similar to that of respective controls throughout the treatment period.

- Body weight
A lower mean body weight gain was observed in males given 220 and 1000 mg/kg bw/day (-17 % and -15 % respectively when compared to controls) and, although there was no dosedependancy, these differences from control were considered to be treatment-related.
The mean body weight gain of males given 50 mg/kg bw/day and females in all treated groups was considered to be similar to that of respective controls.


LABORATORY INVESTIGATIONS

- Haematology
A slight, but statistically significant lower mean haemoglobin concentration and mean cell haemoglobin concentration was noted in females given 220 and 1000 mg/kg bw/day (-7 % and -8 %; -3 % and -3 %, respectively). In addition, a slight but statistically significant lower packed cell volume was noted in females given 1000 mg/kg bw/day (-5%).
However, all these differences from control were minor and all individual values were within the range of background data. Consequently, they were considered to be of no toxicological importance.

A slight, but statistically significant higher fibrinogen level was noted in females given
220 mg/kg bw/day, but in the absence of any difference from control in the high dose group, this finding is considered to be fortuitous.
No perceptible differences from control were noted in the other haematological parameters.

- Blood biochemistry (tables 9 and 10, appendix 10)
A slight but statistically significant lower inorganic phosphorus concentration was noted in females given 1000 mg/kg bw/day (-19 %). The individual values were below the lower limit of background data (2.00 mmol/l) in 4 out of 6 animals. This was considered to be treatment related.
A moderate, but statistically significant lower mean alkaline phosphatase activity was noted in males given 1000 mg/kg bw/day (-40 %). This was due to the fact that 2 individual values in controls were above the upper normal limit of background data (565 IU/l). Consequently, this finding was considered to be of no toxicological importance.
The slight, but statistically significant differences from control noted among the other biochemical parameters (namely: sodium, chloride, creatinine, total proteins, albumin, A/G ratio, bilirubin and alanine aminotransferase activity) were minor and the individual values were within the normal range of our background data. Therefore, they were regarded as being of no toxicological significance.

- Urinalysis
No qualitative or quantitative differences from control were noted among the urinary
parameters.


PATHOLOGIE

- Organ weighs
A higher mean liver weight was noted in treated groups, when compared to respective controls. The higher mean liver weight was considered to be treatment-related in females given 50 mg/kg bw/day and in animals of both sexes given 220 and 1000 mg/kg bw/day.
A slight, but sadistically significant lower (-13%) mean absolute heart weight was noted in males given 220 or 1000 mg/kg bw/day.

A statistically significant higher mean absolute kidney weight was noted in females given 220 or 1000 mg/kg bw/day (respectively +12 % and +13 %), when compared to values of controls.
A statistically significant, but not dose-related, lower absolute and/or relative spleen weight was also noted in males given 220 or 1000 mg/kg bw/day.
As no relevant histopathological findings were noted in the heart, kidneys or spleen of animals given 1000 mg/kg bw/day, these differences from control were considered to be of no toxicological importance.

- Macroscopic examination
Liver enlargement was noted in 1/6 males given 220 mg/kg bw/day and in 1/6 males given 1000 mg/kg bw/day. Paleness of the liver was noted in another male of the high dose group. These findings were considered to be treatment-related.
The liver enlargement noted in the male given 220 mg/kg bw/day was not associated with any relevant histological findings, but was associated with a slightly higher absolute and relative liver weight and was considered to be treatment-related.
Small testes, sometimes with a soft consistency, and small unilateral epididymides were noted in 1/6 control males, 2/6 males given 50 mg/kg bw/day, 1/6 males given 220 mg/kg bw/day and 2/6 males given 1000 mg/kg bw/day. These findings were considered to be of a spontaneous nature and not treatment-related.
The few other macroscopic findings noted were those which are commonly recorded in laboratory rats of this strain and, consequently, they were considered to be of no toxicological importance.

- Microscopic examination
A dose-related minimal to marked hepatocellular hypertrophy, mainly centrolobular, was noted in 1/6 females given 50 mg/kg bw/day, 5/6 females given 220 mg/kg bw/day, 4/6 males and 4/6 females given 1000 mg/kg bw/day. However, there was no evidence of morphological changes indicative of hepatic cell degeneration or necrosis. In the high dose group, the severity was slightly higher in females than in males. This finding was considered to be treatment-related and is most probably attributed to an increased demand for liver function subsequent to test substance administration.
Severe degeneration of seminiferous tubules together with marked to severe inhibition of spermatogenesis in testes (uni- or bilateral) and unilateral oligospermia or aspermia of epididymides were noted 1/6 control males, 2/6 males given 50 mg/kg bw/day, 1/6 males given 220 mg/kg bw/day and 2/3 males given 1000 mg/kg bw/day and were well-correlated with the macroscopic findings in these Organs.
These findings were considered to be of a spontaneous ship to treatment. nature and, therefore, to bear no relationship to treatment. The other microscopic findings noted were those which are commonly recorded in untreated laboratory rats of this strain. Furthermore their incidence, severity and morphological characteristics were similar in treated and control groups. Accordingly, they were considered to be not treatment-related.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: lower body weight gain at 200 and at 1000 mg/kg bw/d
Dose descriptor:
NOAEL
Effect level:
220 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: lower inorganic phosphorous concentration at 1000 mg/kg bw d

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Executive summary:

In a subacute toxicity study (Rhone-Poulenc, 1992) (E)-5[(4 - chlorophenyl)methylene]-2,2 - dimethylcyclopentanone was administered daily by oral route (gavage) to groups of 6 male and 6 female Sprague-Dawley rats at doses 0, 50, 220 or 1000 mg/kg/ bw day for 4 weeks.

The animals were checked regularly each day for clinical signs and mortality. Body weight and food consumption were recorded once a week. Haematological and blood biochemical investigations and urinalysis were performed on week 4. At the end of the treatment period, the animals were sacrificed. Ihe animals were submitted to a macroscopic examination and designated organs were weighed. A range of tissues was preserved for microscopic examination.

The daily administration of the test substance at doses of 50, 220 or 1000 mg/kg bw/ day for 4 weeks induced ptyalism in all treated groups, a lower body weight gain in males given 220 and 1000 mg/kg/day and a decrease in plasma inorganic phosphorous concentration in females given 1000 mg/kg bw/day.

An increase in liver weight was noted in females given 50 mg/kg/day and in males and females given 220 and 1000 mg/kg/day. This was associated with a dose-related, minimal to marked hepatocellular hypertrophy, noted in one female given 50 mg/kg bw/day, in females given 220 mg/kg bw/day and in both males and females given 1000 mg/kg/day. The changes in the liver were clearly treatment-related, but were not associated with any degenerative changes or necrosis. Thus, these findings are attributed to an increased demand for liver function subsequent to test substance administration and, therefore, were considered not to be evidence of toxicity.

Consequently, the NOAEL in this study is 50 mg/kg bw/day for males and 220 mg/kg bw/d for females, respectively.