4-propylcyclohexanone

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:

1190-04-22 to 1990-05-22

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:

1981

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

4-propylcyclohexanone

EC Number:

406-810-4

EC Name:

4-propylcyclohexanone

Cas Number:

40649-36-3

Molecular formula:

C9H16O

IUPAC Name:

4-propylcyclohexan-1-one

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Chbb: THOM

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Dr. Thomae GmbH, Biberach, Germany
- Age at study initiation: 9 weeks
- Weight at study initiation (mean): Males: 302 g (287-319 g); Females: 196 g (184-208 g)
- Housing: individually in type III Makrolon cages
- Diet: Altromin Standard Diet TPF® N 1324; ad libitum
- Water. tap water; ad libitum

DETAILS OF FOOD AND WATER QUALITY: The diet was checked periodically by an independent and German Government approved testing laboratory, according to the regulations of the manufacturer. Analysis includes both qualitative and quantitative evaluation for heavy metals, aflatoxins, pesticides and antibiotics. Drinking water employed in this type of study was regularly investigated microbiologically, physico-chemically, and chemically.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22-28
- Humidity (%): 26-4
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: 0.5 % Methocel® K4M Premium/Tween 80

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was suspended in the vehicle. Every week three test material concentrations were prepared and stored in brown glass bottles. Before application the test material suspensions were stirred intensively.

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

4 weeks

Frequency of treatment:

once daily, 7 times per week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

250 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

500 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5

Control animals:

yes, concurrent no treatment

Details on study design:

- Dose selection rationale: 1000 mg/kg are recommended as highest dose for a 4-week-toxicity-study in the OECD-guidelines. The lower doses were always half of the higher dose.

Positive control:

none

Examinations

Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: twice weekly

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes, once weekly

WATER CONSUMPTION: Yes
- Time schedule for examinations: twice weekly

HAEMATOLOGY: Yes
- Time schedule for collection of blood: in treatment week 4
- Anaesthetic used for blood collection: Yes, halothane anesthesia
- Animals fasted: Not specified
- How many animals: all
- Parameters checked: Erythrocyte count (ERY), Leukocyte count (LEUK), Hemoglobin (HB), Packed cell volume (PCV), Platelet count (PLAT), Reticulocyte count (RET), Differential blood cell count % Absolute number of segmented neutrophilic granulocytes(SE), Absolute number of lymphocytes (LY), Mean corpuscular volume (MCV), Mean corpuscular hemoglobin pg content (MCH), Mean corpuscular hemoglobin g/dl concentration (MCHC)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:
in treatment week 4
- Animals fasted: Not specified
- How many animals: all
- Parameters checked: Sodium, Potassium, Calcium, Chloride, Inorganic phosphorus, Glucose, Urea, Creatinine, Bilirubin Cholesterol, Triglycerides, Alanine aminotransferase, Alkaline phosphatase Aspartate aminotransferase, Protein (total), Albumin

URINALYSIS: Yes
- Time schedule for collection of urine: In study week 4
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes, animals without feed for 18 hours
- Parameters checked: pH value, Protein, Glucose, Urobilinogen, Bilirubin, Blood, Sediment

OPHTHALMOSCOPIC EXAMINATION: Yes
Prior to start of treatment and in study weeks 1 and 4 ophthalmologic examinations were performed in all rats. The anterior chamber of the eye was examined directly with an ophthalmoloscope (Carl Zeiss, No. 29902). The fundus of the eye was examined (indirectly) with a hand slit lamp and a lens.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
The organs below were fixed in ca. 10% formaldehyde solution. In addition a liver sample from the lobus sinister lateralis of the surviving rats was fixed in Lison-Vokaer glycogen fixative. Then the organs for which a stain is given were embedded in paraffin, and sections of them were stained as follows:
Heart, Aorta thoracalis, Lung, Liver, Spleen, Kidneys, Testes, Epididymides, Prostate, Seminal vesicles, Uterus, Ovaries, Urinary bladder, Stomach, Pars proventricularis, Pars fundica, Pars pylorica, Small intestine, Duodenum, Jejunum, Ileum
Large intestine, Caecum, Colon ascendens, Rectum, Mesenterial vessels, Adrenals, Lymph nodes, Lnn. maxillares ant., Lnn. mesenteriales, Thyroids, Parathyroids, Thymus, Pancreas, Bone, Bone marrow (femur), Trachea, Esophagus, Salivary gland Diaphragm, Thigh muscle, Sciatic nerve, Cerebrum, Cerebellum, Medulla oblongata, Spinal cord (I.lumbalis), Pituitary, Adipose tissue (retroperitoneal), Skin with mammary glands, R. thoracalis dextra and/or R. inguinalis sinistra, Eyes, Tongue, Larynx, Nasal and ethmoidal mucosa Middle ear, inner ear

HISTOPATHOLOGY: Yes
The following weights were determined for the surviving rats which were sacrificed at the scheduled times: Body weight (after exsanguination)
Heart, Liver, Kidneys (together), Spleen, Thymus, Testes or ovaries (together) Prostate or uterus, Adrenals (together), Thyroid glands with parathyroids (together), Pituitary, Brain, Eyes (together)
After removing of the ossa frontalia, the skull with brain, pituitary, and eyes, was fixed in ca. 10 % formaldehyde solution for about 2 weeks. The thyroids were left at the larynx and fixed for about 2 weeks in ca. 10% formaldehyde, also. After fixation brain, pituitary, eyes, and thyroids were dissected and weighed. The relative organ weights were calculated per 100 g body weight.

Statistics:

Body weight gain, food consumption, water consumption, organ weights, the clinico-chemical serum parameters and the haematological parameters haematocrit, haemoglobin and numbers of erythrocytes and leucocytes of the dose groups were compared with those of the control (separately for each sex and time) using the multiple two-sided t-test according to DUNNETT (1955).
Different group sizes or variance non- homogeneity between the dose groups and the control were considered by correcting the critical t-values according to DUNNETT (1964), and in case of variance non-homogeneity by WELCH'S correction of the degrees of freedom (cf. WINER, 1970, pp. 36 - 39).
In cases of very low group sizes or extreme variance non-homogeneity no statistical comparison was performed.
For evaluation of the haematology parameters reticulocytes, eosinophilic, basophilic, juvenile/ bandform neutrophilic granulocytes and the leucocytes (which were not classified further) the procedure of STUCKY and VOLLMAR (1976) (which is based on a linear rank test acc. to KRAUTH, 1971) with the two sided alternative was used.
The parameters segmented neutrophilic granulocytes (SE) and lymphocytes (LY) were evaluated together acc. to UNKELBACH (1980), using the same procedure for the values SE/(SE +LY) .

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

All test material treated rats showed salivation and burrowing in the litter directly after treatment, symptoms which are often seen after administration of substances with bad taste. Additional abdominal or lateral position, followed by reduced spontaneous activity, was only observed in the 1000 mg/kg group. In study weeks 3 and 4 these symptoms were only sporadically seen in males, while in some females they lasted up to the end of the study.

Mortality:

no mortality observed

Description (incidence):

There were no mortalities after treatment with the test item.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Up to 1000 mg/kg there were no significant differences in body weight gain, as compared to the controls, neither in males nor in females.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

In all dose groups the food consumption values were comparable to those of the controls.

Food efficiency:

not examined

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

effects observed, treatment-related

Description (incidence and severity):

In the 1000 mg/kg group water consumption was increased in males only at the end of the study, and moderately increased in the females during the whole course of the study.

Ophthalmological findings:

effects observed, non-treatment-related

Description (incidence and severity):

As a finding, which is characteristic for young rats of this rat strain, rests of the membrana pupillaris persis-tens were seen in treated and untreated rats with the same frequency

Haematological findings:

no effects observed

Description (incidence and severity):

There was no indication of effects of the test material on the red or white blood cell counts.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

In males of the highest dose group (group 7) the serum glucose values were slightly decreased. A significant increase in cholesterol and total protein values was found in all male dose groups (groups 5-7). The females of groups 6 and 7 showed slightly increased ALAT activity. The other serum parameters of the test item treated groups were in all normal, inspite of sporadic significant deviations from the controls - as were the urine parameters - and did not indicate test material induced impairment of physiological functions

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

The increased adrenal weights, seen after the highest dose (1000 mg/kg), were considered as adaptation syndrome. The increased liver weight (also seen only in the highest dose group) is a consequence of increased metabolic activity.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Gross pathology findings were reddish-coloured contents in the intestines and increased size of Beyer's plaques in test substance treated females.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Mamma: Test material effects could be excluded after examination of all groups.
Liver: In rats of the highest dose group reduced glycogen contents was seen.
Intestines: Test material-related hyperplasia of the lymphatic tissues with sporadic calcium depositions was seen in the small intestines. Lymphatic hyperplasia is known to occur in the intestinal tract of untreated rats also. Therefore the finding of one affected animal in the 250 mg/kg group is without toxico-logical relevance (see table 1 in section 'Any other information on results incl. tables').

Histopathological findings: neoplastic:

not examined

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

250 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical biochemistry

clinical signs

histopathology: non-neoplastic

Target system / organ toxicity

Key result

Critical effects observed:

no

Any other information on results incl. tables

Intestines:

Test material-related hyperplasia of the lymphatic tissue with sporacid calcium deposition was seen in the small intestines. The frequecy was as follows

Table 1:

Dose	0 mg/kg		250 mg/kg		500 mg/kg		1000 mg/kg
Sex	m	f	m	f	m	f	m	f
Lymphatic hyperplasia	0	0	0	1	0	4	0	3
Calcification	0	0	0	0	0	1	0	1

Applicant's summary and conclusion

Conclusions:

The NOAEL after oral application was determined to be 250 mg/kg bw/day for males and females after 28 days administration to males and female rats.

Executive summary:

A study according OECD TG 407 was conducted to determine the oral toxicity after repeated administration of the test item to Wistar rats by gavage. The test material was administered as suspension in aqueous 0.5 % Methocel® K4M Premium/Tween 80.
Both substances were given to rats in daily oral doses of 250, 500, and 1000 mg/kg bw/day. Each group consisted of 10 animals (5 m, 5 f). Appearance and behaviour of all rats were checked daily. Body weight and water consumption were determined twice weekly and food consumption once weekly. In weeks 1 and 4 ophthalmological inspections were performed. Haematological examinations and clinico-chemical analyses of serum and urine were carried out in all animals in treatment week 4. At the end of the 4-week treatment period all animals were sacrificed and necropsied. 12 organs were weighed from each animal and a maximum of 38 organs and tissues per animal were subjected to histopathological examination.

There were no mortalities after treatment with the test substance. All rats showed salivation and burrowing in the litter after treatment. Abdominal and lateral position, followed by reduced spontaneous activity, were only seen in the 1000 mg/kg group. There were no significant differences to the control in body weight gain and food consumption up to the highest dose group. But water consumption was increased in the 1000 mg/kg group. The hematological parameters gave no indication of test material effects on the blood picture. The clinico-chemical examinations showed a clear increase of serum cholesterol and total protein values in the males of all three dose groups, and in the highest dose group slightly reduced serum glucose values in addition. The females only showed slightly increased alanine aminotransferase activity from 500 mg/kg on. There were no ophthalmological findings related to the test substance. Gross pathology findings were reddish-coloured contents in the intestines and increased size of Beyer's plaques in test substance treated females. The histopathological finding was lymphatic hyperplasia. As this finding was only seen in one rat of the 250 mg/kg group, 250 mg/kg are considered as still tolerated. No histological correlate to the increased adrenal and liver weights of the 1000 mg/kg dose group was found. The weight alterations are considered to be attributable to an adaptation syndrome and increased metabolie activity.

The NOAEL after oral application was determined to be 250 mg/kg bw/day for males and females after 28 days administration to males and female rats.