Methyl 2-hexyl-3-oxocyclopentanecarboxylate

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

02 Mar 2000 to 02 Jun 2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2000

Report date:

2000

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

21 September 1998

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

3-oxo-2-pentylcyclopentaneacetate

Cas Number:

24851-98-7

Molecular formula:

C13H22O3

IUPAC Name:

3-oxo-2-pentylcyclopentaneacetate

Test material form:

liquid

Test animals

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Sprague-Dawley - derived (CD); Crl: CD (SD) IGS BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories; Kingston, New York 12484
- Females nulliparous and non-pregnant: yes
- Age at study initiation: approximately 6 weeks
- Weight at study initiation: 180-231 (male) and 149-207 (female)
- Housing: Animals were doubly housed in elevated, stainless steel, wire mesh cages during the first week of the acclimation period and individually housed thereafter.
- Diet: Certified Rodent Diet, No. 5002; (Meal) (pMI Nutrition International, St. Louis, Missouri) was available without restriction. Fresh feed was presented once weekly.
- Water: Water was available without restriction via an automated watering system.
- Acclimation period: 16 days

DETAILS OF FOOD AND WATER QUALITY: Analysis of each feed lot used during this study was performed by the manufacturer. Water analyses are conducted to ensure that water meets standards specified under the EPA Federal Safe Drinking Water Act Regulations (40 CFR Part 141). In addition, water samples are collected biannually from representative rooms in the Testing Facility; chemical and microbiological water analyses are conducted on these samples by a subcontract laboratory. Results of all water analyses are maintained on file at the Testing Facility.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 - 23
- Humidity (%): 40 - 74
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: From: 02 March 2000 To: 02 June 2000

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

DIET PREPARATION
Appropriately identified dose group buckets were used to weigh and store the test diets. The appropriate amount of Certified Rodent Diet No. 5002 was weighed into each bucket. For each dose group/sex, approximately 100 grams of untreated diet from the bucket was added to a mortar. The test article was poured from the transfer container into the mortar. Additional untreated diet from the bucket was added as needed and mixed in the mortar with a pestle until homogenous. The transfer container was rinsed with several grams of untreated diet from the bucket. The rinse was added to the contents of the mortar and mixed with a pestle until homogenous. Approximately half of the untreated diet from the bucket was placed into the bowl of a Hobart mixer. The contents of the mortar were poured onto the layer of diet in the bowl of the Hobart mixer. The mortar and pestle were rinsed with several grams of untreated diet from the bucket and this rinse was added to the bowl of the Hobart mixer. The remaining untreated diet in the bucket was added to the Hobart mixer. Using a paddle blade, the untreated diet and mortar contents were mixed for approximately 15 minutes. Control animals received untreated diet. Dietary concentrations were adjusted weekly based on body weight and feed consumption data from the preceding week.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

ANALYSIS OF DIETS
Prior to initiation of the study, homogeneity of diet batches prepared using the proposed preparation procedure was demonstrated by taking 3 top, 3 middle and 3 bottom samples (9 samples per batch) from diet batches of concentrations that bracket the expected concentrations for the study. Stability under storage conditions to be used in the study (14 days) was determined previously.
HOMOGENEITY
Prior to initiation of the study, batches of low-concentration and high-concentration diets were prepared. Three samples each from the top, middle and bottom portion of each mixture were taken for analysis.
CONFIRMATION ANALYSIS
All dietary levels were assayed weekly for the first month (2 samples per concentration). Subsequent assays were performed at monthly intervals for the remainder of the study.

Duration of treatment / exposure:

90 days

Frequency of treatment:

continuous

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

10 mg/kg bw/day (nominal)

Remarks:

Group 2

Doses / concentrations 2

Dose / conc.:

50 mg/kg bw/day (nominal)

Remarks:

Group 3

Doses / concentrations 3

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

Group 4

No. of animals per sex per dose:

10

Control animals:

yes, plain diet

Details on study design:

Dose selection rationale: Doses were selected by the Sponsor based on a 2-week preliminary range-finding study in rats (HLS Study No. 99-2620).

Examinations

Observations and examinations performed and frequency:

VIABILITY CHECKS
Animals were observed, in their cages, for mortality and general condition, twice daily (once in the morning and once in the afternoon). Animals in extremely poor health or in a possible moribund condition were identified for further monitoring and possible euthanasia.

CLINICAL OBSERVATIONS (CAGE-SIDE)
Observations were made daily (concurrent with one viability check) for any signs of poor health or toxic or pharmacologic effects (e.g., abnormalities in general condition, appearance, activity, behavior, respiration, etc.). Unusual signs were also recorded.

PHYSICAL EXAMINATIONS
Animals were removed from their cages and examined twice pretest and once weekly during the study period. Examinations included observations of general condition, skin and fur, eyes, nose, oral cavity, abdomen and external genitalia, occurrence of secretions and excretions, and autonomic activity (e.g., lacrimation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypy (e.g., excessive grooming, repetitive circling) or bizarre behavior (e.g., selfmutilation, walking backward) were recorded.

BODY WEIGHT
Animals were removed from their cages and weighed twice pretest, weekly during treatment and at termination. Fasted body weights were obtained just prior to necropsy.

FOOD CONSUMPTION
Feed was available without restriction 7 days/week. Animals were presented with full feeders weighing 570 ± 3 grams (includes weight of feed, jar and lid). After 6 days, feeders were reweighed and the resulting weight was subtracted from the full feeder weight to obtain the grams consumed per animal over the 6-day period. Food consumption was measured (weighed) weekly, beginning one week prior to treatment.
Calculation: food consumption (g/kg/day) = (grams of feed consumed ÷ 6 days / body weight (kg)
The average of the current and previous weight was used for calculations except for Day -1 interval (calculated from the current weight only).

TEST ARTICLE INTAKE
Calculated from food consumption data and based on nominal dietary concentrations.
Calculation: milligrams of test article consumed/kilogram of body weight/day (mg/kg/day) = food consumed (g/kg/day) x dietary concentration (mg test article/g of diet).

OPHTHALMOSCOPIC EXAMINATION
Lids, lacrimal apparatus and conjunctiva were examined visually. The cornea, anterior chamber, lens, iris, vitreous humor, retina and optic disc were examined by indirect ophthalmoscopy. Mydriacyl 1% was used to induce mydriasis. Examination Schedule: Pretest: Study Day -10; Termination: Study Day 90.

NEUROBEHAVIORAL STUDIES
Testing was staggered over four sessions with approximately equal numbers of animals per sex per dose group in each session. Noise level in the functional observational battery room was maintained within a level of 55 to 65 decibels by a White Noise Generator, Model MDF 5001. Temperature, humidity, noise level and illumination of each room were measured and recorded to ensure that variations in environmental conditions were minimal during all evaluations.
- Frequency: Pretest and Week 13
- Temperature: Temperature was monitored and recorded twice daily, just prior to the first evaluation and just after the last evaluation.
- Relative Humidity: Relative humidity was monitored and recorded twice daily, just prior to the first evaluation and just after the last evaluation. Excursions outside the specified range were not considered to have affected the integrity of the study.
- Noise Level: Noise level was monitored using a Digital Sound Level Meter, Model 840029 and recorded twice daily, just prior to the first evaluation and just after the last evaluation. Excursions outside the specified range were not considered to have affected the integrity of the study.
- Illumination: Illumination was monitored using a Photometer 1 and recorded twice daily, just prior to the first evaluation and just after the last evaluation.

MOTOR ACTIVITY
- Method: Using a modified version of Schulze's procedures (Schulze, 1990), the locomotor activity of all animals was monitored using an automated Photobeam Activity System (San Diego Instruments, Inc., San Diego, California). Sessions were 60 minutes in length; each session was divided into 12 5-minute intervals. The time of testing was balanced across treatment groups.

FUNCTIONAL OBSERVATIONAL BATTERY
Method: A functional observational battery (Moser, 1989) was performed on all animals. With the exception of pretest, evaluations were performed "blind", i.e., the observer did not know the identity of the animal's dose group. Time of testing was counter-balanced across treatment groups.
The following evaluations were performed as part of the functional observational battery:
- Home Cage Evaluations: posture, vocalization and palpebral closure.
- Handling Evaluations: reactivity to general stimuli (handling); assessment of signs of autonomic function: lacrimation, salivation, altered fur appearance, or red/crusty deposits around eyes.
- Open Field Evaluations: arousal level and gait; count of urination and defecation; convulsions, tremors, abnormal movements or behaviors, excessive or repetitive actions; piloerection and exophthalmos.
- Reflex Assessments: response to visual (approach response) and auditory (finger snap) stimuli; response to a tail pinch; pupillary function.
- Grip Strength: Grip strength was measured using a Grip Strength Meter.
- Landing Foot Splay: A small dot of paint was applied to each animal's hindpaws. Each animal was then dropped onto a flat surface from a height of one foot. The distance between the marks left by the hindpaws was measured in centimeters.
- Hindlimb Extensor Strength: Animals were held in a vertical position facing the observer with a firm grasp around the thorax. The observer placed one finger against the bottom of each hindpaw and pressed towards the animal. Muscular resistance and pressure exerted by the animals were scored.
- Air Righting Ability: Animals were held upside down and dropped from a height of two feet into a container of bedding. The landing position of each animal was recorded.
- Body Weight: Animals were removed from their cages and weighed using a Mettler Balance.

CLINICAL PATHOLOGY
Clinical pathology procedures and parameters are based on those recommended in guidelines published by the Joint Scientific Committee for International Harmonization of Clinical Pathology Testing in "Harmonization of Animal Clinical Pathology Testing in Toxicity and Safety Studies", Fund. Appl. Tax.: 29, 198-201 (1996). Blood was obtained via puncture of the orbital sinus (retrobulbar) under light CO/O2 anesthesia. Animals were fasted overnight prior to blood collection.

HEMATOLOGY
- Method of Blood Collection: Blood for hematology studies (approximately 0.5 mL) was collected into tubes containing EDTA anticoagulant. Blood was obtained from up to 10 animals/sex/group at Termination (Week 14).
- Analysis of Blood Samples: Erythrocyte count, Hematocrit, Hemoglobin, Mean corpuscular volume, Mean corpuscular hemoglobin, Mean corpuscular hemoglobin concentration, Total leukocyte count, Differential leukocyte count, Platelet count, Reticulocyte count, Erythrocyte morphology, Bone marrow differential count.

COAGULATION
- Method of Blood Collection: Blood for coagulation studies (approximately 1.0 mL) was collected into tubes containing sodium citrate anticoagulant. Blood was obtained from up to 10 animals/sex/group at Termination (Week 14).
- Analysis of Blood Samples: Prothrombin time, Activated partial thromboplastin time.

CLINICAL CHEMISTRY
- Method of Blood Collection: Blood for clinical chemistry studies (approximately 1.5 mL) was collected into tubes with no anticoagulant, allowed to clot, and centrifuged to obtain serum. Blood was obtained from up to 10 animals/sex/group at Termination (Week 14).
- Analysis of Blood Samples: Alanine aminotransferase, Aspartate aminotransferase, Alkaline phosphatase, Gamma-glutamyl transpeptidase, Blood urea nitrogen, Creatinine, Glucose, Cholesterol, Triglycerides, Total protein, Albumin, Total bilirubin, Sodium, Potassium, Chloride, Calcium, Inorganic phosphorus, Globulin, (calculated value; total protein - albumin) Albumin/globulin ratio (calculated value; albumin ÷ globulin)

Sacrifice and pathology:

NECROPSY INFORMATION
Necropsy was performed on up to 10 animals/sex/group after animals had been treated for 3 months. Animals were fasted overnight prior to necropsy. A necropsy schedule was established to ensure that approximately equal numbers of males and females were examined on each day of necropsy and that examination of animals of both sexes were performed at similar times of the day throughout the necropsy period. Method of Euthanasia: Exsanguination following carbon dioxide inhalation.

MACROSCOPIC EXAMINATIONS
A complete macroscopic examination was performed on all animals, including animal humane euthanized; all abnormal observations were recorded. The necropsy consisted of an external examination, including identification of all clinically recorded lesions, as well as a detailed internal examination. Animals were fasted prior to scheduled sacrifices.

ORGAN WEIGHTS
Organs indicated below were weighed for all animals at the scheduled sacrifice interval. Prior to weighing, the organs were carefully dissected and properly trimmed to remove adipose and other contiguous tissues in a uniform manner. Organs were weighed as soon as possible after dissection in order to avoid drying. Paired organs were weighed together. Adrenals, brain, epididymides, heart, kidneys, liver, ovaries, pituitary, prostate, spleen, testes, thymus, thyroid/parathyroids, uterus (with cervix).

TISSUES PRESERVED AND EXAMINED HISTOPATHOLOGICALLY
The tissues indicated in Table 1 of 'Any other information on materials and methods incl. tables' were obtained at termination and preserved for all animals. In addition, slides of the indicated tissues were prepared and examined microscopically for all animals in the 0 and 100 mg/kg/day groups. Any abnormalities not noted during macroscopic examinations that were seen during histology processing were recorded.
- Preservatives: Eyes - glutaraldehyde/paraformaldehyde; Testes/epididymides - Bouin's solution; All other tissues - 10% neutral buffered formalin; Note: Lungs and urinary bladder were infused with formalin to ensure fixation.
- Processing: After fixation, the tissues and organs from all animals from the 0 and 100 mg/kg/day groups were routinely processed, embedded in paraffin, cut at a microtome setting of 4-7 microns, mounted on glass slides, stained with hematoxylin and eosin and examined by light microscopy. The bones were decalcified in formic acid. Larynx sections were prepared from two sites; one was the area of the ventral diverticulum and the other was the area of the ventral seromucous glands at the base of the epiglottis.

Statistics:

Mean values of all dose groups were compared to the mean value for the control group at each time interval. Evaluation of equality of group means was made by the appropriate statistical method, followed by a multiple comparison test if needed. Bartlett's test (Bartlett, 1937; Sokal and Rohlf, 1995) was performed to determine if groups had equal variances. For all parameters except organ weights, if the variances were equal, parametric procedures were used; if not, nonparametric procedures were used. Organ weight data was analyzed only by parametric methods. The parametric method was the standard one way analysis of variance (ANOVA) using the F ratio to assess significance (Armitage, 1971; Dunlap and Duffy, 1975). If significant differences among the means were indicated, additional tests were used to determine which means were significantly different from the control: Dunnett's (Dunlap et aI., 1981; Dunnett, 1955, 1964), Williams (Williams, 1971, 1972), or Cochran and Cox's modified t-test (Cochran and Cox, 1959). The nonparametric method was the Kruskal-Wallis test (Kruskal and Wallis, 1952, 1953) and if differences were indicated, Shirley's test (Shirley, 1977), Dunn's test (Dunn, 1964), or Pairwise Comparison with Bonferroni Correction (Games and Howell, 1976) were used to determine which means differed from control. Bartlett's test for equality of variance was conducted at the 1% significance level; all other statistical tests were conducted at the 5% and 1% significance levels.

The following parameters were analysed statistically: mean body weight values; mean food consumption values; mean haematology values; mean coagulation values; mean clinical chemistry values; mean terminal organ weights, organ/body and organ/brain weight ratios; mean motor activity counts; forelimb and hindlimb grip strength measurements; landing foot splay measurements.

For statistical analysis of motor activity, see 'Any other information on materials and methods incl. tables'.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

Physical observations seen during the study were considered common findings in laboratory rats and were not considered to be due to test article administration.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One female administered 0 mg/kg/day of the test substance was sacrificed for humane reasons, due to an injury, during Month 2 of test article administration.
No test article-related mortality was observed during the study.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Body weights and bodyweight gains were comparable between control and test article-treated groups throughout the study.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption values of test article treated groups were comparable to control values during the study.
Test article intake (Table 1 in 'Any other information on results incl. tables'), calculated from food consumption data and based on nominal dietary concentrations and summarized below, confirm that animals received the desired levels.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

Administration of the test substance did not result in any test article-related ocular findings.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No test article-related effects were seen in the haematology parameters examined. Statistically significant differences from controls in mean haematocrit values of treated males were attributed to normal biological variability and fell within historical control data ranges.
No test article-related effects were seen in the coagulation parameters examined.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

No test article-related effects were seen in the clinical chemistry parameters evaluated. Statistically significant differences from control in mean potassium values of all treated males and a statistically significant decrease in mean globulin value of males fed 100 mg/kg day were considered to be due to normal variability since they occurred in only one sex and fell within historical control data ranges.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

MOTOR ACTIVITY
No test article-related effects were evident in the motor activity for either sex in any of the treatment groups.

FUNCTIONAL OBSERVATION BATTERY
Test article administration did not affect the neurological condition of the animals as measured by a functional observational battery of assessments. Landing foot splay and grip strength for all test article treated groups were comparable to control values or within the range of normal variation. No abnormal movements were observed.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Organ weight data of rats fed the test substance were considered comparable to control weights or were within normal variability at termination of the study. The statistically significant difference in mean organ to body weight ratio of kidneys of males administered 100 mg/kg/day was not considered test article-related since it fell within historical control data ranges and there were no associated microscopic findings in the kidney.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no test article-related macroscopic findings. Findings observed occurred with comparable incidence and severity in the control and test article treated groups or they occurred sporadically. These incidental findings have been seen in rats of this strain and age used in other studies conducted in this facility.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

There were no test article-related microscopic findings. Microscopic findings observed occurred with comparable incidence and severity in the 0 and 100 mg/kg/day groups or they occurred sporadically. These incidental findings have been seen in rats of this strain and age used in similar studies conducted in this facility.

Histopathological findings: neoplastic:

no effects observed

Effect levels

Dose descriptor:

NOAEL

Effect level:

>= 100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

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

Analytical results

Analysis of preliminary mixes confirmed that the preparation procedure used for this study produced homogeneous mixtures and that the test article was stable in the diet, under storage conditions used in this study, for at least fourteen days. Analyses conducted during the treatment period confirmed that diets of appropriate concentration were administered. Mean analytical concentrations, expressed as percent of nominal (desired) concentrations were as follows:

GROUP	NOMINAL CONCENTRATION mg/kg/day	ANALYTICAL CONCENTRATION (% of Nominal)
		Males	Females
2	10	105.7	102.2
3	50	105.5	100.3
4	100	103.6	107.2

Table 1. Test article intake, calculated from food consumption data

Group	Desired Dose (mg/kg/day)	Mean Test Article Intake (mg/kg/day)
		Males	Females
2	10	10	10
3	50	50	50
4	100	100	100

Applicant's summary and conclusion

Conclusions:

Administration of dietary concentrations of 10, 50 and 100 mg/kg/day of the test substance to Sprague-Dawley rats for up to 90 days did not result in any adverse, toxicological effects. The NOAEL (No Observable Adverse Effect Level) was 100 mg/kg/day.

Executive summary:

This study was designed to assess the potential toxicity of the test substance when administered orally, via dietary admixture, to Sprague-Dawley CD® rats (10/sex/group) at dose levels of 10, 50, and 100 mg/kg/day for a period of at least 3 months in accordance with OECD TG 408 and in compliance with GLP. Control animals (10/sex) received untreated standard laboratory diet. Clinical observations and body weight measurements were performed on all animals pretest and weekly during the study period. Ophthalmoscopic examinations were conducted on all animals pretest and at termination of the dosing period. Food consumption was evaluated weekly beginning one week prior to initiation of dosing. Test substance intake measurements were performed weekly beginning one week after initiation of dosing. Motor activity, functional observational battery and ophthalmology observations were performed pretest and during the twelfth week of dosing. After 3 months of treatment, haematology, coagulation and clinical chemistry evaluations were performed, surviving animals were euthanatized, selected organs were weighed and organ/body weight and organ/brain weight ratios calculated. Complete macroscopic postmortem examinations were performed on all animals, and histopathological evaluation of all tissues was conducted for animals in the 0 and 100 mg/kg/day groups.

Administration of dietary concentrations of 10, 50 and 100 mg/kg/day of the test substance to Sprague-Dawley rats for up to 90 days did not result in any adverse toxicological effects.

Therefore, the NOAEL (No-Observable-Adverse-Effect-Level) was 100 mg/kg/day.