2,6-bis(1,1-dimethylethyl)-4-(phenylenemethylene)cyclohexa-2,5-dien-1-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:

1998-07-16 - 1999-01-20

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Guideline study (OECD TG 407) performed under GLP with minor deficiency to current guidelines: Wet weighing of coagulating glands, prostate and seminal vesicles was not performed. Not all recommended tissues/organs were examined histopathologically. However, wet weights of thyroid and ovaries were determined and histopathological endpoints for the detection of endocrine-related effects were included in this reliable study.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1999

Report date:

1999

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)

Version / remarks:

adopted on 27th July 1995

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: Japanese Ministry of Health and Welfare Guidelines (Notification No. 24, Pharmaceutical Affairs Bureau, September 11, 1989, and as amended by Notification No. 88, August 10, 1993) and with Japanese Substance Law (no. 700), MHW (1039) and MITI (1014)

Deviations:

not specified

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

429-460-4

EC Name:

-

Cas Number:

7078-98-0

Molecular formula:

C21 H26 O

IUPAC Name:

2,6-bis(1,1-dimethylethyl)-4-(phenylenemethylene)cyclohexa-2,5-dien-1-one

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, North Carolina, U.S.A.
- Age at study initiation: 6-weeks old
- Weight at study initiation: 170 - 206 g in males; 122 - 164 g in females
- Housing: individually in stainless-steel, hanging, wire-mesh cages (study), two/cage upon receipt
- Diet: Pelleted, certified rodent diet ad libitum
- Water: Tap water ad libitum
- Acclimation period: approximately 2 weeks
- Duration of treatment: 1997-08-05 - 1997-09-02
- Observation period after the end of dosing: 1997-09-03 - 1997-09-22

ENVIRONMENTAL CONDITIONS
- Temperature: 18 – 26 °C
- Humidity: 50 +/- 20 %
- Air changes: > 10 changes/h
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 1998-08-03 To: 1998-09-15

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was weighed on an analytical balance and transferred to a precalibrated beaker. The vehicle, corn oil, was added in small amounts and mixed into a paste. The corn oil was added to the paste until the desired volume was achieved. The formulations were mixed on a magnetic stirrer, sonicated until solution was achieved, subsequently transferred to jars that were suitable for the dosing procedure. Dosing formulations were prepared at least once weekly for the purpose of dosage calculations. Dose concentrations were based on the test material as supplied. All dose preparations were stored at room temperature until dosed. The formulations were stable for up to 11 days at room temperature.

VEHICLE
- Justification for use and choice of vehicle: standard vehicle for studies of this type
- Concentration in vehicle: 1, 3, 10 or 30 mg/mL
- Amount of vehicle: dose factor of 10 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of dose formulations of the pretest and of the test were analysed to check homogeneity, concentration and stability.

Duration of treatment / exposure:

29 days

Frequency of treatment:

once daily

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 10, 30, 100, 300 mg/kg bw/d
Basis:
actual ingested

No. of animals per sex per dose:

- 5 animals/sex/dose level (main study)
- additional 5 animals/sex at 0 and 300 mg/kg bw/d (recovery assessment)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Doses were selected based upon the results of a 7-day gavage range finding study (Dose Selecting Test – Covance Study No. 6623-118).
- Randomization: Computer-generated random method
- Post-exposure recovery period in satellite groups: 14 days

Examinations

Observations and examinations performed and frequency:

MORTALITY AND MORIBUNDITY: Yes
- Time schedule: evaluated twice daily

IN-LIFE OBSERVATIONS: Yes
- Time schedule:
On each day of dosing, each animal was observed approximately 1 hour postdose.
Once before treatment and weekly thereafter, each animal was removed from its cage and examined.

BODY WEIGHT: Yes
- Time schedule for examinations: once prior to treatment, on the first day of treatment, and weekly thereafter.

FOOD CONSUMPTION: Yes
- Time schedule: measured and recorded weekly

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of the treatment period (day 30); additionally at the end of the recovery period (day 44)
- Anaesthetic used for blood collection: No data, blood samples were obtained via the jugular vein
- Animals fasted: Yes, overnight
- How many animals: all animals
- Parameters examined: Red blood cell (erythrocyte) count, hemoglobin, hematocrit, red cell distribution width, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelet count, white blood cell (leukocyte) count, differential blood cell count, blood cell morphology

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of the treatment period (day 30); additionally at the end of the recovery period (day 44)
- Animals fasted: Yes, overnight
- How many animals: all animals
- Parameters examined: glucose, urea nitrogen, creatinine, total protein, albumin, globulin, albumin/globulin ratio, cholesterol, triglycerides, total bilirubin, alkaline phosphatase, alanine aminotransferase, gamma glutamyltransferase, aspartate aminotransferase, calcium, inorganic phosphorus, sodium, potassium, chloride

URINALYSIS: Yes
- Metabolism cages used for collection of urine: yes
- Animals fasted: yes
- Time schedule for collection of urine: at the end of the treatment period
- specimens were obtained during the overnight fast in individual urine collection cages
- Parameters examined: appearance, pH, protein, urobilinogen, glucose, ketones, bilirubin, blood, microscopic examination of sediment

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule: prior to treatment and prior to terminal sacrifice

NEUROBEHAVIOURAL EXAMINATION: Yes
- Expanded Clinical Observations: battery of behavioral tests and observations; performed on all animals during week 4 of treatment and during week 2 of recovery (week 6)
- Hand-Held Observations (e.g. piloerection)
- Open-Field Observations (e.g. locomotor activity)
- Elicited Behaviours, Grip Strength and Nociceptive Reflex

Sacrifice and pathology:

GROSS PATHOLOGY/NECROPSY: Yes
After 29 days of treatment, the first five surviving animals/sex/group were fasted overnight, weighed, anesthetized with sodium pentobarbital, exsanguinated, and necropsied. After at least a 14 day posttreatment recovery period, the last five animals/sex in groups 1 and 4 were sacrificed in the same manner. A necropsy was performed on each animal and all findings were recorded.

The necropsy included examination of the following:
- all orifices
- cervical tissues and organs
- cranial cavity
- external surface of the brain; the external surface of the spinal cord and cut surfaces of the brain and spinal cord were examined whenever tissue trimming was performed
- external surface of the body
- nasal cavity and paranasal sinuses
- thoracic, abdominal, and pelvic cavities and viscera

Organ Weights
At each scheduled sacrifice, the following organs were weighed after careful dissection and trimming of fat and other contiguous tissue:
adrenal (2), ovary (2), liver, heart, spleen, kidney (2), testis with epididymis (2), brain, thymus, thyroid gland (2) with parathyroid

Organ-to-body weight percentages and organ-to brain weight ratios were calculated.

Tissue Preservation
The following tissues from each animal were preserved in 10% neutralbuffered formalin:
adrenal (2), aorta, brain (cerebrum, cerebellum, pons), cecum, colon, duodenum, esophagus, [eye with optic nerve (2)], [femur with bone marrow (articular surface of the distal end)], [Harderian gland], heart, ileum (with Peyer's patches), jejunum, kidney (2), [larynx], lesions, liver, lung, mammary gland (females only), mandibular lymph node, [mandibular salivary gland (2)], mesenteric lymph node, [nasopharynx], ovary (2), pancreas, pituitary gland, prostate gland, rectum, sciatic nerve, [seminal vesicle (2)], [skeletal muscle, thigh], [skin], spleen, spinal cord (cervical, mid-thoracic and lumbar), stomach, sternum with bone marrow, [sublingual salivary gland (2)], testis with epididymis (2), thymus, thyroid (2) with parathyroid, [tongue], trachea, urinary bladder, uterus with cervix and vagina

HISTOPATHOLOGY: Yes

Tissue samples from animals in the control and high-dose group were embedded in paraffin, sectioned, stained with hematoxylin and eosin, and examined microscopically. The slides from tissues in brackets were examined only if indicated by signs of toxicity or target organ involvement. Macroscopic lesions, lungs, liver, and kidney were examined microscopically from each low, mid, and mid-high doses. The liver was identified as a target organ and examined microscopically from recovery-sacrifice animals.

Statistics:

Body weight, food consumption, clinical pathology, and organ weight data were analyzed by analysis of variance techniques. Further exploratory analysis was performed using tests for trend as necessary after a review of the initial analyses. Tests for homogeneity of variances and ANOVA was evaluated at the 5.0 % probability level. Control vs. treated group mean comparisons were evaluated at the 5.0 % twotailed probability level.

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

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry 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:

effects observed, treatment-related

Description (incidence and severity):

transient periportal hepatocellular vacuolation in high dose group

Details on results:

IN-LIFE OBSERVATIONS AND MORTALITY:
Three rats that received 300 mg/kg bw/d (two males, one female) were found dead during the study; these deaths were determined to be gavage-related. In the surviving animals, there were no clinical observations or behavioral changes which were considered test item-related.

BODY WEIGHT:
There were no significant differences in mean body weight values throughout the course of the study.

FOOD CONSUMPTION AND FOOD CONSUMPTION RATIOS:
There were no significant differences in mean food consumption values throughout the course of the study.

HAEMATOLOGY / CLINICAL CHEMISTRY:
Test item-related, toxicologically relevant, changes noted in the clinical pathology data at the time of the terminal sacrifice included slightly elevated alanine aminotransferase activity in rats that received 300 mg/kg bw/d. This value was comparable to the control value after the recovery period.

ORGAN WEIGHTS:
Mean liver weights at terminal sacrifice were significantly elevated in the females that received 100 (liver-to-body weight percentage only) or 300 mg/kg bw/d (with noted increases in the incidence and severity of periportal vacuolation in the livers of the 30, 100, and 300 mg/kg/day females).

GROSS PATHOLOGY:
Observations at necropsy were those noted routinely in rats of this age.

HISTOPATHOLOGY:
The incidence and severity of periportal vacuolation in the livers of the 30, 100, and 300 mg/kg bw/d females were increased. After the recovery period, the incidence of periportal hepatocellular vacuolation in the 300 mg/kg bw/d rats was not remarkably different from that of the control group.

OPHTHALMOSCOPIC EXAMINATION:
None of the observations noted at Day 29 were attributed to test item administration; therefore, examinations were not performed on the recovery animals.

NEUROBEHAVIOURAL EXAMINATION:
None of the observations were consistent with a test item-related effect on neurobehavioral functions. There were no marked differences in the quantitative parameters between the control and treated rats or abnormal observations in the subjective evaluations.

Effect levels

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Based upon the lack of adverse test item-related effects up to and including the high dose level of 300 mg/kg bw/d.

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

DOSE ANALYSIS

Homogeneity analyses (low- and high-dose levels) indicated that the test item was homogeneously mixed. Results of stability analyses indicated that the formulations were stable for 11 days at room temperature. All values were within 4.3 % of initial concentration. Results of routine concentration analyses indicated that all formulations were within 9 % of target.

RANGE-FINDING STUDY (Dose Seleting Test):

- Species: rat

- Number of animals: 5/sex

- Method: oral gavage

- Dose Levels: 0, 10, 100 and 1000 mg/kg bw/d

- Duration of treatment: 7 days

- Testing Facility: Covance Laboratories Inc.

- Study No.: 6623-118