1,1'-(1,1-dimethyl-3-methylene-1,3-propanediyl)bisbenzene

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study performed according to GLP and guideline

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

1,1'-(1,1-dimethyl-3-methylene-1,3-propanediyl)bisbenzene

EC Number:

228-846-8

EC Name:

1,1'-(1,1-dimethyl-3-methylene-1,3-propanediyl)bisbenzene

Cas Number:

6362-80-7

Molecular formula:

C18H20

IUPAC Name:

(4-methyl-4-phenylpent-1-en-2-yl)benzene

Details on test material:

- Name of test material (as cited in study report): , 2,4-diphenyl-4-methyl-1-pentene [English name: 1,1’-(1,1-dimethyl-3-methylene-1,3-propanediyl)bisbenzene
- Analytical purity: 96.97%
- Impurities (identity and concentrations): 2,4-Diphenyl-4-methyl-2-pentene 2.54%)
- Storage condition of test material: Stored in a cabinet in the test substance storage room of the testing facility at room temperature (nominal value: 23°C, measured values 18.9 - 25.9°C) and protected from light.

Test animals

Species:

rat

Strain:

Crj: CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories Japan (Hino Breeding Center)
- Age at study initiation: 7 weeks of age (upon receipt)
- Weight at study initiation: was 237-271 g in males, 154-184 g in test group females and 168-179 g in recovery group females (upon receipt)
- Diet: Free access to pellet diet (CRF-1, Oriental Yeast Co., Ltd.)
- Water: Free access to tap water in water bottles
- Acclimation period: A five day quarantine period was followed by a 16 day acclimation period (17 days for the recovery group females

ENVIRONMENTAL CONDITIONS
- Temperature: Nominal temperature of 23°C (measured values: 22-24°C)
- Humidity: Nominal humidity of 55% (measured values: 40-60%)
- Air changes: 12 times/hour (filter-sterilized fresh air).
- Photoperiod: 12-hour light/dark cycle (illumination: 6:00 am to 6:00 pm)

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The required amount of 2,4-diphenyl-4-methyl-1-pentene was weighed (electronic balance PM2500, Mettler-Toledo) and dissolved in corn oil to a concentration of 144 mg/mL. Dosing preparations at 36 and 9 mg/mL were prepared by serial dilution of the 144 mg/mL solution. Correction for the content of test substance was made at preparation


VEHICLE
- Amount of vehicle: 5 mg/mL
- Lot/batch no: Lot No. V4M3466 (expiration date: October 4, 2009), Lot No. V5F6057 (expiration date: May 29, 2010) and Lot No. V5P7775 (expiration date: October 25, 2010), Nacalai Tesque Inc

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Test substance concentrations in the dosing preparations used on the starting day of administration to males and females and on the final day of administration to males were determined by gas chromatography (GC-14B, Shimadzu Corp.). The results showed that the test substance content was 91.6-100.7% of the nominal concentration, which was within the specified range (100±10%). Therefore, the concentration of each dosing preparation was acceptable

Duration of treatment / exposure:

The administration period was set based on the OECD Guideline for Testing of Chemicals for Combined Repeat Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test (422). Administration to males was for 14 days before the mating period and 28 days thereafter, a total of 42 days. Administration to test group females was for 14 days before the mating period, during the mating period and gestation period, and until day 6 of lactation, a total of 44 to 56 days. Half the animals in each group of males were assigned to a 14-day recovery period following the 42-day administration period. For the recovery group females, a 14-day recovery period followed a 42-day administration period. The starting day of administration was day 1 of administration, and the day following the final administration was day 1 of recovery.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Vehicle control

Doses / concentrations 2

Dose / conc.:

45 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

180 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

720 mg/kg bw/day (nominal)

No. of animals per sex per dose:

The number of animals in each test group was 12 males and 12 females, of which half the males in each group belonged to a recovery group. Recovery groups of 6 females separate from the test group were provided for the control, 180 and 720 mg/kg groups.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
Dose levels were selected from the results of a preliminary combined repeat dose toxicity study with reproduction/developmental toxicity screening test by oral administration of 2,4-diphenyl-4-methyl-1-pentene to rats (dose levels: 0, 250, 500 and 1000 mg/kg, 5 males per group).3) In the 1000 mg/kg group, 1 animal died and decrease in body weight was observed. However, no abnormalities in clinical signs, body weight, food consumption or necropsy findings were observed in the 500 mg/kg group. Therefore, in this study an intermediate dose between 1000 mg/kg and 500 mg/kg of 720 mg/kg was selected as the high dose, and lower doses were set at 180 and 45 mg/kg in a common ratio of 4. The control group received the vehicle (corn oil) at the same volume.

Examinations

Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Males were observed for clinical signs and mortality twice daily during the administration period, before and after administration, once daily during the recovery period, and once on the day of necropsy. Females were observed for clinical signs and mortality twice daily during the administration period, before and after administration, and once on the day of necropsy. Recovery group females were observed for clinical signs and mortality twice daily during the administration period, before and after administration, once daily during the recovery period, and once on the day of necropsy.

BODY WEIGHT: Yes
- Time schedule for examinations: Bodyweight was measured twice a week

FOOD CONSUMPTION:
Food consumption was measured twice a week for 14 days before the start of the mating period and from after the end of the mating period

HAEMATOLOGY: Yes
- Time schedule for collection of blood: On the day after the final administration and after the end of the recovery period
- Anaesthetic used for blood collection: Yes (sodium pentobarbital)
-Parameters examined: Red blood cell count (RBC), hemoglobin content (HGB), hematocrit value (HCT), platelet count (PLT) and white blood cell count (WBC) for blood treated with EDTA-2K were measured using an automated hematology analyzer (Sysmex K-4500, Sysmex Corporation). Mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC) were also calculated.

For calculation of reticulocyte ratio (RET), blood treated with EDTA-2K was stained supravitally by the Brecher method and smeared on a glass slide, then a Giemsa-stained specimen was prepared and the number of reticulocytes in 1000 red blood cells was counted under a microscope.

For differential blood count, blood treated with EDTA-2K was smeared on a glass slide, a May-Giemsa-stained specimen was prepared, and 100 white blood cells were classified under a microscope.

For prothrombin time (PT), activated partial thromboplastin time (ATT) and fibrinogen concentration (Fbg), blood was treated with 3.2% sodium citrate, centrifuged [about 4°C, 3000 rpm (about 1972×g), 15 min, centrifuge: CF8DL, Hitachi Koki Co., Ltd.], and the plasma obtained was used for measurement by light scattering detection with a fully automated coagulation analyzer (CA-530, Sysmex Corporation).


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: On the day after the final administration and after the end of the recovery period
- Animals fasted: No data
- Parameters examined. AST was measured by MDH-UV method, ALT by LDH-UV method, ALP by p-nitrophenal phosphate substrate method, γ-GTP by L-γ-glutamyl-3-carboxy-4-nitroanilide substrate method, total protein (TP) by Biuret method, total bilirubin (T-Bil) by diazo method, urea nitrogen (UN) by urease-GIDH method, creatinine (CRE) by creatinase F-DAOS method, Glucose (Glu) by hexokinase G-6-PDH method, Total cholesterol (T-Cho) by COD-HDAOS method, triglyceride (TG) by GPO-HDAOS method, calcium by o-CPC method, inorganic phosphorus (IP) by PNP-XDH method, and sodium, potassium and chlorine by ion selective electrode method. All measurements were done using an automated chemistry analyzer (AU 400, Olympus Corp.).
Albumin (Alb) was calculated from total protein and the protein fraction (electrophoresis method, automated electrophoresis system (AES 310, Olympus Corp.) and A/G (albumin/globulin) from the protein fractions.


URINALYSIS: Yes
- Time schedule for collection of urine: Prior to dosing before the end of the administration period (day 37 of administration) for animals necropsied at the end of the administration period, and before the end of the recovery period (day 9 of recovery) for animals necropsied at the end of the recovery period
- Metabolism cages used for collection of urine: No data
- Animals fasted: Yes
- Parameters examined: Fresh urine: Color was examined by appearance. Examination of pH, protein, glucose, ketone body, bilirubin, occult blood and urobilinogen was done by reflectance spectrophotometry using an automated urine analyzer (US-2100, Eiken Chemical Co., Ltd.) after dropping urine on urinalysis test paper (Eiken Chemical Co., Ltd.). Urinary sediments were examined under a microscope after staining with urine sediment stain (New Sternheimer method, International Reagents Corporation).

24-hour urine: Urine volume (UV) was calculated from urine specific gravity and weight (electronic balance: PG2002-S, Mettler-Toledo). Specific gravity (SG) was measured from the refractive index using a specific gravity meter (Uripet-II D, Nikon Corporation).


NEUROBEHAVIOURAL EXAMINATION: Yes

Sensory response: Animals necropsied at the end of the administration period were examined for pupillary reflex, approaching behavior, tactile reflex, auditory reflex and pain reflex after the functional observational battery (FOB) on day 41 of administration.

Grip strength: Animals necropsied at the end of the administration period were examined 5 times for grip strength of the forelimbs and hindlimbs using a CPU gauge (Aikoh Engineering Co., Ltd.) after test of sensory response on day 41 of administration, and the mean of 3 measurements excluding the highest and lowest values was adopted.

Motor activity: Animals necropsied at the end of the administration were examined on day 40 of administration for ambulation and rearing, measured in 10-minute intervals for 1 hour after administration, using an Activity Monitor (MED Associates Inc.).


OTHER:
All animals were observed for the following items 1) - 3) before the start of administration and on days 7, 14, 21, 28, 35 and 41 of administration. Observation time was 1 hour after administration. The observer was usually the same and blind method was applied.

1) Animals were observed in the cage for posture, biting behavior, palpebral closure and convulsions.

2) Ease of removal from cage, each of handling, muscle tone, fur condition, lacrimation, salivation and respiration were observed holding the animal on the palm.

3) Frequency of rearing, frequency of grooming, gait, palpebral closure, consciousness, behavioral abnormalities and righting reflex were observed in the open-field test.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Males: The brain (cerebrum, cerebellum, medulla oblongata), pituitary, thyroids, thymus, heart, liver, spleen, kidneys, adrenals, testes and epididymis were weighed (electronic balance: AB204, Mettler-Toledo). The relative weight of each organ was calculated by dividing its weight by the final body weight. The pituitary and thyroids were weighed after being fixed overnight in 20 vol% neutral buffered formalin. These organs and the lungs, trachea, pancreas, salivary glands (sublingual and submandibular), esophagus, stomach, duodenum, jejunum, ileum, cecum, colon, rectum, lymph nodes (submandibular and mesenteric), urinary bladder, seminal vesicles, prostate, parathyroid, spinal cord, sciatic nerve, eyeballs, Harderian gland, sternum and femur were fixed in 20 vol% neutral buffered formalin. However, the testes and epididymis were fixed in Bouin’s solution for 2-3 hours, then re-fixed in 90 vol% ethanol, and eyeballs were fixed overnight in glutaraldehyde/formalin, then re-fixed in 20 vol% neutral buffered formalin.

Females: Were sacrificed by exsanguination and necropsied after determining the numbers of corpora lutea and implantations. In each group, the animals in which urinary, hematological and blood chemical examinations were not conducted were sacrificed by exsanguination from the abdominal aorta under anesthesia by intraperitoneal administration of sodium pentobarbital (40 mg/kg), and necropsied after determining the numbers of corpora lutea and implantations. The brain (cerebrum, cerebellum, medulla oblongata), pituitary, thyroids, thymus, heart, liver, spleen, kidneys, adrenals, ovaries and uterus were weighed (electronic balance: AB204, Mettler-Toledo). The relative weight of each organ was calculated by dividing its weight by the final body weight. The pituitary and thyroids were weighed after being fixed overnight in 20 vol% neutral buffered formalin. These organs and the lungs, trachea, pancreas, salivary glands (sublingual and submandibular), esophagus, stomach, duodenum, jejunum, ileum, cecum, colon, rectum, lymph nodes (submandibular and mesenteric), urinary bladder, vagina, parathyroid, spinal cord, sciatic nerve, eyeballs, Harderian gland, sternum, femur and mammary glands were fixed in 20 vol% neutral buffered formalin. However, the eyeballs were first fixed overnight in glutaraldehyde/formalin, then re-fixed in 20 vol% neutral buffered formalin.

The dam which did not deliver by 10:00 on day 25 of pregnancy was necropsied after exsanguination from the abdominal aorta under anesthesia by intraperitoneal administration of sodium pentobarbital (40 mg/kg), and pregnancy status was confirmed by the presence or absence of implantations. The organs and tissues listed above were fixed in 20 vol% neutral buffered formalin and preserved in 10 vol% neutral buffered formalin. However, the eyeballs were first fixed overnight in glutaraldehyde/formalin, then re-fixed in 20 vol% neutral buffered formalin.





HISTOPATHOLOGY: Yes
Males: For animals necropsied at the end of the administration period, paraffin-embedded specimens of the heart, lung, trachea, liver, pancreas, salivary glands (sublingual and submandibular), esophagus, stomach, duodenum, jejunum, ileum, cecum, rectum, colon, thymus, spleen, lymph nodes (submandibular and mesenteric), kidney, urinary bladder, testis, epididymis, seminal vesicle, prostate, pituitary, adrenal, thyroid, parathyroid, brain (cerebrum, cerebellum, medulla oblongata), spinal cord, sciatic nerve, eyeball, Harderian gland, bone marrow (sternum and femur) and bone (sternum and femur) were prepared. The excised organs and tissues were preserved in 10 vol% neutral buffered formalin. For the 720 mg/kg group and control group, HE-stained tissue specimens were prepared and examined histopathologically. PAS-hematoxylin-stained tissue specimens of testes were prepared. HE-stained tissue specimens of the liver and thyroid, which showed difference between the 720 mg/kg group and control group in the number of animals with abnormality, were also prepared for the 180 and 45 mg/kg groups and examined histopathologically. HE-stained specimens of the kidney, in which effects of test substance administration were suspected in the 720 mg/kg group, were also prepared for the 180 and 45 mg/kg group and examined histopathologically. PAS-hematoxylin-stained tissue specimens of the kidneys of all animals were also prepared and examined histopathologically.

Females: For 6 animals with the lowest animal numbers in each group and the animals that died in the 720 mg/kg group, paraffin-embedded specimens of the heart, lung, trachea, liver, pancreas, salivary glands (sublingual and submandibular), esophagus, stomach, duodenum, jejunum, ileum, cecum, rectum, colon, thymus, spleen, lymph nodes (submandibular and mesenteric), kidney, urinary bladder, ovary, uterus, vagina, pituitary, adrenal, thyroid, parathyroid, brain (cerebrum, cerebellum, medulla oblongata), spinal cord, sciatic nerve, eyeball, Harderian gland, bone marrow (sternum and femur), bone (sternum and femur) and mammary gland were prepared. The excised organs and tissues were preserved in 10 vol% neutral buffered formalin. Then for 6 animals with the lowest animal numbers in the 720 mg/kg group and control group and the animals that died in the 720 mg/kg group, HE-stained tissue specimens were prepared and examined histopathologically. HE-stained tissue specimens of the liver, kidney and thyroid, which showed difference between the 720 mg/kg group and control group in the number of animals with abnormality, were also prepared for 6 animals with the lowest animal numbers in 180 and 45 mg/kg groups and examined histopathologically. PAS-hematoxylin-stained tissue specimens of the kidneys of 6 animals with the lowest animal numbers in each group were also prepared and examined histopathologically

Statistics:

The significance of differences between the control group and each administration group was tested as follows, with a hazard ratio of 5% and indicated as less than 5% (p<0.05) or less than 1% (p<0.01). Clinical signs, body weight, food consumption and functional observational battery (FOB) of infertile dams were excluded after mating.
Mean and standard deviation in each group were calculated for body weight (parent animals, pups), food consumption, grooming and rearing counts in functional observational battery (FOB), grip strength, motor activity, urine volume, urine specific gravity, hematological test results, blood chemical test results, absolute and relative organ weight.
Equality of variance was tested by Bartlett’s test, and Dunnett’s test was used if equality of variance was observed. However, if equality of variance was not observed, a Duunett-type rank test was used.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

effects observed, treatment-related

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Details on results:

CLINICAL SIGNS:

Males

There were no deaths or moribundity in any group.

During the administration period, transient salivation immediately after administration was observed in the 720 mg/kg group in all 12 animals, and soiled hair was observed in 1 animal. Transient salivation immediately after administration was observed in the 180 mg/kg group in all 12 animals. No abnormalities in clinical signs were observed in the 45 mg/kg group or control group.

During the recovery period, no abnormalities in clinical signs were observed in any group.

Test Group Females:

In the 720 mg/kg group, 1 animal (No.F04457) died on day 9 of administration. There were no deaths or moribundityin the 180 and 45 mg/kg groups or in the control group.

The animal that died in the 720 mg/kg group (No.F04457) showed transient salivation immediately after administration on days 2-7 of administration, soiled hair on days 4-8 of administration, hypothermia on days 5-8 of administration and decrease in locomotor activity on day 8 of administration.

Before the start of mating and during the mating period, among survivors in the 720 mg/kg group, transient salivation immediately after administration was observed in all 11 animals and soiled hair in 3 animals. Transient salivation immediately after administration was observed in the 180 mg/kg group in all 12 animals. No abnormalities in clinical signs were observed in the 45 mg/kg group or control group.

During the gestation period, transient salivation immediately after administration was observed in the 720 mg/kg group in 10 animals, and soiled hair was observed in 1 animal. Transient salivation immediately after administration was observed in the 180 mg/kg group in 9 animals. No abnormalities in clinical signs were observed in the 45 mg/kg group or control group.

During the lactation period, transient salivation immediately after administration was observed in the 720 mg/kg group in all 10 animals. Transient salivation immediately after administration was observed in the 180 mg/kg group in 4 animals. No abnormalities in clinical signs were observed in the 45 mg/kg group or control group.

Recovery Group Females:

In the 720 mg/kg group, 1 animal (No.F04476) died on day 14 of administration. There were no deaths or moribundity in the 180 group or in the control group.

The animal that died in the 720 mg/kg group (No.F04476) showed transient salivation immediately after administration on days 3, 5, 6 and 8-10 of administration, soiled hair on days 2 and 11-13 of administration, and decrease in locomotor activity, soiled perinasal area and perioral smudge on day 13 of administration.

During the administration period, transient salivation immediately after administration was observed in the 720 mg/kg group in all 5 animals, and soiled hair was observed in 1 animal. Transient salivation immediately after administration was observed in the 180 mg/kg group in 5 animals. No abnormalities in clinical signs were observed in the control group.

During the recovery period, no abnormal clinical signs were observed in any group.


BODY WEIGHT:

Males:
During the administration period, significantly low body weight was seen in the 720 mg/kg group compared to the control group on days 4-42 of administration. The 180 and 45 mg/kg groups showed no significant difference in body weight from the control group on any measurement day.

During the recovery period, significantly low body weight was seen in the 720 mg/kg group compared to the control group on day 4 of recovery. The 180 and 45 mg/kg groups showed no significant difference from the control group on any measurement day.

Test Group Females:
Before the start of mating and during the mating period, significantly low body weight was seen in the 720 mg.kg group compared to the control group on day 4 of administration. The 180 and 45 mg/kg groups showed no significant difference in body weight from the control group on any measurement day.

During the gestation period, significantly low body weight was seen in the 720 mg.kg group compared to the control group on days 0, 14 and 21 of pregnancy. The 180 and 45 mg/kg groups showed no significant difference in body weight from the control group on any measurement day.

During the lactation period, there was no significant difference in body weight between any administration group and the control group on any measurement day.

Recovery Group Females:
During the administration period, there was no significant difference in body weight between the 720 mg/kg group and the control group on any measurement day. In the 180 mg/kg group, significantly low body weight compared to the control group was seen on day 36 of administration, but it was not considered to be an effect of administration because the change lacked dose relation.

During the recovery period, there was no significant difference in body weight between the 720 mg/kg group and the control group on any measurement day. In the 180 mg/kg group, significantly low body weight compared to the control group was seen on day 4 of recovery, but it was not considered to be an effect of administration because the change lacked dose relation.

FOOD COMSUMPTION:

Males:
During the administration period, significantly high food consumption was seen in the 720 mg/kg group compared to the control group on days 33, 37 and 40 of administration, but it was not considered to be a toxic effect since it was not associated with body weight changes. The 180 and 45 mg/kg groups showed no significant difference in food consumption from the control group on any measurement day.

During the recovery period, significantly high food consumption was seen in the 720 mg/kg group compared to the control group on days 2 and 12 of recovery, but it was not considered to be a toxic effect since it was not associated with body weight changes. The 180 and 45 mg/kg groups showed no significant difference in food consumption from the control group on any measurement day.

Test Group Females:
Before the start of mating, no significant difference in food consumption from the control group was observed in any administration group on any measurement day.

During the gestation period, significantly high food consumption was seen in the 720 mg/kg group compared to the control group on days 2 of pregnancy, but it was not considered to be a toxic effect because it was a transient change not associated with body weight change. The 180 and 45 mg/kg groups showed no significant difference in food consumption from the control group on any measurement day.

During the lactation period, there was no significant difference in food consumption between any administration group and the control group on any measurement day.

Recovery Group Females:
During the administration period, significantly high food consumption was seen in the 720 mg/kg group compared to the control group on days 19, 23 and 26 of administration, but it was not considered to be a toxic effect since it was not associated with body weight changes. In the 180 mg/kg group, significantly low food consumption compared to the control group was seen on day 9 of administration, but it was not considered to be an effect of administration because the change lacked dose relation.

During the recovery period, there was no significant difference in food consumption between any administration group and the control group on any measurement day.

FUNCTIONAL OBSERVATION BATTERY:

Males:
Salivation was observed in the 720 and 180 mg/kg groups, and the incidence was significantly higher than in the control group on days 7, 14, 21, 28, 35 and 41 of administration in the 720 mg/kg group and on days 21 and 41 of administration in the 180 mg/kg group, but it was considered to be the continuation of transient salivation immediately after administration. In the 720 mg/kg group, a significantly low value in the frequency of rearing compared to the control group was observed on day 7 of administration, but it was not considered to be a toxic change because it was a transient change and no abnormalities were observed in other observation items. In the 45 mg/kg group, no abnormalities were observed in any item on any measurement day.

Test Group Females:
Salivation was observed in the 720 and 180 mg/kg groups, and the incidence was significantly higher in the 720 mg/kg group than in the control group on days 8 and 15 of administration, on days 1, 8 and 15 of pregnancy and on day 3 of lactation, but it was considered to be the continuation of transient salivation immediately after administration. In the 720 mg/kg group, a significantly high value for fur condition compared to the control group was observed on day 8 of administration, but it was considered to be a continuation of the soiled hair observed before administration. In the 180 and 45 mg/kg groups, a significantly low value for frequency of grooming compared to the control group was observed on day 3 of lactation, but it was not considered to be due to administration because the change lacked dose relation.

Recovery Group Females:
Salivation was observed in the 720 and 180 mg/kg groups, and the incidence was significantly higher than in the control group on days 8, 15, 29 and 36 of administration in the 720 mg/kg group and on day 29 of administration in the 180 mg/kg group, but it was considered to be the continuation of transient salivation immediately after administration.

SENSORY RESPONSE:

Males:
No abnormalities were observed in any item in any administration group.

Test Group Females:
No abnormalities were observed in any item in any administration group.

GRIP STRENGTH:

Males:
No significant differences in grip strength of forelimbs or hindlimbs were observed in any administration group compared to the control group.

Test Group Females:
No significant differences in grip strength of forelimbs or hindlimbs were observed in any administration group compared to the control group.

MOTOR ACTIVITY:

Males:
No significant difference from the control group was observed in any measurement item in any administration group.

Test Group Females:
No significant difference from the control group was observed in any measurement item in any administration group.

URINARY EXAMINATION:

Before the End of the Administration Period

Males:
In the 720 mg/kg group, significantly high urine volume and significantly low specific gravity compared to the control group were observed. In the 180 and 45 mg/kg groups, no significant differences from the control group were observed in urine volume or specific gravity.

Color, pH, protein, glucose, ketone body, bilirubin, occult blood, urobilinogen and sediments were comparable with the control group in each administration group.

Test Group Females:
In the 720 mg/kg group, although there were no significant differences from the control group, there was a tendency toward high urine volume. Urine volume was markedly high in 1 animal in the 720 mg/kg group (No.F04452). In the 180 and 45 mg/kg groups, no significant differences from the control group were observed in urine volume or specific gravity.

Color, pH, protein, glucose, ketone body, bilirubin, occult blood, urobilinogen and sediments were comparable with the control group in each administration group.

Before the End of the Recovery Period

Males:
No significant differences from the control group were observed in urine volume or specific gravity in any administration group.

Color, pH, protein, glucose, ketone body, bilirubin, occult blood, urobilinogen and sediments were comparable with the control group in each administration group.

Recovery Group Females:
No significant differences from the control group were observed in urine volume or specific gravity in any administration group.

Color, pH, protein, glucose, ketone body, bilirubin, occult blood, urobilinogen and sediments were comparable with the control group in each administration group.

HEMATOLOGICAL EXAMINATION:

End of the Administration Period

Males:
In the 720 mg/kg group, significantly high values for prothrombin time, activated partial thromboplastin time and fibrinogen concentration compared to the control group were observed. In the 180 mg/kg group, significantly high values for prothrombin time and activated partial thromboplastin time compared to the control group were observed. In the 45 mg/kg group, there were no significant differences from the control group in any measurement item.

Test Group Females:
In the 720 mg/kg group, significantly low values for red blood cell count and hematocrit and significantly high values for activated partial thromboplastin time compared to the control group were observed. In addition, significantly low values for prothrombin time compared to the control group were observed in 720, 180 and 45 mg/kg group, but it was judged not to be a change due to administration because the difference from the control group was slight, and it was close to the value of the background data of this facility (PT: 14.0±0.8 sec). In the 180 mg/kg group, significantly high white blood cell count compared to the control group were observed, but it was not considered to be an effect of administration because it lacked dose relation.

End of the Recovery Period

Males:
No significant difference from the control group was observed in any measurement item in any administration group.

Recovery Group Females:
No significant difference from the control group was observed in any measurement item in any administration group.

BLOOD CHEMICAL EXAMINATION:

End of the Administration Period

Males:
In the 720 mg/kg group, significantly high values for γ-GTP, total protein, albumin, A/G, total bilirubin, total cholesterol and calcium and a significantly low value for chlorine were observed compared to the control group. In the 180 mg/kg group, a significantly high value for calcium was observed compared to the control group. In addition, significantly low value for AST was observed in the 720 mg/kg group compared to the control group, but it was not considered to be a toxic change because it was judged to be due to a high value in 1 control animal (No. M01101). In the 180 mg/kg group, a significantly low value for potassium compared to the control group was observed, but it was not considered to be an effect of administration because it lacked dose relation. In the 45 mg/kg group, no significant difference from the control group was observed in any measurement item.

Test Group Females:
In the 720 mg/kg group, a significantly low value for glucose and significantly high values for γ-GTP, total protein and total bilirubin were observed compared to the control group. In the 180 mg/kg group, a significantly low value for total protein compared to the control group was observed. Otherwise, significantly low values for A/G and chlorine were observed in the 180 mg/kg group compared to the control group, but they were not considered to be effects of administration because they lacked dose relation. In the 45 mg/kg group, no significant difference from the control group was observed in any measurement item.

End of the Recovery Period

Males:
In the 720 mg/kg group, a significantly high value for γ-GTP compared to the control group was observed. No significant difference from the control group was observed in any measurement item in the 180 or 45 mg/kg groups.

Recovery Group Females:
In the 720 mg/kg group, a significantly low value for chlorine and significantly high value for total cholesterol compared to the control group were observed, but they were judged not to be due to administration because they were close to the value of the background data of this facility (chlorine: 106.2±1.9 mEq/L, T-Cho: 74.8±13.3 mg/dL), and such trends were not observed at the end of the administration period. In the 180 mg/kg group, no significant difference from the control group was observed in any measurement item.

NECROPSY:

Animals that Died

Test Group Females:
Atrophy of the thymus and spleen was observed in the animal that died in the 720 mg/kg group.

Recovery Group Females:
Atrophy of the thymus and enlargement of the adrenals were observed in the animal that died in the 720 mg/kg group.

End of the Administration Period:

Males:
No abnormalities were observed in any group.

Test Group Females:
No abnormalities were observed in any group.

End of the Recovery Period

Males:
Adhesion of the liver was observed in 1 animal in the 45 mg/kg group, but it was judged to be an incidental change because it lacked dose relation. There were no abnormalities in the 720 and 180 mg/kg groups, or in the control group.

Recovery Group Females:
No abnormalities were observed in any group.


ORGAN WEIGHT:

End of the Administration Period

Males:
Body weight on the day of necropsy was significantly low in the 720 mg/kg group compared to the control group. In the 180 and 45 mg/kg groups, there was no significant difference from the control group in body weight on the day of necropsy.

In organ weight of the 720 mg/kg group, significantly high absolute and relative weights of the thyroids, liver and kidneys, significantly high relative weight of the testes, and tendency toward high absolute weight of the testes without statistical significance were observed compared to the control group. In the 180 mg/kg group, significantly high absolute and relative weights of the liver and kidneys were observed compared to the control group. In the 45 mg/kg group, significantly high absolute and relative weights of the liver were observed compared to the control group. In addition, significantly high relative weight of the brain was observed in the 720 mg/kg group compared to the control group, but it was considered to be due to the difference in body weight from the control group because there was no significant difference in absolute weight.

Test Group Females:
There was no significant difference in body weight on the day of necropsy between any dose group and the control group.

In organ weight of the 720 and 180 mg/kg groups, significantly high absolute and relative weights of the liver were observed compared to the control group. In the 720 mg/kg group, significantly high relative weight of the thyroids and tendency toward high absolute weight of the thyroids without statistical significance were observed compared to the control group. In addition, significantly low absolute weight of the ovaries was observed in the 720 mg/kg group compared to the control group, but it was not considered an effect of administration since there was no significant difference in relative weight. In the 45 mg/kg group, there were no significant differences in absolute or relative weight of each organ compared to the control group.

End of the Recovery Period

Males:
There was no significant difference in body weight on the day of necropsy between any dose group and the control group.

In organ weight of the 720 mg/kg group, significantly high absolute and relative weights of the thyroids, significantly high relative weight of the liver and kidneys, and tendency toward high absolute weight of the liver and kidneys without statistical significance were observed compared to the control group. In the 180 mg/kg group, there were no significant differences in absolute or relative weight of each organ compared to the control group. In addition, significantly high absolute and relative weight of the thyroids were observed in the 45 mg/kg group compared to the control group, but it was judged not to be due administration because there was no significant difference in the 180 mg/kg group, and this tendency was not seen at the end of the administration period.

Recovery Group Females:
There was no significant difference in body weight on the day of necropsy between any dose group and the control group.

In organ weight of the 720 mg/kg group, significantly high relative weights of the liver and kidneys, and tendency toward high absolute weights of the liver and kidneys without statistical significance were observed compared to the control group. In the 180 mg/kg group, significantly high relative weight of the pituitary was observed compared to the control group, but it was judged not to be due administration because there was no significant difference in the 720 mg/kg group, and this tendency was not seen at the end of the administration period.

HISTOPATHOLOGICAL EXAMINATION:

Animals that Died

Female in the 720 mg/kg Test Group:
Liver: Swelling of centrilobular hepatocytes was observed, and the grade was mild.
Mild postmortal changes were observed in all tissues, but no other findings were confirmed.

Female in the 720 mg/kg Recovery Group:
Liver: Swelling of centrilobular hepatocytes was observed, and the grade was mild.
Mild postmortal changes were observed in all tissues, but no other findings were confirmed.

End of the Administration Period

Males:
Liver: Swelling of centrilobular hepatocytes was observed in all 6 animals in the 720 and 180 mg/kg groups and 1 animal in the 45 mg/kg group, graded mild or moderate in the 720 mg/kg group, slight or mild in the 180 mg/kg group, and slight in the 45 mg/kg group. Basophilic change in hepatocytes was observed in all 6 animals in the 720 and 180 mg/kg groups and 1 animal in the 45 mg/kg group, graded mild in the 720 mg/kg group, and slight in the 180 and 45 mg/kg groups. Swelling of centrilobular hepatocytes and basophilic change in hepatocytes showed significant difference from the control group in the 720 and 180 mg/kg groups, and dose response was confirmed.

Thyroid: Diffuse follicular cell hyperplasia was observed in all 6 animals in the 720 mg/kg group, 3 animals in the 180 mg/kg group, 1 animal in the 45 mg/kg group, and 2 animals in the control group, graded slight or mild in the 720 and 180 mg/kg groups, slight in the 45 mg/kg group, and slight or mild in the control group. Since diffuse follicular cell hyperplasia showed significant difference from the control group in the 720 mg/kg group, the change was considered to be an effect of administration of 2,4-diphenyl-4-methyl-1-pentene in the 720 mg/kg group.

Kidney: Hyaline droplets in tubular epithelium were observed in 3 animals in the 720 mg/kg group, graded slight. Hyaline droplets in tubular epithelium showed significant difference from the control group in the 720 mg/kg group, and dose response was also confirmed. Degeneration of tubular epithelium was observed in 1 animal each in the 720 and 180 mg/kg groups, but it was graded slight in each case and there was no significant difference from the control group.

The following findings were observed as other changes.

Heart: Cellular infiltration in 2 animals in the control group

Lung: Mineralization of vascular wall in 3 animals in the 720 mg/kg group

Liver: Focal necrosis of hepatocytes in 1 animal in the control group

Jejunum: Mineralization of Peyer’s patch in 2 animals in the 720 mg/kg group and 1 animal in the control group

Prostate: Cellular infiltration in 3 animals in the control group

These changes were judged to be incidental because they are often observed in control groups, they were all graded slight, and their incidence in administration groups showed no difference from the control group.

Otherwise, no abnormalities were observed in the trachea, pancreas, sublingual gland, submandibular gland, esophagus, stomach, duodenum, ileum, cecum, colon, rectum, thymus, spleen, submandibular lymph node, mesenteric lymph node, urinary bladder, testis, epididymis, seminal vesicle, pituitary, adrenal, parathyroid, cerebrum, cerebellum, medulla oblongata, spinal cord, sciatic nerve, eyeball, Harderian gland, bone marrow (sternum and femur) or bone (sternum and femur) in the 720 mg/kg group or control group.

Test Group Females:
Liver: Swelling of centrilobular hepatocytes was observed in all 6 animals in the 720 and 180 mg/kg groups, graded mild in the 720 mg/kg group and slight in the 180 mg/kg group. Basophilic change in hepatocytes was observed in all 6 animals in the 720 and 180 mg/kg groups, graded slight in both the 720 and 180 mg/kg groups. Swelling of centrilobular hepatocytes and basophilic change in hepatocytes showed significant difference from the control group in the 720 and 180 mg/kg groups, and dose response was confirmed.

Kidney: Proliferation of collecting tubule epithelium and dilatation of urinary tubules were observed in 1 animal in the 720 mg/kg group (No. F04452, which had markedly high urine volume in urinary examination), with proliferation of collecting tubule epithelium graded mild and dilatation of urinary tubules graded slight.

Thyroid: Diffuse follicular cell hyperplasia was observed in 3 animals in the 720 mg/kg group, 1 animal in the 180 mg/kg group, and 2 animals in the 45 mg/kg group, graded slight in each case. Since diffuse follicular cell hyperplasia showed a high incidence in the 720 mg/kg group, the change was considered to be an effect of administration of 2,4-diphenyl-4-methyl-1-pentene in the 720 mg/kg group.

The following finding was observed as another change.

Spleen: Extramedullary hematopoiesis in 4 animals in the 720 mg/kg group and 3 animals in the control group

This change was judged to be incidental because it is often observed in control groups, the grade was slight in each case, and the incidence in administration groups showed no difference from the control group.

Otherwise, no abnormalities were observed in the heart, lung, trachea, pancreas, sublingual gland, submandibular gland, esophagus, stomach, duodenum, jejunum, ileum, cecum, colon, rectum, thymus, spleen, submandibular lymph node, mesenteric lymph node, urinary bladder, ovary, uterus, vagina, pituitary, adrenal, parathyroid, cerebrum, cerebellum, medulla oblongata, spinal cord, sciatic nerve, eyeball, Harderian gland, bone marrow (sternum and femur) , bone (sternum and femur) or mammary gland in the 720 mg/kg group or control group.

End of the Recovery period

Males:
Liver: Swelling of centrilobular hepatocytes was observed in all 6 animals in the 720 mg/kg group and 1 animal in the 180 mg/kg group, graded slight in each case. Swelling of centrilobular hepatocytes showed significant difference from the control group in the 720 mg/kg group, and dose responsewas confirmed. In addition, adhesion was observed in 1 animal in the 45 mg/kg group, but this change was judged to be incidental because it is often observed in control groups, the grade was slight, and the incidence in administration groups showed no difference from the control group.

Kidney: Degeneration of tubular epithelium was observed in 1 animal in the 720 mg/kg group, graded slight. Hyaline droplets in tubular epithelium were observed in 1 animal each in the 180 mg/kg group and control group, graded slight in each case, and judged to be an incidental change.

Thyroid: Diffuse follicular cell hyperplasia was observed in 3 animals in the 720 mg/kg group, 2 animals in the 180 mg/kg group, and 1 animal in the 45 mg/kg group, graded slight in each case. Since diffuse follicular cell hyperplasia showed a high incidence in the 720 mg/kg group, the change was considered to be an effect of administration of 2,4-diphenyl-4-methyl-1-pentene in the 720 mg/kg group. Otherwise, ectopic thymic tissue was observed in 1 animal in the 45 mg/kg group, but this change was judged to be incidental because it is often observed in control groups, the grade was slight, and the incidence in administration groups showed no difference from the control group.

Recovery Group Females:
Liver: Swelling of centrilobular hepatocytes was observed in 2 animals in the 720 mg/kg group, graded slight.

Kidney: Cyst was observed in 1 animal in the 180 mg/kg group, but this change was judged to be incidental because it is often observed in control groups, the grade was slight, and the incidence in administration groups showed no difference from the control group.

Thyroid: No abnormality was observed in the 720 and 180 mg/kg groups, or in the control group.

Effect levels

1

Key result

Dose descriptor:

NOEL

Effect level:

< 45 mg/kg bw (total dose)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Based on the results of this study, the NOEL of the test material administered orally by gavage to Crj: CD(SD) rats, could be established at > 45 mg/kg bw/day.

2

Key result

Dose descriptor:

NOEL

Effect level:

45 mg/kg bw (total dose)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: Based on the results of this study, the NOEL of the test material administered orally by gavage to Crj: CD(SD) rats, could be established at 45 mg/kg bw/day.

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Regarding toxicity due to repeat administration, deaths occurred in the 720 mg/kg group, in 1 test group female (day 9 of administration) and 1 recovery group female (day 14 of administration). The animals that died showed soiled hair, decrease in locomotor activity, hypothermia, soiled perinasal area or perioral smudge. In histopathological examination of the animals that died, swelling of centrilobular hepatocytes was observed in the liver, but it was a mild change and not serious. In other organs and tissues, examination could not be done due to postmortal changes, but from the soiled hair, decrease in locomotor activity, hypothermia, soiled perinasal area or perioral smudge observed immediately before death, the deaths were thought to be due to deterioration of general condition by 2,4-diphenyl-4-methyl-1-pentene. In a single oral dose toxicity study of 2,4-diphenyl-4-methyl-1-pentene in rats,⁵⁾mortality and soiled lower abdomen or diarrhea were observed at 2000 mg/kg. In a preliminary combined repeat dose toxicity study with reproduction/developmental toxicity screening test by oral administration of 2,4-diphenyl-4-methyl-1-pentenein rats,³⁾mortality and prone position, decrease in locomotor activity, soiled hair of the lower abdomen and bradypnea were observed at 1000 mg/kg. Therefore, the changes in general condition observed in this study were also thought to be effects of 2,4-diphenyl-4-methyl-1-pentene administration.

 

As changes in general condition, soiled hair was seen in surviving males and females of the 720 mg/kg group. Salivation was observed in males and females of the 720 and 180 mg/kg groups, but it was only seen transiently immediately after administration, and since nervous symptoms such as convulsion or morphological changes in the salivary glands were not observed, it was judged to be a change due to irritation by the test substance, and not a toxic sign.

 

Body weight was low in males of the 720 mg/kg group throughout the administration period, transiently low in females before the start of mating and low on days 0, 14 and 21 of gestation. The body weight of males recovered during the recovery period. Food consumption showed no changes due to administration in males or females.

 

No changes due to administration were observed inFunctional observational battery (FOB), sensory response, grip strength or motor activity.

 

In urinary examination at the end of the administration period, males of the 720 mg/kg group showed high urine volume and low specific gravity, and females of the 720 mg/kg group showed a tendency toward high urine volume. At the end of the recovery period, no changes due to administration were observed in either males or females. At the end of the administration period, males of the 720 and 180 mg/kg groups showed high absolute and relative kidney weight, but no change due to administration was observed in females. At the end of the recovery period, high relative weight of the kidneys was seen in males of the 720 mg/kg group. Histopathological examination of the kidney at the end of the administration period revealed hyaline droplets in tubular epithelium and degeneration of tubular epithelium in males, and proliferation of collecting tubular epithelium and dilatation of urinary tubules in females of the 720 mg/kg group. Hyaline droplets in tubular epithelium are reported to be often seen in male rats.⁴⁾In this study as well, hyaline droplets in tubular epithelium of the same grade were seen in 1 male of the control group at the end of the recovery period. In the 720 mg/kg group, it is possible that spontaneous lesions were exacerbated by 2,4-diphenyl-4-methyl-1-pentene or a metabolite, since hyaline droplets in tubular epithelium were observed in 3 animals at the end of the administration period. In addition, slight degeneration of tubular epithelium was seen in 1 animal each in the 720 and 180 mg/kg groups, and since this change is not ordinarily seen in the control group, it is possibly an effect of 2,4-diphenyl-4-methyl-1-pentene or a metabolite. When diphenylthiazole is administered to rats, the division capacity of collecting tubules in the kidney is increased and cystic lesions are observed, but it is reported that a decrease in urine reabsorption capacity is observed before morphological changes occur.⁶⁾Therefore, it is possible that urine reabsorption capacity was decreased by 2,4-diphenyl-4-methyl-1-pentene or a metabolite. However, only high relative weight of the kidneys was observed at the end of the recovery period in males of the 720 mg/kg group, and a trend of recovery is possible.

 

In hematological examination at the end of the administration period, males of the 720 and 180 mg/kg group showed high values in prothrombin time and activated partial thromboplastin time and males of the 720 mg/kg group showed a high value for fibrinogen concentration, while females of the 720 mg/kg group showed a high value for activated partial thromboplastin time. At the end of the recovery period, no changes due to administration were observed in either males or females. In blood chemical examination at the end of the administration period, males showed high values for γ-GTP, total protein, albumin, A/G, total bilirubin, total cholesterol and calcium with a low value for chlorine in the 720 mg/kg group and a high value for calcium in the 180 mg/kg group, while females showed high values for total protein in the 720 and 180 mg/kg groups and low values for glucose and high values for γ-GTP and total bilirubin in the 720 mg/kg group. In liver weight at the end of the administration period, males in the 720, 180 and 45 mg/kg groups and females in the 720 and 180 mg/kg groups had high absolute and relative weights. In histopathological examination of the liver, swelling of centrilobular hepatocytes and basophilic change in hepatocytes were observed in males of the 720, 180 and 45 mg/kg groups and females of the 720 and 180 mg/kg groups at the end of the administration period. Basophilic change in hepatocytes is considered to be a condition of enhanced stainability of hematoxylin in cytoplasm due to increase in rough endoplasmic reticulum which increases protein synthesis, or increase in ribosomes which increase proteins for growth used by the cell itself.⁷⁾Since high values for total protein, albumin and A/G were observed in blood chemical examination, it is possible that basophilic change in hepatocytes is due to increased rough endoplasmic reticulum increasing protein synthesis. Swelling of hepatocytes is possibly due to increased protein synthesis and increase in subcellular organelles including rough endoplasmic reticulum. However, because blood chemical examination revealed high values for γ-GTP and total protein, and hematological examination revealed high values for prothrombin time, activated partial thromboplastin time and fibrinogen concentration, impaired liver function accompanying swelling of hepatocytes is possible. At the end of the recovery period, a trend of recovery was suggested, even though swelling of centrilobular hepatocytes was observed in males of the 720 and 180 mg/kg groups and females of the 720 mg/kg group.

 

In thyroid weight at the end of the administration period, high values or tendency toward high values in absolute and relative weight were observed in males and females of the 720 mg/kg group. In histopathological examination of the thyroid at the end of the administration period, the incidence of diffuse follicular cell hyperplasia was high in the 720 mg/kg group in both males and females. Therefore, it is possible that that the high thyroid weight and diffuse follicular cell hyperplasia of the thyroid which were seen in males and females of the 720 mg/kg group were due to 2,4-diphenyl-4-methyl-1-pentene administration. Ordinarily, follicles in an active state have little colloid, and their diameter is reported to decrease.⁸⁾However, it is reported that by administration of chemical substances, thyroid hormone decreases in circulating blood, and due to sustained stimulation of follicular epithelial cells involving a negative-feedback mechanism, the diameter of the follicle shrinks and the follicular epithelial cells are enlarged cuboidally or cylindrically, showing diffuse follicular cell hyperplasia.⁸⁾It is also reported that when thyroid hormone is decreased, high cholesterol values are observed.⁹⁾Reduced thyroid function decreases the proportion of cholesterol catabolized to bile acid, and is thought to increase total cholesterol level.⁹⁾In this study, high values for total cholesterol were observed in blood chemical examination of males. Therefore, the diffuse follicular cell hyperplasia of the thyroid observed in this study is presumably involved decreased thyroid hormone and a negative-feedback mechanism. At the end of the recovery period, diffuse follicular cell hyperplasia was observed in males of the 720 mg/kg group at high frequency, but it was not observed in females of any group, suggesting a recovery trend.

 

In hematological examination at the end of the administration period, high values for red blood cell count and hematocrit were observed in females of the 720 mg/kg, and although no histopathological changes were observed in the spleen or bone marrow, it is possible that 2,4-diphenyl-4-methyl-1-pentene has slight effects on hematopoietic function.

Applicant's summary and conclusion

Conclusions:

The no-effect level of 2,4-diphenyl-4-methyl-1-pentene was less than 45 mg/kg/day in males because high values in the absolute and relative weight of the liver, swelling of centrilobular hepatocytes and basophilic change in hepatocytes were observed at 45 mg/kg, and was 45 mg/kg/day in females because high values in the absolute and relative weight of the liver, swelling of centrilobular hepatocytes and basophilic change in hepatocytes were observed at 180 mg/kg.