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

Endpoint summary

Description of key information

90 -Day Repeat Dose Toxicity Study: It was concluded, following 13 weeks of oral gavage administration of NOFMER MSD to Sprague-Dawley rats at doses of 100, 300 or 800 mg/kg/day that the no-adverse-effect-level (NOAEL) was 800 mg/kg/day; it was not possible to determine the no-observed-effect-level (NOEL).

28 day repeat dose oral toxicity study combined with a reproductive/ developmental toxicity screening test:

The NOEL of the test material for male and female rats can be considered as <45 mg/kg bw and 45 mg/kg bw, respectively.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

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

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

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)

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.

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Vehicle control

Dose / conc.:

45 mg/kg bw/day (nominal)

Dose / conc.:

180 mg/kg bw/day (nominal)

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.

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.

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.

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.

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.

Critical effects observed:

not specified

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.

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.

Reference 2

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental start date (pre-study chemistry): 22 June 2016 ; Experimental completion date: 06 January 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

The rat was chosen as the test species because it is accepted as a predictor of toxic change in man and the requirement for a rodent species by regulatory agencies. The Sprague-Dawley Crl:CD(SD) strain was used because of the historical control data available at the laboratory.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Animals:
Strain/Species: Crl:CD(SD) rat.
Supplier: Charles River (UK) Ltd.
Number of animals: 45 males and 45 females
Duration of acclimatization: 15 days before commencement of treatment.
Age of animals at start of treatment: 44 to 50 days.
Weight range of animals at the start of treatment: Males - 220 to 279 g; Females - 156 to 220 g

Environmental Control:
Rodent facility: Limited access - to minimize entry of external biological and chemical agents and to minimize the transference of such agents between rooms.
Air supply: Filtered fresh air which was passed to atmosphere and not recirculated.
Temperature and relative humidity: Monitored and maintained within the range of 20-24ºC and 40-70%.
Lighting: Artificial lighting, 12 hours light : 12 hours dark.

Animal Accomodation:
Cages: Polycarbonate body with a stainless steel mesh lid, changed at appropriate intervals.
Cage distribution: Males and females were blocked by sex and the cages constituting each group were dispersed in batteries so that possible environmental influences arising from their spatial distribution were equilibrated, as far as was practicable. The positions of the cage batteries in the room were changed weekly, following a rotation plan, to further minimize possible effects of spatial variations.
Number of animals per cage: Five of the same sex.
Bedding: Wood based bedding which was changed at appropriate intervals each week.

Environmental enrichment: Aspen chew block and plastic shelter.

Diet Supply:
Diet: Teklad 2014C pelleted diet.
Availability: Non-restricted (removed overnight before blood sampling for hematology and blood chemistry and during the period of urine collection).

Water Supply:
Supply: Potable water from the public supply via polycarbonate bottles with sipper tubes. Bottles were changed at appropriate intervals.
Availability: Non-restricted (except during urine collection).

Route of administration:

oral: gavage

Details on route of administration:

The oral gavage route of administration was chosen to simulate the conditions of potential human exposure.

Vehicle:

corn oil

Details on oral exposure:

TEST ITEM PREPARATION:
Formulation:
Group 1: Control (Dose:0 mg/kg/day; Nominal concentration: 0 mg/mL; Formulated concentration: 0 mg/mL; Volume dose: 5 mL/kg)
Group 2: Test item (Dose:100 mg/kg/day; Nominal concentration: 20 mg/mL; Formulated concentration: 21.2 mg/mL; Volume dose: 5 mL/kg)
Group 3: Test item (Dose:300 mg/kg/day; Nominal concentration: 60 mg/mL; Formulated concentration: 63.6 mg/mL; Volume dose: 5 mL/kg)
Group 4: Test iem (Dose:800 mg/kg/day; Nominal concentration: 160 mg/mL; Formulated concentration: 169.6 mg/mL; Volume dose: 5 mL/kg)

Correction factor:
A correction factor of 1.06 was used for purity of the test item, when calculating quantities of test item used during dose preparation.

Method of Preparation:
Starting with the lowest concentration, approximately 50% of the final volume of vehicle was added to the required amount of test item. This was magnetically stirred until all of the test item was uniformly mixed. The remaining vehicle was added to achieve the required volume. The formulation was then mixed using a high shear homogenizer until homogenous.
The remaining concentrations were made using the same method in ascending order.

Frequency of preparation:
Weekly. All formulations were administered within the known stability period.

Storage of formulation:
Refrigerated (2 to 8°C).

Test item accounting:
Detailed records of test item usage were maintained. The amount of test item necessary to prepare the formulations and the amount actually used were determined on each occasion. The difference between these amounts was checked before the formulations were dispensed.

DOSE ADMINISTRATION:
The study consisted of one control and three treated groups identified as follows:

Group 1: Control
Dose: 0 mg/kg/day
Number of animals: 10 Male / 10 Female

Group 2: Test item
Dose: 100 mg/kg/day
Number of animals: 10 Male / 10 Female

Group 3: Test item
Dose: 300 mg/kg/day
Number of animals: 10 Male / 10 Female

Group 4: Test item
Dose: 800 mg/kg/day
Number of animals: 11* Male / 10 Female

*One male was found dead on Day 2 and was replaced with a spare animal of suitable weight from the same batch that commenced treatment from Day 2.

Administration:
Route: Oral, by gavage, using a suitably graduated syringe and a rubber catheter inserted via the mouth.
Treated at: Constant doses in mg/kg.
Volume dose: 5 mL/kg bodyweight
Individual dose volume: Calculated from the most recently recorded scheduled body weight.
Formulation: A daily record of the usage of formulation was maintained based on weights. This balance was compared with the expected usage as a check of correct administration. No significant discrepancy was found. Formulations were stirred using a magnetic stirrer before and throughout the dosing procedure.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Stability and homogeneity: Formulations from 2 mg/mL to 200 mg/mL have been shown to be stable in corn oil for up to seven days when stored refrigerated (nominally +4°C) and six hours at ambient temperature (15-25°C), when protected from light.

Achieved concentration: Samples of each formulation prepared for administration in Weeks 1 and 12 of treatment were analyzed for achieved concentration of the test item.

Method of analysis: High performance liquid chromatography (HPLC)

Results:
The analytical procedure was successfully validated with respect to specificity of chromatographic analysis, limit of detection and quantification, linearity of detector response, repeatability, method accuracy and precision.

The mean concentrations of test item in test formulations analyzed for the study were within ±4% of nominal concentrations, confirming accurate formulation.

Duration of treatment / exposure:

Minimum period: 13 weeks

Frequency of treatment:

Once daily at approximately the same time each day.

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Control

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

Group 2

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

Group 3

Dose / conc.:

800 mg/kg bw/day (nominal)

Remarks:

Group 4

No. of animals per sex per dose:

10 per sex per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The doses used in this study (0, 100, 300 and 800 mg/kg/day) were selected in conjunction with the Sponsor, and were based on the results of a 7-day repeat dose preliminary toxicity study conducted in CD rats, in which three groups of five males were treated at 250, 500 or 1000 mg/kg/day. At 1000 mg/kg/day, one male died and the remaining animals showed overall mean body weight loss. Subsequently, dose levels of 45, 180 or 720 mg/kg/day in corn oil were administered to CD rats in the OECD 422 Main study. Treatment in that study was well-tolerated and there were no in-life findings or death at any dose; however post-life investigations at 720 mg/kg/day revealed, high testes weight and microscopic changes in the kidney, liver and thyroid.

It was therefore considered that a high dose of 800 mg/kg/day would be tolerated in this 13-week toxicity study and that the low and intermediate doses would be 100 and
300 mg/kg/day.

Positive control:

No.

Observations and examinations performed and frequency:

MORTALITY:
A viability check was performed near the start and end of each working day. A complete necropsy was performed in all cases of mortality.

CLINICAL OBSERVATIONS:
Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of animal ill-health amongst the occupants. Any deviation from normal was recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as appropriate.

During the acclimatization period, observations of the animals and their cages were recorded at least once per day.

SIGNS ASSOCIATED WITH DOSING:
Detailed observations were recorded on each day during the treatment period, at the following times in relation to dose administration:
- Pre-dose
- One to two hours after completion of dosing of all groups
- As late as possible in the working day #
# Week 1 only

DETAILED PHYSICAL EXAMINATION AND ARENA OBSERVATIONS:
Before treatment commenced and during each week of treatment, detailed physical examination and arena observations were performed on each animal. On each occasion, the examinations were performed at approximately the same time of day (before dosing), by an observer unaware of the experimental group identities.

After removal from the home cage, animals were assessed for physical condition and behavior during handling and after being placed in a standard arena. Any deviation from normal was recorded with respect to the nature and, where appropriate, degree of severity. Particular attention was paid to possible signs of neurotoxicity, such as convulsions, tremor and abnormalities of gait or behavior.

Findings were either reported as "present" or assigned a severity grade - slight, moderate or marked.

SENSORY REACTIVITY AND GRIP STRENGTH:
Sensory reactivity and grip strength assessments were performed (before dosing) on all animals during Week 12. Animals were tested by an observer who was unaware of the treatment group to which each animal belonged. Before the start of observations, cage labels showing the treatment group were replaced by labels stating only the study, animal and cage numbers. Animals were not necessarily all tested on the same day, but the numbers of animals and the times of testing were balanced across the groups on each day of testing.

The following measurements, reflexes and responses were recorded:

- Approach respnse:
A blunt probe was brought towards the animal’s head until it was close to the animal’s nose (but not touching the whiskers). The animal’s reaction was recorded as:
1 - No reaction or ignores probe/walks past probe
2 - Normal awareness and reaction e.g. approaches and/or sniffs probe
3 - Active avoidance, abnormally fearful or aggressive reaction

- Pinna reflex:
The inside of one ear was touched lightly with a nylon filament and the reaction recorded as:
1 - No response
2 - Normal response e.g. ear twitches/flattens or animal shakes its head
3 - Abnormally fearful or aggressive response

Auditory startle reflex:
The animal’s response to a sudden sharp noise was assessed and scored as:
1 - No response
2 - Weak response e.g. ear twitch only
3 - Normal response e.g. obvious flinch or startle
4 - Exaggerated response e.g. all feet off floor

Tail pinch response:
The animal’s tail was pinched sharply with forceps approximately one third from the tip and the response graded as:
1 - No response
2 - Weak response e.g. turns around slowly or weak vocalization without moving away
3 - Normal response e.g. jumps forward or turns around sharply, usually with vocalization
4 - Exaggerated response e.g. excessive vocalization, body movement or aggression

Grip strength:
Forelimb and hindlimb grip strength was measured using Mecmesin Basic Force Gauges. Three trials were performed.
At any point during the observations, additional comments were made as free text where considered appropriate.

MOTOR ACTIVITY:
During Week 12 (before dosing), the motor activity of each animal was measured using a Rodent Activity Monitoring System (Version 2.0.6).
Animals were tested individually in clear polycarbonate cages and motor activity was measured by counting infra-red beam breaks over ten 6-minute intervals (one hour total).
Ten beams were set at two height levels (five low and five high) to detect cage floor and rearing activity respectively. Animals were not necessarily all tested on the same day, but the numbers of animals and the times of testing were balanced across the groups on each day of testing.

BODY WEIGHT:
The weight of each animal was recorded one week before treatment commenced, on the day that treatment commenced (Week 0), weekly throughout the study and before necropsy.
More frequent weighings were instituted, when appropriate, for animals displaying ill-health, so that the progress of the observed condition could be monitored.

FOOD CONSUMPTION:
The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded for the week before treatment started and for each week throughout the study.

WATER CONSUMPTION:
Fluid intake was assessed by daily visual observation on each day.
Water consumption was formally recorded by weight during Weeks 4, 5 (See Section 4), 6, 8, 10 and 12, over a 3 day period on each occasion.

OPHTHALMIC EXAMINATION:
All animals at pretreatment and all animals in groups 1 and 1 at week 12.

The eyes of the animals were examined by means of a binocular indirect ophthalmoscope.
Prior to each examination, the pupils of each animal were dilated using tropicamide ophthalmic solution (Mydriacyl). The adnexae, conjunctiva, cornea, sclera, anterior chamber, iris (pupil dilated), lens, vitreous and fundus were examined.

HEMATOLOGY, PERIPHERAL BLOOD:
All animals at week 13.

Animals were held under light general anesthesia induced by isoflurane. Blood sampling was performed on the morning after overnight collection of urine. Animals were, therefore, deprived of food and water overnight but were allowed access to water for a minimum period of one hour prior to the commencement of the blood sampling procedures. Blood samples (nominally 0.5 mL) were withdrawn from the sublingual vein, collected into tubes containing EDTA anticoagulant and examined for the following characteristics using a Bayer Advia 120 analyzer:

-Hematocrit (Hct)
-Hemoglobin concentration (Hb)
-Erythrocyte count (RBC)
-Absolute reticulocyte count (Retic)
-Mean cell hemoglobin (MCH)
-Mean cell hemoglobin concentration (MCHC)
-Mean cell volume (MCV)
-Red cell distribution width (RDW)
-Total leucocyte count (WBC)
-Differential leucocyte count:
Neutrophils (N)
Lymphocytes (L)
Eosinophils (E)
Basophils (B)
Monocytes (M)
Large unstained cells (LUC)
-Platelet count (Plt)

Blood film (prepared for all samples) - Romanowsky stain, examined for abnormalities by light microscopy, in the case of flags from the Advia 120 analyzer.

Additional blood samples (nominally 0.5 mL) were taken into tubes containing citrate anticoagulant and examined using an ACL series analyzer and appropriate reagent in respect of:
Prothrombin time (PT) - using IL PT Fibrinogen reagent.
Activated partial thromboplastin time (APTT) - using IL APTT reagent.

BLOOD CHEMISTRY:
All animals at week 13.

Animals were held under light general anesthesia induced by isoflurane. Blood sampling was performed on the morning after overnight collection of urine. Animals were, therefore, deprived of food and water overnight but were allowed access to water for a minimum period of one hour prior to the commencement of the blood sampling procedures. Blood samples (nominally 0.7 mL) were withdrawn from the sublingual vein and collected into tubes containing lithium heparin as anticoagulant. After separation, the plasma was examined using a Roche P Modular Analyzer in respect of:

-Alkaline phosphatase (ALP)
-Alanine aminotransferase (ALT)
-Aspartate aminotransferase (AST)
-Total bilirubin (Bili)
-Bile acids (BiAc)
-Urea
-Blood urea nitrogen (BUN)
-Creatinine (Creat)
-Glucose (Gluc)
-Total cholesterol (Chol)
-Triglycerides (Trig)
-Sodium (Na)
-Potassium (K)
-Chloride (Cl)
-Calcium (Ca)
-Inorganic phosphorus (Phos)
-Total protein (Total Prot)
-Albumin (Alb)

Albumin/globulin ratio (A/G Ratio) was calculated from total protein concentration and analyzed albumin concentration.

URINALYSIS:
All animals at week 13.

Animals were placed in an individual metabolism cage overnight, without food or water. The individual samples were examined for the following characteristics:

Using manual methods:
-Clarity and Color (App) - by visual assessment
-Volume (Vol) - using a measuring cylinder
-pH - using a pH meter
-Specific gravity (SG) - by direct refractometry using a SG meter

Using Multistix reagent strips interpreted using the Clinitek®500 instrument:
-Ketones (Keto)
-Bile pigments (Bili)
-Blood pigments (UBld)

Using a Roche P Modular Analyzer:
-Protein (T-Prot)
-Creatinine (T-Creat)
-Glucose (T-Gluc)
-Sodium (T-Na)
-Potassium (T-K)
-Chloride (T-Cl)

A microscopic examination of the urine sediment was performed. An aliquot of the urine sample was centrifuged, stained with Kova stain and the resulting deposit spread on a microscope slide. The number of elements seen in nine high or low power fields (HPF or LPF) was recorded in the raw data and entered onto the database and the number seen /HPF or /LPF was derived from these data as described below.

-Epithelial cells (Epi)
-Leucocytes (WBC)
-Erythrocytes (RBC)
-Casts
-Other abnormal components (A)

The slide was also examined for abnormalities in spermatozoa and crystals.

Sacrifice and pathology:

METHOD OF KILL:
Carbon dioxide asphyxiation with subsequent exsanguination.

NECROPSY:
All study animals were subject to a detailed necropsy. After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative.
The retained tissues were checked before disposal of the carcass.

Schedule: Main study animals were killed following 13 weeks of treatment
Sequence: To allow satisfactory inter-group comparison.

The organs weighed, tissue samples fixed and sections examined microscopically are detailed:
Tissue and regions examined:
Abnormalities
Adrenals
Aorta
Brain (cerebellum, cerebrum, midbrain)
Cecum
Colon
Duodenum
Epididymides
Esophagus
Eyes
Femur (femorotibial joint)
Head
Heart (including auricular and ventricular regions)
Ileum (including Peyers’ patch)
Jejunum
Kidneys
Liver (section from two lobes)
Lungs (section from two major lobes including bronchi)
Lymph nodes;
- mesenteric
- left axillary
Ovaries
Pancreas
Pituitary
Prostate
Salivary glands;
- submandibular
- sublingual
- parotid
Sciatic nerves
Seminal vesicles
Skin with mammary glands (inguinal area)
Spinal cord (transverse and longitudinal sections at the cervical, thoracic and lumbar levels)
Spleen
Sternum (with bone marrow)
Stomach
Testes
Thymus
Thyroid with parathyroids
Trachea
Urinary bladder
Uterus with cervix
Vagina

ORGAN WEIGHTS:
Weighed organs: adrenals, brain, epididymides, heart, kidneys, liver, ovaries, spleen, testes, thymus, uterus with cervix.
For bilateral organs, left and right organs were weighed together, unless specified above. Requisite organs were weighed for animals killed at the end of the treatment period.

FIXATION:
Tissues were routinely preserved in 10% Neutral Buffered Formalin with the exception of those detailed below:
Testes: In modified Davidson’s fluid.
Eyes: In Davidson’s fluid.

The kidneys from all male animals, at scheduled termination, were fixed in 10% Neutral Buffered Formalin for 24-72 hours, to assist with the subsequent immunohistochemistry investigation

HISTOLOGY:
Processing: Tissue samples were dehydrated, embedded in paraffin wax and sectioned at a nominal four to five micron thickness. For bilateral organs, sections of both organs were prepared. A single section was prepared from each of the remaining tissues required.

Full list: All animals killed or dying prematurely. All terminal animals of Groups 1 - 4.

Routine staining: Sections were stained with hematoxylin and eosin.

IMMUNOHISTOCHEMISTRY:
Immunohistochemistry for diagnostic assessment of α2μ-globulinglobulin was undertaken on the kidneys from males in Groups 1 and 4.

LIGHT MICROSCOPY:
Tissues preserved for examination were examined as follows:

Category: Terminal sacrifice
Animals: All males from Groups 1 and 4
Tissues: All specified in necropsy section and immunohistochemistry section.


Category: Terminal sacrifice
Animals: All females from Groups 1 and 4 and all animals from groups 2 and 3.
Tissues: All specified in necropsy section.

Findings were either reported as "present" or assigned a severity grade. In the latter case one of the following five grades was used - minimal, slight, moderate, marked or severe. A reviewing pathologist undertook a peer review of the microscopic findings.

Statistics:

All statistical analyses were carried out separately for males and females using the individual animal as the basic experimental unit.

The following data types were analyzed at each timepoint separately:
-Grip strength and motor activity
-Body weight, using gains over appropriate study periods
-Hematology
-Blood chemistry
-Urinalysis
-Organ weights, absolute and adjusted for terminal body weight

The following comparisons were performed:
-Group 1 vs 2, 3 and 4

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

There was a higher incidence, when compared with Control, of yellow staining of the ventral surface, including the lower ventral surface, in males and females treated at 800 mg/kg/day. There were no other signs at the weekly physical examination considered to be related to treatment.

Transient salivation was evident on some occasions in one or more animals from the end of the second week of treatment at 300 or 800 mg/kg/day, with or without chin rubbing; this was attributed to the palatability of the test item and not of toxicological significance. One male treated at 100 mg/kg/day had piloerection on Day 31 and, in the absence of any other findings at 100, 300 or 800 mg/kg/day was considered not to be toxicological significant.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One male receiving 800 mg/kg/day was found cannibalised on Day 2. The animal had shown no evidence of toxicity following the single administration on Day 1. The subsequent macroscopic examination was limited to few tissues, as the animal had been cannibalised and the cause of death could not be determined. No histopathological investigations were performed on this animal. An additional animal, supplied with the Study animals as a spare animal, was allocated to study as a replacement.

One Control female was euthanized in Week 13, as it was not possible to staunch blood flow from the sublingual vein shortly after blood collection for the hematology and biochemical investigation. The animal was overtly normal prior to sampling; however became prostrate and pale and had irregular respiration shortly afterwards. Macroscopic examination revealed dark contents, considered to be blood, in the stomach and jejunum. The microscopic examination revealed no findings and histologically, the cause of death was clearly not related to treatment.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weight gain was high in females treated at 800 mg/kg/day, during the first four weeks of treatment (127% of Control). Body weight gain in males and females treated at 300 or 800 mg/kg/day was significantly low (71 or 64% and 78 or 44%, respectively), during Weeks 4-13 and resulted in low overall body weight gain in males at 800 mg/kg/day (80%).

There was no effect of treatment on body weight gain at 100 mg/kg/day.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Overall food intake at 800 mg/kg/day was high in males and females (119 and 124% of Control, respectively).

Food intake at 100 or 300 mg/kg/day was unaffected by treatment.

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

effects observed, treatment-related

Description (incidence and severity):

Water intake (when visually assessed) appeared to be higher than Control in animals receiving 300 or 800 mg/kg/day, from Week 1. Formal water consumption was performed on three consecutive days during Weeks 4, 5, 6, 8, 10 and 12.

When compared with Control, overall water consumption (Weeks 4-12) at 800 mg/kg/day was markedly high in males and females (145% and 173%, respectively) and was slightly high at 300 mg/kg/day (109% and 123% respectively).

There was no effect of treatment on water consumption at 100 mg/kg/day.

Ophthalmological findings:

no effects observed

Description (incidence and severity):

There were no treatment-related ophthalmic findings.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

The hematological examination in Week 13 did not identify any differences from Control that were attributable to treatment.

All differences from Control were minor or were confined to one sex and were therefore attributed to normal biological variation. In males and females at 300 or 800 mg/kg/day, hemoglobin concentration was marginally but statistically significantly, low (97 or 97% and 94 or 94%, respectively). In males only at 800 mg/kg/day, neutrophil, lymphocyte, monocyte, large unstained cell and overall white cell counts were statistically significant high (149%, 130%, 141%, 150% and 133%, respectively) and platelet count was marginally high (111%) and in females only at 300 or 800 mg/kg/day, hematocrit and red cell counts were marginally low (95% or 95% and 95% or 95%, respectively) and reticulocyte count was slightly low (80% or 85%, respectively), prothrombin time was slightly short (95 or 88%, respectively) and activated partial thromboplastin time was slightly short at 800 mg/kg/day (67%).

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

The biochemical examination of the blood plasma in Week 13 indicated several statistically significant differences from Control, namely; increased alanine amino-transferase activity in males and females treated at 800 mg/kg/day (124% and 117%, respectively). Bilirubin concentration was high in males and females at 800 mg/kg/day (both 300%) and was also evident in females at 300 mg/kg/day (200%), but at this dose individual values were within the Control range and creatinine concentration in males and females at 800 mg/kg/day was high (112% and 116%, respectively); however the majority of individual values were with the Control range. Plasma glucose concentration was low in males and females treated at 100, 300 or 800 mg/kg/day (79, 79% or 69% and 84, 85% or 77%, respectively).

At 100, 300 or 800 mg/kg/day, total protein concentration was marginally high (males 105, 108 or 111% and females 104, 107 or 116%, respectively) and was reflected in marginally high albumin concentration in males at 100, 300 or 800 mg/kg/day and females at 800 mg/kg/day (103, 105 or 108% and 109%, respectively). The albumin/globulin ratio was marginally low in males and females at 100, 300 or 800 mg/kg/day (95%, 95% or 92% and 89%, 88% or 88%, respectively).

All other differences from Control were minor or were confined to one sex and were therefore attributed to normal biological variation, these included; a dose-related decrease in aspartate amino-transferase activity in females at 100, 300 or 800 mg/kg/day (92, 86 or 74%, respectively), high urea/BUN and cholesterol concentrations in males at 800 mg/kg/day (134% and 155%, respectively); cholesterol concentrations were also higher than the Control range in four females treated at 800 mg/kg/day, high bile acid concentration in females treated at 800 mg/kg/day (240%) and low triglyceride concentration in males at 300 or
800 mg/kg/day (50% and 57%, respectively) and potassium concentration was low in males at 800 mg/kg/day (90%). The differences in plasma chloride concentration (marginally low in males and females treated at 300 or 800 mg/kg/day (98% or 96% and 97% or 93, respectively)) and phosphorus concentration (slightly high in males and females at 800 mg/kg/day (115% and 130%, respectively)) were considered not to be biologically relevant and therefore not toxicologically significant.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

Examination of the composition of the urine in Week 13 indicated a marked reduction in total glucose output in males and females treated at 300 or 800 mg/kg/day (47% or 2% and 58% or 40% of Control, respectively) and an increase in output of total protein, sodium, potassium and chloride in both sexes at 800 mg/kg/day (males 202%, 219%, 160% and 302%; females 215%, 196%, 158% and 263%). Urine volume was markedly high in males and females at 800 mg/kg/day (310% or 215%, respectively) and correlated with the increase in water intake at this dose; however specific gravity was not adversely affected (males 99%; females 100% of Control).

All other statistically significant differences from Control were minor, lacked dose-relationship, was confined to one sex or individual animal values were within the Control range and were therefore attributed to normal biological variation. Such differences included at 100 or 300 mg/kg/day; high specific gravity in females (both 101%) and low sodium output in males (65 or 77%, respectively), marginally low urinary pH in females at 100, 300 or 800 mg/kg/day (94, 95 or 94%, respectively) and low plasma creatinine in females treated at 300 mg/kg/day.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Sensory Reactivity and Grip Strength: There was no effect of treatment on sensory reactivity or grip strength.
Motor Activity: There was no effect of treatment upon motor activity.
A slight dose related increase in activity was observed in males treated at 100, 300 or 800 mg/kg/day; however the difference was not statistically significant and not evident in females and was therefore considered not to be toxicologically significant.

Immunological findings:

no effects observed

Description (incidence and severity):

Immunohistochemistry for alpha 2u globulin nephropathy showed no increase in this protein in males treated at 800 mg/kg/day.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Evaluation of organ weights after 13 weeks of treatment revealed, when compared with Control, high adjusted mean liver weight in males and females treated at 100, 300 or
800 mg/kg/day (128, 145% or 175% and 120, 141% or 200%, respectively). Adjusted mean kidney weight in males treated at 100, 300 or 800 mg/kg/day and females treated at 300 or 800 mg/kg/day were high (123, 141% or 135% and 109% or 116%, respectively).

All other differences from Control, including those that attained statistical significance, were confined to males only, considered minor and had no microscopic correlate and were therefore considered not to be toxicologically significant. These were high adjusted mean spleen weight at 800 mg/kg/day (121%) and high adjusted mean thymus weight, without relationship to dose at 100, 300 or 800 mg/kg/day (122%, 128% and 115%).

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

The macroscopic examination performed after 13weeks of treatment revealed the following changes in the liver and kidneys.

Liver:
Dark abnormal color of the liver was seen in males and females receiving 300 or 800 mg/kg/day.

Kidneys:
Dark abnormal color of the kidney was seen in seven females receiving 800 mg/kg/day. Pale areas were seen in one male and one female receiving 800 mg/kg/day, one male receiving 300 mg/kg/day and one male receiving 100 mg/kg/day.


The incidence and distribution of all other findings were considered incidental and unrelated to the test item.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

TREATMENT RELATED FINDINGS:
Changes related to treatment with test item were seen in the liver, kidneys and thyroid glands.

Liver:
Dose-related centrilobular hepatocellular hypertrophy was seen in males and females of all treatment groups.

Kidneys:
Increased incidence of pigment accumulation was seen in males and females at all doses with clear dose-relationship and was characterized by a golden brown appearance. The pigment stained positive for lipofuscin with Schmorls special stain. Immunohistochemistry for alpha 2u globulin nephropathy showed no increase in this protein in males treated at 800 mg/kg/day. A dose-related increase in the incidence of tubular basophilia was seen in males receiving 300 or 800 mg/kg/day and was evident in a few females receiving 800 mg/kg/day.

Thyroid glands:
Dose-related diffuse follicular cell hypertrophy, with or without colloid alteration was seen in males and females of all treated groups.

Incidental Findings:
All other microscopic findings are considered incidental and unrelated to the test item.

Histopathological findings: neoplastic:

not examined

Dose descriptor:

NOAEL

Effect level:

800 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no 'adverse' effects observed in parameters measured.

Critical effects observed:

no

Macropathology:

Liver:

Summary of findings in the liver for animals killed after 13 weeks of treatment

 

Group/sex	1M	2M	3M	4M	1F	2F	3F	4F
Dose (mg/kg/day)	0	100	300	800	0	100	300	800
Abnormal color, Dark	0	0	2	10	0	0	5	10
Number of tissues examined	10	10	10	10	9	10	10	10

Kidneys:

Summary of findings in the kidneys for animals killed after 13 weeks of treatment

 

Group/sex	1M	2M	3M	4M	1F	2F	3F	4F
Dose (mg/kg/day)	0	100	300	800	0	100	300	800
Abnormal color, Dark	0	0	0	0	0	0	0	7
Pale area(s)	0	1	1	1	0	0	0	1
Number of tissues examined	10	10	10	10	9	10	10	10

Histopatholgy:

Liver:

Summary of treatment related findings in the liver of animals killed after 13 weeks of treatment

 

Group/sex	1M	2M	3M	4M	1F	2F	3F	4F
Dose (mg/kg/day)	0	100	300	800	0	100	300	800
Hypertrophy, Hepatocellular, Centrilobular
Minimal	0	5	7	10	0	6	6	5
Slight	0	0	0	0	0	0	2	5
Total	0	5	7	10	0	6	8	10
Number of tissues examined	10	10	10	10	9	10	10	10

 

Kidneys:

Summary of treatment related findings in the kidneys of animals killed after 13 weeks of treatment

 

Group/sex	1M	2M	3M	4M	1F	2F	3F	4F
Dose (mg/kg/day)	0	100	300	800	0	100	300	800
Accumulation, Pigment
Minimal	2	4	7	9	0	2	4	5
Slight	0	0	0	0	0	0	0	5
Total	2	4	7	9	0	2	4	10
Basophilia, Tubular
Minimal	1	1	6	4	0	0	0	2
Slight	0	0	0	3	0	0	0	1
Moderate	0	0	0	1	0	0	0	0
Total	1	1	6	8	0	0	0	3
Number of tissues examined	10	10	10	10	9	10	10	10

Thyroid glands:

Summary of treatment related findings in the thyroid glands of animals killed after 13 weeks of treatment

 

Group/sex	1M	2M	3M	4M	1F	2F	3F	4F
Dose (mg/kg/day)	0	100	300	800	0	100	300	800
Hypertrophy, Follicular Cell, Diffuse/Alteration, Colloid
Minimal	0	7	2	2	0	1	2	5
Slight	0	0	4	8	0	1	1	4
Total	0	7	6	10	0	2	3	9
Number of tissues examined	10	10	10	10	9	10	10	10

Conclusions:

It was concluded, following 13 weeks of oral gavage administration of NOFMER MSD to Sprague-Dawley rats at doses of 100, 300 or 800 mg/kg/day that the no-adverse-effect-level (NOAEL) was 800 mg/kg/day; it was not possible to determine the no-observed-effect-level (NOEL).

Executive summary:

Summary:

The objective of this study was to assess the systemic toxic potential of the test item, when administered orally, by gavage, to Sprague-Dawley rats for 13 weeks. Three groups, each comprising ten males and ten females, received doses of 100, 300 or 800 mg/kg/day. A similarly constituted group received the vehicle (corn oil) at the same volume-dose (5 mL/kg body weight).

During the study, clinical condition, detailed physical and arena observations, sensory reactivity, grip strength, motor activity, body weight, food consumption, water consumption, ophthalmoscopy, hematology (peripheral blood), blood chemistry, urinalysis, organ weight, macropathology and histopathology investigations were undertaken.

Results:

One male was found dead after a single administration at 800 mg/kg/day. The cause of this death was undetermined; however as other animals at this dose remained in good general condition throughout the study period, it was considered that it was un-related to treatment. One control animal was euthanized, this death was not related to treatment.

The incidence of yellow ventral staining (urine) was high in animals treated at 800 mg/kg/day.

Overall mean body weight gain was low in males at 800 mg/kg/day. Food consumption was high in animals treated at 800 mg/kg/day and water consumption was high at 300 or 800 mg/kg/day.

Alanine amino-transferase activity, bilirubin and creatinine concentrations were high in animals treated at 800 mg/kg/day; albumin and total protein concentrations were marginally high and albumin/globulin ratios were marginally low, at 100 mg/kg/day or more; glucose concentrations were low at 300 mg/kg/day, or more.

Water consumption was markedly high at 800 mg/kg/day and slightly high at 300 mg/kg/day and urine output was markedly high at 800 mg/kg/day and correlated with low urinary glucose concentrations at 300 or 800 mg/kg/day. Total protein output was high at 800 mg/kg/day.

Treatment-related centrilobular hepatocellular hypertrophy and high adjusted liver weights were evident in animals treated at 100 mg/kg/day or more and there was a treatment-related increase in diffuse follicellular cell hypertrophy in the thyroid, with or without colloid alteration. Adjusted kidney weights were high in males treated at 100 mg/kg.day or more and females at 300 mg/kg/day or more and there was a treatment-related increase in lopfuscin at 100 mg/kg/day or more and tubular basophilia was evident in males at 300 mg/kg/day or more and in a few females at 800 mg/kg/day.

Conclusion:

It was concluded, following 13 weeks of oral gavage administration of NOFMER MSD to Sprague-Dawley rats at doses of 100, 300 or 800 mg/kg/day that the no-adverse-effect-level (NOAEL) was 800 mg/kg/day; it was not possible to determine the no-observed-effect-level (NOEL).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

800 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

90 -Day Repeat Dose Toxicity Study:

The objective of this study was to assess the systemic toxic potential of test item to rats for 13 weeks, at doses of 100, 300 or 800 mg/kgday, when administered once daily by oral gavage to Sprague-Dawley rats for 13 weeks.

One male treated at 800 mg/kg/day was found cannibalized on the morning after the first administration. Macroscopic examination was limited to few tissues and there was no pathological examination of the remaining tissues. The cause of the death of this animal could not be determined; however as other animals at this dose remained in good general condition throughout the study period, it was considered not to be related to treatment. It is however noted that one female, in an embryo-fetal main study conducted, was found dead on the morning after one treatment at 600 mg/kg/day. One animal from the Control group was euthanized, as it was not possible to staunch the bleeding from the sampling site (sublingual vein), shortly after blood collection for the hematology and biochemical investigations in Week 13. This death was attributed to the sampling procedure and not related with treatment.

 

Females receiving 800 mg/kg/day showed high weight gain, during the first four weeks of treatment and weight gain waslowin both sexes receiving 300 or 800 mg/kg/day from Week 4. Overall body weight gain in males receiving 800 mg/kg/day was low, but was unaffectedinfemales at 800 mg/kg/day or in either sex at 300 mg/kg/day. Conversely, food consumption in males and females treated at 800 mg/kg/day was decreased, but water intake was increased at 300 mg/kg/day or more.

 

Pathological examination revealed test item related findings in the liver, thyroids and kidneys. There was in the liver, in animals treated at 100 mg/kg/day or more, a dose-related increase in centrilobular hypertrophy that correlated at 300 mg/kg/day or more, with high adjusted liver weight and the macroscopic observation of dark livers. Also at 100 mg/kg/day or more, albumin and total protein concentrations were marginally high and the albumin/globulin ratio was correspondingly low, but clotting parameters were unaffected. Alanine amino-transferase activity and bilirubin concentrations were high at 800 mg/kg/day. At 100 mg/kg/day or more, there were diffuse follicular cell hypertrophy/colloid alterations in the thyroid. It was therefore considered that the hypertrophy of the liver was due to P450 microsomal enzyme induction, responsible for metabolism of most xenobiotics and intrinsic substances such as hormones. The metabolism and clearance of hormones, especially thyroglobulins, in cases of induced liver microsomal enzymes, results in a compensatory feedback and an increase in the production of thyroid stimulating hormone (TSH) by the pituitary. The increase in TSH levels stimulates the thyroid gland, leading to follicular cell hypertrophy. Changes, secondary to the liver/thyroid changes were evident in the kidney and were considered due to the excretion of large protein molecules. Kidney weight was high in males at 100 mg/kg/day and females at 300 mg/kg/day or more and the kidneys of the majority of females at 800 mg/kg/day were dark. In addition, there was a clear treatment related increase in the accumulation of lipofuscin, indicating cellular wear and tear, at 100 mg/kg/day or more and an increase in the incidence of tubular basophilia in males at 300 mg/kg/day or more and in females at 800 mg/kg/day. The concentration of total protein was increased at 800 mg/kg/day in both sexes. Water intake was increased at 300 or 800 mg/kg/day and correlated with low urinary glucose concentrations at these doses. Urinary volume was increased at 800 mg/kg/day and there was a high incidence of animals with urine staining of the ventral surface at this dose. The immunohistochemistry investigation for alpha 2u globulin nephropathy in the kidneys of males treated at 800 mg/kg/day revealed no increase in this protein, when compared with Control.

 

It is considered that the stimulation of the thyroid gland, as a result of the liver hypertrophy and the resultant changes in the kidneys are specific to the rodent and therefore not relevant to man.

28 day repeat dose oral toxicity study combined with a reproductive/ developmental toxicity screening test:

A 28 day repeat dose oral toxicity study combined with a reproductive/ developmental toxicity screening test was performed in the rat in accordance with GLP and OECD guideline 422 (Anon, 2007). The test material was administered by gavage to three groups, each of 12 male and 12 female Crj: CD(SD) strain rats for up to 8 weeks (including a 2 week pre-pairing phase, pairing, gestation and early lactation for females), at dose levels of 45, 180 and 720 mg/kg bw/day.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 the test item. Changes in general condition were 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, 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 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 the test item 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 the test item or a metabolite. It is possible that urine reabsorption capacity was decreased the test item 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. 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 test item administration. 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 the test item has slight effects on hematopoietic function. The no-effect level of the test item 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.

Justification for classification or non-classification

Based a 28 -day repeat dose oral toxicity study combined with a reproductive/developmental toxicity sceening test, The no-effect level of the test item was less than 45 mg/kg/day in males and 45 mg/kg/day in females because of effects on the liver. As such, the substance should be classified as Specific Target Organ Toxicity Repeated Exposure Category 2 in accordance with Regulation 1272/2008.