Benzeneacetic acid, 4-[4-[4-(hydroxydiphenylmethyl)-1-piperidinyl]-1-butyn-1-yl]-.alpha.,.alpha.-dimethyl-

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2006-2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP, Guideline study.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Version / remarks:

OECD Guidelines for the Testing of Chemicals, Section 4, Health Effects, Number 407, July 27, 1995

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

FEXO-07

IUPAC Name:

FEXO-07

Test material form:

other: White/apparently white solid powder

Details on test material:

Purity: > 99%
Stability of test item: stable under storage conditions
Storage conditions: at room temperature (20 ±5 °C) in the original container protected from light

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

Rationale: recognized by the international guidelines as the recommended test system.

Test system: Rat, HanRcc:WIST (SPF)
Group allocation: groups 1 to 4: 5 males and 5 females
Total number of animals used: 20 males and 20 females
Total number of animals ordered 21 males and 21 females
Age at delivery: 6 weeks
Body weight range (at acclimatization): Males: 142.5-163.0 g (mean 156.2 g); Females: 120.9-129.7 g (mean 125.9 g)


Identification
Acclimatization: Cage card and tail mark (later ear tattoo)
Treatment: Cage card and individual ear tattoo
Randomization: Computer-generated random algorithm
Acclimatization: Under test conditions after health examination. Only animals without any visible signs of illness were used for the study

Conditions: Standard Laboratory Conditions. Air-conditioned with 10-15 air changes per hour, and continuously monitored environmental conditions (temperature range: 22 ± 3 °C; relative humidity range: 30-70 %). There was 12-hour fluorescent light/12-hour dark cycle with music during the light period.

Accommodation: in groups of five in Makrolon type-4 cages with wire mesh tops and standardized softwood bedding ('Lignocel' Schill AG, CH-4132 Muttenz/Switzerland).

Diet: pelleted standard Provimi Kliba 3433 rat maintenance diet (Provimi Kliba AG, CH-4303 Kaiseraugst/ Switzerland) was available ad libitum. The feed batch was analyzed for contaminants.

Water: community tap-water from ltingen was available ad libitum in water bottles. Results of bacteriological assay, chemical and contaminant analyses of representative samples are attached to the report. None of the contaminants analyzed in the water and diet is considered to have been present at a concentration that would have affected the validity of the results.

The dose formulations were prepared weekly. FEX0-07 was weighed into a glass beaker on a tared Mettler balance and the vehicle added. The mixtures were prepared using a magnetic stirrer and stored at room temperature ( 15-25°C).
Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

Rationale: administration by gavage is a common and accepted route of exposure for studies of this type.

Daily dose levels:
Group 1: 0 mg/kg body weight
Group 2: 50 mg/kg body weight
Group 3: 150 mg/kg body weight
Group 4: 1000 mg/kg body weight
Dose levels are expressed in terms of the test item as supplied.

Rationale for dose level selection: based upon the results of a non-GLP 5-Day Dose Range-Finding Study in which FEX0-07 was administered by gavage to 2 rats per group and sex.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Concentration, homogeneity and stability (after 2 hours and 7 days) of the dose formulations were determined in samples taken after experimental start. In addition, concentration, homogeneity and stability (stability in Group 2 and 3 only) of the dose formulations were determined in samples taken during Week 3 of the treatment.

Duration of treatment / exposure:

Duration of treatment:
Groups 1 to 3: 28 days
Group 4: 8 to 9 days

Frequency of treatment:

Frequency of administration: daily
(Dose volume: 5 mL/kg body weight).

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
Group 1: 0 mg/kg body weight
Basis:
other: Dose levels are expressed in terms of the test item as supplied

Doses / concentrations 2

Remarks:

Doses / Concentrations:
Group 2: 50 mg/kg body weight
Basis:
other: Dose levels are expressed in terms of the test item as supplied

Doses / concentrations 3

Remarks:

Doses / Concentrations:
Group 3: 150 mg/kg body weight
Basis:
other: Dose levels are expressed in terms of the test item as supplied

Doses / concentrations 4

Remarks:

Doses / Concentrations:
Group 4: 1000 mg/kg body weight
Basis:
other: Dose levels are expressed in terms of the test item as supplied

No. of animals per sex per dose:

FEX0-07 was administered by gavage to 2 rats per group and sex.

Control animals:

yes

Positive control:

no data

Examinations

Observations and examinations performed and frequency:

Mortality/Viability: observations for mortality/viability were recorded twice daily.

General Cageside Observations (Daily): the animals were observed for clinical signs once before commencement of administration; twice daily on Days 1 - 3, as well as once daily on days 4-28.

Detailed Clinical Observations (Weekly): the animals were observed in their home cages, outside their home cages in a standard arena and in the hand. These observations were performed in random sequence once before commencement of administration and once weekly (Weeks 1-3) thereafter.

The following hematology parameters were determined:
erythrocyte count, hemoglobin, hematocrit, mean corpuscular volume, red cell volume distribution width, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, hemoglobin concentration distribution width, Platelet (thrombocyte) count, reticulocyte count, reticulocyte maturity index, methemoglobin, total leukocyte count, differential leukocyte count, coagulation: (i) thromboplastin time and (ii) coltivated partial thromboplastin time.

The following clinical biochemistry parameters were determined:
glucose, urea, creatinine, bilirubin, total cholesterol, total triglycerides, phospholipids, aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase, glutamate dehydrogenase, creatine kinase, alkaline phosphatase, gamma-glutamyl-transferase, sodium, potassium, chloride, calcium, phosphorus inorganic protein, total albumin, globulin and albumin/globulin ratio.

Sacrifice and pathology:

Pathology

Necropsy
Sacrifice: after 4 weeks, on day 8 (one rat), on day 9 (nine rats).
All animals were weighed and necropsied.

All animals surviving to scheduled necropsy and all moribund animals were anesthetized by intraperitoneal injection of sodium pentobarbitone and killed by exsanguination.
Samples of the following tissues and organs were collected from all animals at necropsy and fixed in neutral phosphate buffered 4 % formaldehyde solution (unless otherwise indicated): adrenal glands, aorta, bone (sternum, femur including joint), bone marrow (femur), brain (al least 3 levels), cecum, colon, duodenum, epididymides (fixed in Bouin's solution), esophagus, ovaries, pancreas, pituitary gland, prostate gland (incl coagulating gland), rectum, salivary glands (mandibular, sublingual), sciatic nerve, seminal vesicles, skeletal muscle, skin, eyes with optic nerve (fixed in Davidson's solution), harderian gland (fixed in Davidson's solution), heart, ileum (with Peyer's patches), jejunum (with Peyer's patches), kidneys, larynx, lacrimal gland (exorbital), liver, lungs (infused with formalin at necropsy), lymph nodes (mesenteric, mandibular), mammary gland area, nasal cavity, spinal cord (cervical, midthoracic, lumbar), spleen, stomach, sestes (fixed in Bouin's solution), thymus, thyroid (incl. parathyroid gland), tongue, trachea, urinary bladder (infused with formalin at necropsy), uterus, vagina, gross lesions.

Other examinations:

Summary of parameters observed weekly:
- appearance: piloerection, salivation and hunched eosture
- motor: ataxia, tremor/twitching, prostration, circling and spasm
- behavior: hyperactivity, somnolence, increased exploration, reduced grooming and vocalization
- respiration: dyspnea, tachypnea and bradypenea
- reflexis: blink, pinna, iridic light reflex, push-off (hind leg), pain response, startle/hearing and righting reflex
- miscellanous: lacrimation, limbs cyanotic, mydriasis, miosis, exophthalmos and reduced muscle tane

Moreover, they were observed:
- food consumption: was recorded once during the acclimatization period and weekly thereafter
- body weights: body weights were recorded weekly during acclimatization, treatment and before necropsy
- functional observational battery: during week 4, relevant parameters presented earlier from a modified lrwin screen test were evaluated in all animals (Groups 1 to 3)
- grip strength: forelimb and hind limb grip strength measurements were performed
- locomotor activity: locomotor (decreased or increased) activity was measured quantitatively.

Statistics:

The following statistical methods were used to analyze (i) grip strength, (ii) locomotor activity, (iii) body weight, (iv) clinical laboratory investigations, (v) organ weights and (vi) ratios:
- the Dunnett-test (many to one t-test) based on a pooled variance estimate were applied if the variables could be assumed to follow a normal
distribution for the comparison of the treated groups and the control groups for each sex
- the Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data can not be assumed to follow a normal distribution
- Student's t-test was applied to locomotor activity and grip strength.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

All animals of Groups 1 to 3 survived until scheduled necropsy.

Mortality:

no mortality observed

Description (incidence):

All animals of Groups 1 to 3 survived until scheduled necropsy.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The mean body weight and the mean body weight gain of test item-treated rats receiving 50 mg/kg/day (Group 2) and 150 mg/kg/day (Group 3), respectively, were similar to the respective control data (Group 1 ).

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

The mean daily food consumption (absolute and body weight relative) was unaffected by the treatment with the test item at 50 mg/kg/day (Group 2) and 150 mg/kg/day (Group 3) in both sexes throughout the treatment period.

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

no effects observed

Description (incidence and severity):

No test item-related differences of toxicological relevance were noted in hematology parameters at any dose level after the treatment period.

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not specified

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Mean absolute organ weights were unaffected by the treatment with the test item.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No test item-related macroscopic changes were noted at any dose level.

Histopathological findings: non-neoplastic:

not specified

Histopathological findings: neoplastic:

not specified

Details on results:

Viability/Mortality
All animals of Groups 1 to 3 survived until scheduled necropsy.
Animals of Group 4 treated with 1000 mg/kg/day had to be killed in extremis on Day 8 (Female 40) and on Day 9 (Males 16 to 20 and Females 36 to 39) of the treatment period, respectively.
The exact cause of morbidity of the above mentioned decedents could not be established from the tissues and organs examined.

Observations
(i) Generai Cageside Observations (Daily): no test item-related changes of toxicological relevance were noted in Group 2 (50 mg/kg/day) and Group 3 (150 mg/kg/day) during daily cageside observations. From Day 21 slight local alopecia on the right cheek was noted in Male 12 receiving 150 mg/kg/day and persisted during the treatment period. Due to the low incidence of hair loss and in absence of any related observation this finding was considered to be of no toxicological relevance.

(ii) Detailed Clinical Observations (Weekly): no test item-related clinica! signs were noted during the weekly detailed observations (Weeks 1 - 3) in males and females administered 50 mg/kg/day (Group 2) and 150 mg/kg/day (Group 3). Slight local alopecia on the right side of the head was record ed in Male 12 treated with 150 mg/kg/day (Group 3). This finding was considered to be unrelated to the treatment with the test item.

(iii) Functional Observational Battery: no test item-related changes of toxicological relevance were noted during functional observational battery (Week 4) in rats at any dose level. In accordance with the daily general cageside observations, slight local alopecia on the right cheek of Male 12 was the only clinical sign observed and was considered to be without toxicological relevance.

(iv) Grip Strength: no test item-related differences in fore- and hindlimb grip strength of toxicological relevance were noted al any dose level.

(v) Locomotor Activity: no test item-related alterations in mean locomotor activity were noted al any dose level.

(vi) Food Consumption: as reported above, the mean daily food consumption (absolute and body weight relative) was unaffected by the treatment with the test item at 50 mg/kg/day (Group 2) and 150 mg/kg/day (Group 3) in both sexes throughout the treatment period.

(vii) Body Weights: as reported above, the mean body weight and the mean body weight gain of test item-treated rats receiving 50 mg/kg/day (Group 2) and 150 mg/kg/day (Group 3), respectively, were similar to the respective control data (Group 1 ).

Clinical Laboratory lnvestigations
(i) Hematology: the mean cell hemoglobin concentration was significantly lower in females treated with 50 mg/kg/day (Group 2, p<0.05) as well as 150 mg/kg/day (Group 3, p<0.01) exceeding the tolerance limit of the historical control data. lnsofar as the mean cell hemoglobin concentration of the respective controls (Group 1) was even higher and since no changes in related parameters were noted, this finding was considered to be incidental. The absolute lymphocyte count was significantly increased in males receiving 150 mg/kg/day (Group 3, p<0.05) and marginally exceeded the tolerance limit of the historical control data. Since this difference was not observed in females and unsupported by commensurate changes in related parameters, it was considered not to be of toxicological relevance. Minor statistically significant differences observed in other hematology parameters
remained within the tolerance limits of the historical reference data and were considered to be incidental.

(ii) Clinical Biochemistry: in males, increased calcium (p<0.05), protein and globulin concentrations (p<0.01) were recorded at 50 mg/kg/day (Group 2) and at 150 mg/kg/day (Group 3). Males showed decreased chloride concentrations (p<0.05) at 50 mg/kg/day, and reduced NG ratios (p<0.05) at both, 50 mg/kg/day and 150 mg/kg/day. In females, reduced creatinine (p<0.05) and elevated total bilirubin levels (p<0.05) were noted at 50 mg/kg/day (Group 2) and at 150 mg/kg/day (Group 3). At 50 mg/kg/day, increased NG ratio was noted (p<0.05). Although these differences attained statistical significance, they all remained within the limits of their historical reference data. Therefore, these findings may be considered not to be test item-related.

Pathology

(i) Organ weights: in males, significantly reduced mean brain weight (body weight relative: p<0.01) was noted at 50 mg/kg/day. Mean heart weight was significantly reduced at 50 mg/kg/day (body weight relative: p<0.01) and at 150 mg/kg/day (body weight relative and brain weight relative: p<0.05). Mean liver weight was significantly increased at 50 mg/kg/day, and significantly reduced at 150 mg/kg/day (brain weight relative: both p<0.05).
In females, a significant reduction in mean spleen weight was noted at 150 mg/kg/day (body weight relative: p<0.05). Overall, the few significant deviations in mean organ weight were considered to be incidental reflecting the usual individual variability.

(ii) Macroscopic findings: all rats were without gross lesions.

(iii) Microscopic Findings: there were a number of microscopic lesions, which distinguished test item-treated animals at 50 mg/kg/day (Group 2), and 150 mg/kg/day (Group 3) from controls (Group 1 ):
Liver: lncreased fatty change
Testes: Sertoli celi vacuolation and tubular atrophy.
Kidneys: lncreased mineralization of the cortico-medullary junction in females.
Due to the high incidence and due to the presence of a dose relationship, the above mentioned changes in liver, testes and female kidneys were considered to be test item-related. Additionally, a variety of other changes was found in this study. They commonly occur in laboratory rats of this strain and age under the conditions of this study type. Neither their incidences nor their distribution or morphologic appearance gave any indication of a treatment-related association.

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Microscopic findings

The findings that distinguished low and mid dose treated animals from controls were:

- liver: increased fatty change

- kidneys: increased mineralization at the cortico-medullary junction, only in female animals

- testes: Sertoli cell vacuolation and tubular atrophy.

Table 1. Incidence and mean severity of findings in liver

	Group 1		Group 2		Group 3
Finding	Males 5	Females  5	Males 5	Females  5	Males 5	Females  5
Fatty change Incidence/Mean severity	3/1.3	3/1.0	5/1.4	3/1.3	5/1.4	5/1.8

Table 2. Incidence and mean severity of findings in kidney

	Group 1		Group 2		Group 3
Finding	Males 5	Females  5	Males 5	Females  5	Males 5	Females  5
Mineralization - Incidence/Mean severity	-	1/1.0	-	4/1.0	-	4/1.3

Table 3. Incidence and mean severity of findings in testes

	Group 1		Group 2		Group 3
Finding	Males 5	Females  5	Males 5	Females  5	Males 5	Females  5
Sertoli cell vacuolation - Incidence/Mean severity	1/1.0	-	3/1.0	-	3/1.0	-
Tubular atrophy -  Incidence/Mean severity	-	-	1/1.0	-	3/1.0	-

Table 4. Incidence and mean severity of findings in animals of group 4

		Group 4
Organ	Finding	Males 5	Females 5
Liver	Hepatocyte necrosis Incidence/Mean severity	1/1.0	-
	Fatty change Incidence/Mean severity	-	5/1.0
	Hepatocellular hypertrophy Incidence/Mean severity	1/1.0	2/1.0
	Bile duct proliferation Incidence/Mean severity	1/1.0	2/1.0
Kidneys	Mineralization Incidence/Mean severity	-	4/1.5
Thymus	Atrophy Incidence/Mean severity	-	3/1.0
Jejunum	Lymphangiectasias lncidence/Mean severity	3/1.0	5/2.0

Table 5. Incidence and mean severity of findings in animals of group 4

Testes	Sertoli cell vacuolation Incidence/Mean severity	5/2.2	-
Epididymides	Hypospermia Incidence/Mean severily	4/1.3	-
	Cellular debris Incidence/Mean severily	5/1.0	-
Prostate gland	Atrophy Incidence/Mean severity	5/1.0	-

Table 6. Incidence and mean severity of findings in adrenal cortices

	Group 1		Group 2		Group 3
Finding	Males 5	Female 5	Males 5	Female 5	Males 5	Female 5
Fatty change Incidence/Mean severity	3/1.0	-	2/1.0	-	5/1.2	-

Applicant's summary and conclusion

Conclusions:

Under the conditions of this study all male and female animals of the high dose group had to be killed in extremis prematurely. Animal 40 was killed o n the eighth day of the study and all other animals of this group were killed on the ninth day.
The exact cause of morbidity of the above mentioned decedents could not be established from the tissues and organs examined. In spite of this, several findings were recorded in these animals that distinguished them from the other animals in this study. Due to their short time under treatment, the interpretation and meaning of these findings are controversial. At necropsy, performed at the end of treatment period, no macroscopic findings were recorded.
Microscopically, the following lindings were recorded in animals of groups 2 and 3:
- liver: increased latty change
- kidneys: increased mineralization at the cortico-medullary junction, only in female animals
- testes: Sertoli cell vacuolation and tubular atrophy
The above-mentioned changes in liver, female kidneys and testes can be present as incidental findings in 28-Day rat oral toxicity studies. However, in the present study either their incidence or mean severity or both are slightly increased in groups 2 and 3 when compared to control group 1. Consequently, they are considered as being treatment-related.

Executive summary:

The purpose of this subacute oral toxicity study was to assess the cumulative toxicity of FEX0-07 when administered daily to rats by gavage for a period of 28 days. This study should provide a rational basis for a risk assessment in man: the results of this study should indicate potential target organs and should identify chemicals with neurotoxic potential.

FEX0-07 was administered daily by oral gavage to 40 SPFbred Wistar rats (assigned to four dose groups each containing 5 animals per sex) at dose levels of 50 mg/kg body weight day (Group 2) and 150 mg/kg body weight day (Group 3) for a period of 28 days. A control group (Group 1) was treated similarly with the vehicle, corn oil, only. The animals of Groups 1 to 3 were sacrificed after 28 days of treatment. All animals of Group 4, treated with 1000 rng/kg body weight day, had to be killed in extremis on Days 8 and 9 of the treatment period, respectively. Clinical signs, outside cage observations, food consumption and body weights were recorded periodically during acclimatization and the treatment period. Functional observational battery, locomotor activity and grip strength were performed during Week4. After the treatment period, blood samples were withdrawn for hematology and plasma chemistry analyses. All animals were killed, necropsied and examined post mortem. Histological examinations were performed on organs and tissues from all control and mid dose animals. The same applied to all animals of Group 4, that had to be killed in extremis. From the animals of the low dose group, livers of males and females, kidneys of females as well as testes and adrenal cortices of males were examined to establish a no-effect level.