4-Ethoxy-2,3-difluoro-4'-(trans-4-propylcyclohexyl)-1,1'-biphenyl

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Jan 22, 2009 - Jan 26, 2010

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted October 03, 2008

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 (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

4-ethoxy-2,3-difluoro-4'-(trans-4-propylcyclohexyl)-1,1'-biphenyl

EC Number:

921-136-5

Cas Number:

189750-98-9

Molecular formula:

C₂₃H₂₈F₂O

IUPAC Name:

4-ethoxy-2,3-difluoro-4'-(trans-4-propylcyclohexyl)-1,1'-biphenyl

Test material form:

solid

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Species: Rat
Strain: Crl: WI (Han)
Breeder: Charles River, Germany

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Germany
- Age at study initiation: 7 weeks
- Weight at study initiation: 201 (m), 154 (f)
- Fasting period before study: no
- Housing: grouped
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22.4 - 25.1
- Humidity (%): 41.6 - 55.9
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12 h / 12 h

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

The test item was administered orally by gavage, once daily, 7 times a week for 4 weeks. The volume of administration was 5 mL/kg body weight. The volume of administration per animal was calculated by means of the LIM-System.

Vehicle:

other: 0.25 % aqueous hydroxypropyl methylcellulose (Methocel® K4M Premium)

Details on oral exposure:

VEHICLE
- Justification for use and choice of vehicle (if other than water): lab standard vehicle
- Concentration in vehicle: 0.25 % aqueous hydroxypropyl methylcellulose (Methocel® K4M Premium)
- Amount of vehicle (if gavage): 5 mL/kg bw
- Lot/batch no. (if required): -
- Purity: analytical grade

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The quantification of the test item in the dosing formulations was performed using a HPLC method with UV detection. During the course of the study each dosing formulation (including control) of two preparations periods were sampled and analyzed at the beginning and the end of usage, resulting in 4 time points of formulation analysis.

Duration of treatment / exposure:

4 weeks

Frequency of treatment:

once daily, 7 times a week for 4 weeks

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

0 mg/kg bw/d: 20 (10 m/ 10 f)
100 mg/kg bw/d: 10 (5 m/ 5 f)
300 mg/kg bw/d: 10 (5 m/ 5 f)
1000 mg/kg bw/d: 20 (10 m/ 10 f)

Control animals:

yes, concurrent vehicle

Details on study design:

- Rationale for animal assignment (if not random): random
- Rationale for selecting satellite groups: recovery
- Post-exposure recovery period in satellite groups: 2 weeks
- Section schedule rationale (if not random): random

Positive control:

no

Examinations

Observations and examinations performed and frequency:

Clinical Signs
Mortality, the behavior and appearance of each animal were checked twice daily at working days and once at off days, preferably at the same time each day. Symptoms were recorded with the LIM-System

Body Weight
Each rat was weighed before treatment and thereafter once a week. The parameter was recorded with the LIM-System.

Food consumption
Food consumption was determined once a week by weighing the food per cage which had not been consumed. The parameter was recorded with the LIM-System.

Gross Pathology
At the time scheduled the rats were anesthetized by a carbon dioxide air mixture and exsanguinated by opening the abdominal vessels. They were necropsied and examined for gross
pathological alterations. All findings were recorded with the LIM System.

Body and Organ Weights
The body and organs weights were recorded with the LIM-System. Based on the absolute organ weights the relative organs weights (related to 100 g body weight) were calculated. For all animals the following weights were determined:
Terminal body weight (after exsanguination)
Heart
Liver
Kidneys (together)
Spleen
Thymus
Testes (together)
Prostate
Uterus
Ovaries (together)
Adrenals (together after fixation)
Thyroids with Parathyroids (together after fixation)
Brain (after fixation)
Seminal vesicles
Epididymides (together)

Histopathology
The organs and tissues of main kill animals were fixed, histotechnically processed and examined
as listed below.

Main kill
Adrenal (2)
Aorta
Bone with knee joint (os femoris)
Bone with bone marrow (sternum, femur)
Brain (cerebrum, cerebellum, brain stem)
Esophagus
Eye (2)
Heart
Intestine, large
Cecum
Colon
Rectum
Intestine, small
Duodenum
Jejunum
Ileum
Kidney (2)
Larynx
Liver (left lateral and right medial lobe)
Lung (with mainstem bronchi)
Lymph nodes
mandibular (2)
mesenteric
Mammary gland (inguinal)
Micro transponder
Muscle, skeletal (thigh)
Nasal turbinates
Nerve, optic (2)
Nerve, sciatic
Pancreas
Parathyroid (2)
Peyer’s Patches
Pituitary
Reproductive organs, female
Ovary (2)
Oviduct (2)
Uterus (cornu/corpus/cervix)
Vagina
Reproductive organs, male
Epididymis (2)
Prostate
Seminal vesicle
Testis (2)
Salivary gland (2)
(submandibular, parotid, sublingual)
Skin (inguinal)
Spinal cord (cervical, thoracal, lumbal)
Spleen
Stomach (proventricular, fundic, pyloric)
Thymus
Thyroid (2)
Tongue
Trachea
Ureter (2)
Urinary bladder
Zymbal's gland (2)
All tissues showing abnormalities

For recovery animals tissue fixation was performed as for the main kill group.
The adrenals, female reproductive organs (ovary, uterus, vagina) and pituitary were investigated in all dose groups of the main kill and in recovery animals.
All histopathology findings were recorded with the LIM-System.

Sacrifice and pathology:

The animals were necropsied, examined for gross pathological alterations, the weights of selected organs were recorded and histotechnical procedures and histopathological examinations were performed.

Statistics:

All parameters were analyzed separately for each sex and time. To take the number of dose groups into account, all the test procedures used maintain a multiple significance level of alpha= 0.05.

Absolute body weight, body weight gain (differences to baseline values on day 0), food consumption, and organ weights - relative and absolute - of the dose groups were compared with those of the control, using the multiple two-sided Dunnett-Test (Dunnett 1955, 1964). When the parameters were compared in the recovery period between the control (group 1) and high dose (group 4), the standard t-test (Winer, 1970) was used.
Software: Body weight gain, food consumption and organ weights were evaluated within
the LIM-System.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 1000 mg/kg bw/d male animals showed slight significant increases of body weight on days 7, 21, 28, and 42 not correlated with a significant body weight gain. Females of this dose group were not affected.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption showed some slight significant changes of dose groups versus control (increase and decrease), however, the changes were small and mostly not dose-dependent. The high dose males (1000 mg/kg bw/d) showed a slight significant increase of food consumption that correlates with the slight increase of body weight.

Food efficiency:

no effects observed

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

effects observed, treatment-related

Description (incidence and severity):

Water consumption showed some slight significant increases of low dose groups (males and females, 100 mg/kg bw/d) versus control at a few time points. High dose males (1000 mg/kg bw/d) showed a significant increase of water consumption during the recovery period. No histopathological correlate was observed.

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

Hematological evaluation at the end of the 28-day treatment period (week 5) and the recovery period (week 7) showed no relevant changes of hematological or coagulation parameters. Slight variations of the platelet number in week 7 were within the normal internal laboratory range of values.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Clinical chemistry at the end of the 28-day treatment period (week 5) and the recovery period (week 7) showed no relevant changes of serum-electrolytes, -substrates, -proteins, and -enzymes. A few parameters showed slight changes that were minimal and within the normal internal laboratory range of values.

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

The functional observational battery (FOB) was performed in designated animals on day 0, day 7 and day 28 of treatment. No significant changes were noted in the autonomous, neuromuscular, sensomotoric or central nervous domain at any time point. Motor activity was measured in designated animals on day 28. No dose-dependent significant changes of locomotor activity in both genders versus control were noted.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

At gross pathology only sporadic and spontaneous alterations were diagnosed. The body weights were not affected at all dose levels. Males showed decreased liver weights at 100 and 1000 mg/kg bw/d without clear dose relationship. In females the liver weights were significantly decreased at 1000 mg/kg bw/d.

Gross pathological findings:

no effects observed

Description (incidence and severity):

At necropsy only spontaneous alterations were observed. Toxicological relevant body or organ weight deviations were not observed in any dose group of main kill or recovery animals.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

At histopathology a slightly increased incidence of microgranuloma in the liver of group 4 rats (1000 mg/kg bw/d) of both sexes were observed. This finding is considered to be of questionable toxicological relevance as microgranulomas are known to occur spontaneously in laboratory rats. After a 2-week recovery period, the incidence of liver alterations did not differ between the control and treatment groups.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no treatment related alterations were present at 100, 300 and 1000 mg/kg bw/day for 4 weeks in rats of both genders

Target system / organ toxicity

Key result

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

1000 mg/kg bw/d were considered to be the NOAEL (no observed adverse effect level) as no treatment related alterations were present at 100, 300 and 1000 mg/kg bw/day for 4 weeks in rats of both genders.

Executive summary:

A study according to OECD 407 was conducted to evaluate the test item for repeated dose toxicity in rats. The test material was administered orally by gavage, once daily, 7 times a week for 4 weeks to 3 groups of male and female Crl:WI (Han) rats at doses of 100, 300 or 1000 mg/kg bw/d. A similarly constituted control group received the vehicle, 0.25 % aqueous hydroxypropyl methylcellulose (Methocel® K4M Premium), and served to generate contemporary control data. The control and high dose groups consisted of 10 male and 10 female rats each. The low dose and mid dose groups consisted of 5 male and 5 female rats each. At the end of the treatment period, 10 (5 males and 5 females) rats were scheduled for necropsy. The remaining rats of groups 1 and 4 were scheduled for a 2-week recovery period. The rats were gang-housed under conventional conditions. All rats were subjected to macroscopic and histopathological examinations. Selected organs were weighed from each rat at the end of the treatment period.

Formulation analysis revealed that the dose groups received the anticipated concentrations and no test material was detected in the control formulations.

All animals survived the treatment period and recovery period until their scheduled death.

No clinical symptoms in the animals were noted.

The functional observational battery (FOB) was performed in designated animals on day 0, day 7 and day 28 of treatment. No significant changes were noted in the autonomous, neuromuscular, sensomotoric or central nervous domain at any time point. Motor activity was measured in designated animals on day 28. No dose-dependent significant changes of locomotor activity in both genders versus control were noted.

Body temperature did not show any dose-related changes from control on days 0, 7, and 28. Body weight and body weight gain were not impaired by the treatment with the test material at doses of up to 300 mg/kg bw/d. At 1000 mg/kg bw/d male animals showed slight significant increases of body weight on days 7, 21, 28, and 42 not correlated with a significant body weight gain. Females of this dose group were not affected.

Food consumption showed some slight significant changes of dose groups versus control (increase and decrease), however, the changes were small and mostly not dose-dependent. The high dose males (1000 mg/kg bw/d) showed a slight significant increase of food

consumption that correlates with the slight increase of body weight.

Water consumption showed some slight significant increases of low dose groups (males and females, 100 mg/kg bw/d) versus control at a few time points. High dose males (1000 mg/kg bw/d) showed a significant increase of water consumption during the recovery period. No histopathological correlate was observed.

Hematological evaluation at the end of the 28-day treatment period (week 5) and the recovery period (week 7) showed no relevant changes of hematological or coagulation parameters. Slight variations of the platelet number in week 7 were within the normal internal laboratory range of values.

Clinical chemistry at the end of the 28-day treatment period (week 5) and the recovery period (week 7) showed no relevant changes of serum-electrolytes, -substrates, -proteins, and -enzymes. A few parameters showed slight changes that were minimal and within the normal internal laboratory range of values.

The semi-quantitative urinalysis showed that no toxicologically relevant differences between treatment groups and controls.

At gross pathology only sporadic and spontaneous alterations were diagnosed. The body weights were not affected at all dose levels. Males showed decreased liver weights at 100 and 1000 mg/kg bw/d without clear dose relationship. In females the liver weights were significantly decreased at 1000 mg/kg bw/d.

At histopathology a slightly increased incidence of microgranuloma in the liver of group 4 rats (1000 mg/kg bw/d) of both sexes were observed. This finding is considered to be of questionable toxicological relevance as microgranulomas are known to occur spontaneously in laboratory rats. After a 2-week recovery period, the incidence of liver alterations did not differ between the control and treatment groups.

1000 mg/kg bw/d were considered to be the NOAEL (no observed adverse effect level) as no treatment related alterations were present at 100, 300 and 1000 mg/kg bw/day for 4 weeks in rats of both genders.