4-[difluoro(3,4,5-trifluorophenoxy)methyl]-2',3,5-trifluoro-4''-propyl-1,1':4',1''-terphenyl

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:

Jul 27, 2009 - Feb 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 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

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: Chinese Guidelines for the Testing of Chemicals (HJ/T153-2004)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

4-{4-[difluoro(3,4,5-trifluorophenoxy)methyl]-3,5-difluorophenyl}-3-fluoro-4'-propyl-1,1'-biphenyl

EC Number:

619-490-8

Cas Number:

303186-36-9

Molecular formula:

C27H22F8O2

IUPAC Name:

4-{4-[difluoro(3,4,5-trifluorophenoxy)methyl]-3,5-difluorophenyl}-3-fluoro-4'-propyl-1,1'-biphenyl

Test material form:

solid: bulk

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: 8w
- Weight at study initiation: mean: 241.7 (m), 176.6 (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): 22.3 - 24.8°C
- Humidity (%): 41.6 - 77.4 %
- Photoperiod (hrs dark / hrs light): 12 h / 12 h

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

Administration was done orally by gavage, once daily, 7 times a week. 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:

The test material was administered orally by gavage. The control rats received the vehicle, 0.25% aqueous hydroxypropyl methylcellulose (Methocel® K4M Premium), at the same frequency as the animals treated with the test item.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The test material in 0.25% aqueous hydroxypropyl methylcellulose (Methocel® K4M Premium), was stable for at least 14 days and homogenous as was shown in a prestudy. Therefore, in the beginning of the study the preparations were done weekly. Samples were scheduled to be taken from all dose groups at six different time points at least, once directly after delivery and once at the end of three preparation periods (under use conditions) for determination of concentrations at the Institute of Toxicology using an HPLC method with UV detection. No test item was detected in the formulation samples of the control group. The test item was detected in all samples of the test item groups. However, the concentration levels of several samples were outside the predefined acceptance limit of ± 15% of the nominal concentrations, which could be attributed to the large vessels used for the preparation of the suspensions. Consequently, based on the results of this formulation analysis it was decided to produce daily preparations of the test item using the smaller vessels from 27 August 2009 onwards.

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.:

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

Doses / concentrations 3

Dose / conc.:

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

Doses / concentrations 4

Dose / conc.:

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

No. of animals per sex per dose:

0 mf/kg: 20 (10 m / 10 f)
30 mg/kg: 10 (5 m / 5 f)
100 mg/kg: 20 (10 m / 10 f)
300 mg/kg: 20 (10 m / 10 f)

Control animals:

yes, concurrent vehicle

Details on study design:

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

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(s) 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.

Water consumption
Water consumption was determined twice a week by measuring the water per cage which had not been consumed. The parameter was recorded with the LIM-system.

Laboratory tests
In weeks 5 and 7 blood was taken under inhalation anesthesia. Except for the animals that were kept for recovery, blood was taken before necropsy in week 5. The blood samples were divided for hematological and clinico-chemical examinations. Before blood sampling the animals were kept in metabolism cages for the collection of urine for approximately 18 hours without food.

Gross Pathology
Rats were necropsied and examined for gross pathological alterations. Animals were killed by anesthesia with a carbon dioxide air mixture and exsanguination after opening of the abdominal vessels. 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 100g 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 following organs and tissues were fixed, histotechnically processed and examined:
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

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).
For the evaluation of the clinico-chemical parameters and the hematological parameters, the Wilcoxon rank sum test (Hollander, 1973) was used to make pairwise comparisons of the dose groups with the control group. The correction for multiple testing was done according to Bonferroni-Holm (Holm, 1979).
Software: Body weight gain, food and water consumption, organ weights, hematological parameters, clinico-chemical parameters, were evaluated within the LIM-system.

Results and discussion

Results of examinations

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Hair loss was observed on day 32, 16 and 24 in each one female of the control, 100 mg/kg and 300 mg/kg dose group, respectively. Hair loss is not an unusual observation in Wistar-Han rats. Since no dose-dependency could be observed, the symptom is not considered substance-related.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Body weight and body weight gain were not significantly changed when comparing dose groups with concurrent controls.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption in males did not show any changes between treated animals and controls on any day measured. In the females a slight increase of food consumption in group 3 (100 mg/kg) versus control was observed on day 7 (statistically significant) and a slight decrease in group 4 (300 mg/kg) versus control was noted on day 35 (recovery period, statistically significant). The differences to control were significant but very slight and of no biological relevance.

Food efficiency:

not examined

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

effects observed, treatment-related

Description (incidence and severity):

Water consumption was slightly reduced at several time points (days 17, 24, 28, 35, 38, and 42) in male group 3 animals (100 mg/kg) versus control. No changes were seen in the other dose groups or the female animals. As the changes were very slight and no dosedependency could be observed, the changes are not considered biologically relevant.

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

In week 5, at the end of the treatment, the group 3 females (100 mg/kg) showed a slight (but statistically significant) decrease of WBC. However, the values were within the normal internal laboratory range of values. In group 4 females (300 mg/kg) the relative number of neutrophilic granulocytes was increased together with a reduced relative number of lymphocytes. The absolute numbers were still unchanged and comparable to the control animals. At the end of the recovery period, no statistically significant changes were observed any more. No changes of red blood cells were observed in week 5 and 7. No changes of coagulation parameters were observed in week 5 and 7.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Overall, the changes of serum electrolytes, serum-substrates and –proteins, and serum enzymes are very slight and always in the range of the normal internal laboratory range of values and without toxicological relevance.

Urinalysis findings:

no effects observed

Description (incidence and severity):

Urinalysis revealed no pathological alteration in test weeks 5 and 7.

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 treatment-related relevant changes were observed at any time point in both genders in the different domains (autonomous, neuromuscular, sensomotoric and central nervous system). The locomotor activity was measured on day 28 of treatment. All tested parameters did not reveal any significant changes between treated animals versus concurrent controls on day 28 in both genders.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Organ weight deviations that achieved statistical significance were not observed in main kill animals. Toxicologically relevant organ weight deviations were not observed in the recovery group. Decreased absolute ovary weights in group 3 and increased epididymis weights in group 4 depend on single animals. The individual ovary and epididymides organ weights of these animals did not exceed those seen in controls.

Gross pathological findings:

no effects observed

Description (incidence and severity):

At gross pathology only spontaneous and sporadic findings were noted.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

Treatment-related organ alterations were not observed.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Effect levels

Key result

Dose descriptor:

NOEL

Effect level:

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

Sex:

male/female

Basis for effect level:

other: No treatment-related changes during the inlife observations, behavior parameters, clinical pathology evaluation or organ alterations at necropsy, organ weighing and histopathology examination.

Target system / organ toxicity

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

Administration of the test item for 4 weeks followed by a 2 week treatmentfree recovery period did not induce any treatment-related changes during the inlife observations, behavior parameters, clinical pathology evaluation or organ alterations at necrospy, organ weighing and histopathology examination. Therefore, the dose of 300 mg/kg is considered the NOEL (no effect level).

Executive summary:

The informtion for this endpoint study record was obtained from an experimental study. The OECD GLP criteria were met and the methods applied are fully compliant with OECD TG 407.

Administration of the test item for 4 weeks followed by a 2 week treatmentfree recovery period did not induce any treatment-related changes during the inlife observations, behavior parameters, clinical pathology evaluation or organ alterations at necrospy, organ weighing and histopathology examination. Therefore, the dose of 300 mg/kg is considered the NOEL (no effect level).