4-Ethyl-2',3'-difluor-4''-propyl-1,1':4',1''-terphenyl

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Aug 07, 2012 - Feb 20, 2013

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

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'-ethyl-2,3-difluoro-4-(4-propylphenyl)-1,1'-biphenyl

EC Number:

605-092-1

Cas Number:

157248-25-4

Molecular formula:

C₂₃H₂₂F₂

IUPAC Name:

4'-ethyl-2,3-difluoro-4-(4-propylphenyl)-1,1'-biphenyl

Test material form:

solid: bulk

Specific details on test material used for the study:

Purity: >99.90%

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: 233 (m), 165 (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

DETAILS OF FOOD AND WATER QUALITY:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 1°C
- Humidity (%): 37 - 65 %
- 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:

PREPARATION OF DOSING SOLUTIONS:


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
- 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.:

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

Doses / concentrations 3

Dose / conc.:

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

Doses / concentrations 4

Dose / conc.:

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

No. of animals per sex per dose:

0 mf/kg: 20 (10 m/ 10 f)
3 mg/kg: 10 (5 m/ 5 f)
10 mg/kg: 10 (5 m/ 5 f)
30 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
- 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 (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.

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 100g body weight) were calculated. For all
animals the following weights were determined:
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 F
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:

no effects observed

Description (incidence and severity):

No treatment-related clinical signs, changes of body weight, and body weight gain were
observed in any dose group of both genders during the treatment and recovery period.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption was unchanged during the treatment period in both genders, and in
females during the recovery period. The males showed a slightly decreased food
consumption during the recovery period that is not considered biologically relevant.

Food efficiency:

no effects observed

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

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, treatment-related

Description (incidence and severity):

At histopathology examination the female rats of all groups exhibited cytoplasmatic vacuolation in the zona fasciculata of the adrenal gland. The males were not affected. There was a clear dose dependency
with regard to incidence and severity as one female at 3 mg/kg was affected to a mild degree; all females at 10 mg/kg showed a minimal to mild degree and all females at 30 mg/kg exhibited a moderate degree. The high dose recovery females showed a vacuolation of the zona fasciculata which was more pronounced than the main kill females as a moderate to massive degree was noted in all animals. Two females of the recovery group showed minimal to mild degeneration/ necrosis of inner cortical cells in addition.
In the ovary, minimal cytoplasmic vacuolation of interstitial cells was detected in one rat at 3 mg/kg, a mild degree was seen in four rats at 10 mg/kg and a moderate degree in all 5 rats at
30 mg/kg. In recovery animals at 30 mg/kg incidence and degree of cytoplasmic vacuolation of interstinal cells was consistent with the main kill animals at this dose.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Effect levels

Key result

Dose descriptor:

NOEL

Effect level:

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

Sex:

male

Basis for effect level:

histopathology: non-neoplastic

Target system / organ toxicity

Critical effects observed:

no

Lowest effective dose / conc.:

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

System:

urinary

Organ:

adrenal glands

Applicant's summary and conclusion

Conclusions:

Daily oral administration of 3, 10, or 30 mg/kg of 4-Ethyl-2',3'-difluor-4''-propyl-1,1':4',1''-terphenyl to rats for 4 weeks was clinically tolerated with no mortality, clinical signs, changes of body weight, or
relevant changes of food consumption.

However, at histopathology, alterations in the adrenal gland and ovary in female rats were observed at all dose levels. Dose-dependent vacuolation in the adrenal cortex and
vacuolation of interstitial cells of the ovary were observed, at the low dose of 3 mg/kg one rat was affected. No signs of reversibility were discernable after the 2-week treatment free
period. The cytoplasmic vacuolation in the adrenal and ovary might reflect persistent metabolic changes due to impairment of steroidogenesis and/or retention of the xenobiotic.

Therefore, for the females a NOEL (no effect level) or NOAEL (no observed adverse effect level) could not be established. For the males, a NOEL could be established at 30
mg/kg under the conditions of this study.

Executive summary:

 

Objective

4-Ethyl-2',3'-difluor-4''-propyl-1,1':4',1''-terphenyli s an industrial chemical which needs to be registered in the European Union according to Annex VIII of the REACH regulation (>10-100 tons per annum). Such registrations require data on repeat dose toxicity, i.e. a subacute toxicity study in rats with oral administration.

 

Study Design

4-Ethyl-2',3'-difluor-4''-propyl-1,1':4',1''-terphenyl 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 3, 10 or 30 mg/kg. 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.

 

Results

 

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 and recovery period.

 

No treatment-related clinical signs, changes of body weight, and body weight gain were observed in any dose group of both genders during the treatment and recovery period.

Food consumption was unchanged during the treatment period in both genders, and in females during the recovery period. The males showed a slightly decreased food

consumption during the recovery period that is not considered biologically relevant.

At necropsy only spontaneous alterations were observed. Body and organ weight determinations revealed no relevant deviations in any dose group of main kill or recovery

animals.

At histopathology the female rats of all dose groups (3, 10, and 30 mg/kg) exhibited a cytoplasmatic vacuolation in the zona fasciculata of the adrenal gland. Incidence and

severity were clearly dose-dependent as one female at 3 mg/kg was affected to a mild degree; all females at 10 mg/kg showed a minimal to mild degree and all females at

30 mg/kg exhibited a moderate degree. The males were not affected.

At the end of recovery, the high dose (30 mg/kg) females showed a moderate to massive vacuolation of the zona fasciculate. Severity was more pronounced in the main kill

females. Two females of the recovery group showed minimal to mild degeneration/necrosis of inner cortical cells in addition.

In the ovary, minimal cytoplasmic vacuolation of interstitial cells was detected in one rat at 3 mg/kg, a mild degree was seen in four rats at 10 mg/kg and a moderate degree in all 5

rats at 30 mg/kg. At the end of recovery, high dose (30 mg/kg) recovery animals had the same incidence and degree of cytoplasmic vacuolation of interstitital cells as the main kill

animals of this dose level.

 

 

Conclusions

Daily oral administration of 3, 10, or 30 mg/kg of 4-Ethyl-2',3'-difluor-4''-propyl-1,1':4',1''-terphenyl to rats for 4 weeks was clinically tolerated with no mortality, clinical signs, changes of body weight, or

relevant changes of food consumption.

 

However, at histopathology, alterations in the adrenal gland and ovary in female rats were observed at all dose levels. Dose-dependent vacuolation in the adrenal cortex and

vacuolation of interstitial cells of the ovary were observed, at the low dose of 3 mg/kg one rat was affected. No signs of reversibility were discernable after the 2-week treatment free

period. The cytoplasmic vacuolation in the adrenal and ovary might reflect persistent metabolic changes due to impairment of steroidogenesis and/or retention of the xenobiotic.

 

Therefore, for the females a NOEL (no effect level) or NOAEL (no observed adverse effect level) could not be established. For the males, a NOEL could be established at 30

mg/kg under the conditions of this study.