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

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016-05-04 to 2017-06-13

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

Principles of method if other than guideline:

Health Effects guidelines, OPPTS 870.3550, Reproduction/ Developmental Toxicity Screening Test. EPA 712-C-00-367, July 2000

Commission Regulation (EC) No. 440/2008, L 142, Appendix Part B, May 30, 2008

GLP compliance:

yes (incl. QA statement)

Remarks:

(Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany)

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: 99.93 %

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

Test System
Species/strain: healthy Wistar rats, Crl: WI(Han) (Full Barrier)
Source: Charles River, 97633 Sulzfeld, Germany
Sex: male and female; the female animals were non-pregnant and nulliparous.
Age at the start of the treatment period: approx. 14-15 weeks old
Body weight at the allocation of the animals to the experimental groups:
males: 348 - 409 g (mean: 361.36 g, ± 20% = 289.09 – 433.63 g)
females: 205 - 250 g (mean: 229.53 g, ± 20% = 183.62 – 275.43 g)

The animals were derived from a controlled full-barrier maintained breeding system (SPF). According to Art. 9.2, No. 7 of the German Act
on Animal Welfare the animals were bred for experimental purposes.
This study was performed in an AAALAC-accredited laboratory. According to German animal protection law, the study type has been reviewed and accepted by local authorities. Furthermore, the study has been subjected to Ethical Review Process and was authorised by the Bavarian animal welfare administration.
The animal study was authorized by local government under file no. 55.2-1-54-2532.0-31-2014.

Housing and Feeding Conditions
- Full barrier in an air-conditioned room
- Temperature: 22 +/- 3°C
- Relative humidity: 55 +/- 10%
- Artificial light, sequence being 12 hours light, 12 hours dark
- Air change: 10 x / hour
- Free access to Altromin 1324 maintenance diet for rats and mice
- Free access to tap water, sulphur acidified to a pH of approximately 2.8 (drinking water, municipal residue control, microbiological controls at regular intervals). Acidification of the water applies to the drinking water for mice and rats only. The purpose of the acidification is to provide additional protection against microbial contamination. This is a standard veterinary practice at BSL and should not be associated with any risk for long-term studies.
- Animals were housed in groups of 5 animals / sex / cage in IVC cages (type IV, polysulphone cages) during the premating period for both males and females and during postmating period for males depending on the mating status. During mating period males and females were housed together in ratio 1:1 (male to female). After the confirmation of mating, females were kept individually during gestation/lactation period and males were returned to their original cage. In each cage Altromin saw fibre was used as bedding.
- Certificates of food, water and bedding are filed for two years at BSL Munich and afterwards archived at Eurofins Munich
- Adequate acclimatisation period (at least 5 days) under laboratory conditions

Preparation of the Animals
Prior to the start of the treatment period a detailed clinical observation outside the home cage was made. None of the animal showed showing pathological signs before the first administration.
Before dosing all females were screened for two weeks for regular estrous cyclicity and animals (10 females/ group) with regular estrous cycle (4-5 day cycle) were used in the study.
Before the first administration all animals to be used for the study were weighed and assigned to the experimental groups with achieving a most homogenous variation in body weight throughout the groups of males and females, respectively.
Each animal was marked with its identification number by individual ear tattoo or tail marking.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

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

Remarks:

Specifications provided by the supplier Manufacturer: Methocel® K4M Premium Batch No.: # ZE 10012NO2 Physical State: liquid Storage Conditions: at room temperature Expiry Date: not applicable

Details on exposure:

Preparation of the Test Item Formulations

The vehicle was selected based on the test item’s characteristics and testing guideline.
The test item, as delivered, was weighed into a tared plastic vial on a suitable precision balance. The vehicle aqueous hydroxypropyl methylcellulose (Methocel® K4M Premium) was added to give the appropriate final concentration. The formulation sample was dispersed by ultraturrax for at least 3 minutes.
 
According to the results obtained from stability investigations (Eurofins Munich Study no. 160287 the test item formulation was prepared once a week. The test item formulation was stored at approximately 2 to 8 °C in refrigerator.
Homogeneity of the test item in the vehicle was maintained by stirring the prepared suspension thoroughly before and during every dose administration.
The vehicle was also used as control item.


Experimental Groups and Doses

In consultation with the sponsor and available data from earlier studies, the following doses were selected for the 3 dose groups (LD = low dose, MD = medium dose, HD = high dose) and 1 control group (C). The animals were treated with the test item formulation or vehicle on 7 days per week for a maximum period of 63 days, i.e. during 14 days of pre-mating and maximum 14 days of mating in both males and females, during the gestation period and up to post-natal day 12 in females. Males were dosed after the mating period until the minimum total dosing period of 28 days was completed.

C 0 mg/kg bw/ day (Male No.:1-10/ Female No.:41-50)
LD 1 mg/kg bw/ day (Male No.:11-20/ Female No.:51-60)
MD 3 mg/kg bw/ day (Male No.:21-30/ Female No.:61-70)
HD 10 mg/kg bw/ day (Male No.:31-40/ Female No.:71-80)

The highest dose level was chosen with the aim of inducing toxic effects, but not death or severe suffering. Thereafter, a descending sequence of dose levels was selected with a view to demonstrate any dosage related response and NOAEL. The animals in the control group were handled in an identical manner to the test group subjects and received corn oil using the same volume as used for the dose groups.



Administration of Doses

The test item formulation and vehicle were administered at a single dose to the animals by oral gavage. The application volume for all groups was 5 mL/kg body weight (depending on the solubility). For each animal the individual dosing volume was calculated on the basis of the body weight most recently measured.
Concentration: C: 0 mg/mL; LD: 0.2 mg/mL; MD: 0.6 mg/mL; HD: 2 mg/mL

Details on mating procedure:

Mating was performed using a ratio of 1:1 (male to female). The vaginal smear of the females was checked every morning after the start of the mating period to confirm the pregnancy. If the vaginal smear of a particular female was not found to be sperm-positive, the actual stage of the estrous cycle on that day was documented. The day of the vaginal plug and/or sperm was considered as day ‘0’ of gestation. The cages were arranged in such a way that possible effects due to cage placement were minimised.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Before beginning of the treatment period, formulation samples were prepared and analysed in order to obtain knowledge about stability and homogeneity of the
test item in the chosen vehicle at Eurofins Munich as part of a separate GLP study (study no. 160287).
During the study samples were collected for the investigation of homogeneity and nominal substance concentration.
Samples for homogeneity analysis were taken from the top, middle and bottom of the high dose and the low dose formulation in study week 1 and
5 (12 samples).
Samples for the nominal concentration verification were taken in study week 1 (first day of pre-mating period), 3 (first week of mating),
5 (gestation) and in the last week of the study (gestation/lactation) from all groups (16 samples).
All formulation samples collected during the study were stored only under conditions according to stability analysis (160287) and analysed within 10 days.
These samples were analysed at Eurofins Munich under the BSL study no. 160288.
Each sample was retained twice (sample A and B) with a volume of approx. 5 mL. The samples B were retained at BSL Munich as back-up until the analysis was performed, and discarded after completion of the final study report.
All samples were stored between -15 °C and -35 °C (conditions according to stability analysis (160287). The A samples were delivered to Eurofins Munich.
The exact procedure was described in a phase plan which was amended to the study plan. The results are reported in the appendix of the final report.

Duration of treatment / exposure:

The test item was administered daily in graduated doses to 3 groups of test animals, one dose level per group for a treatment period up to 63 days, i.e. during 14 days of pre-mating and maximum 14 days of mating in both males and females, during the gestation period and up to post-natal day 12 in females. Males were dosed after the mating period until the minimum total dosing period of 28 days was completed.

Frequency of treatment:

7 days/week

Details on study schedule:

Arrival of the Test Item: 20 January 2016
Study Initiation Date: 04 May 2016
Delivery of Animals: 12 May 2016
Acclimatisation Period: 12 May to 18 May 2016
Experimental Starting Date: 18 May 2016
Experimental Completion Date: 25 July 2016
Completion Date of Delegated Phase (Histopathology): 31 May 2017
Completion Date of Delegated Phase (Formulation Analysis): 29 March 2017

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

1 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

3 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

10 mg/kg bw/day (nominal)

No. of animals per sex per dose:

100 animals (40 males and 60 females) were included in the study. 60 females were screened for regular estrous cycles for 14 days before the treatment initiation and only 40 females (10 females/ group) showing regular estrous cycles were continued in the study. Remaining not selected 20 females were discarded without any observations or used for other appropriate studies/procedures.

Control animals:

yes, concurrent vehicle

Details on study design:

Justification for Dose Selection
Based on the results of earlier 28 day toxicity studies performed by the sponsor with this test item, dose levels had been selected for this study. In one 28 day toxicity study, test item was orally administered to male and female animals for 28 days at the dose levels of 100, 300 and 1000 mg/kg bw/day. These dose levels were well tolerated by the animals with no mortality, effect on clinical signs, body weight change, food and water consumption, hematology and clinical biochemistry at the end of treatment and recovery period. Histopathological examination of the dose groups revealed cytoplasmic vacuolation of the adrenal cortex, especially in female rats, combined with slight parenchymal degeneration and single cell necrosis. Cytoplasmic vacuolation was also observed in interstitial cells of the ovary. All investigated dose groups were affected in a comparable manner. No signs of reversibility were discernable after the 2 week treatment free period.
In another 28 day repeated dose toxicity study with this test item at dose levels of 3, 10 and 30 mg/kg bw/day, 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. 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 of the findings were clearly dose-dependent. The males were not affected. 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. The histopathology findings were not reversible after 2 weeks recovery period.
The aim of the present study was to provide initial information on the effects on reproduction and / or development, at doses that cause the histopathological findings as observed in the previous studies performed by the sponsor (i.e. 1, 3 and 10 mg/kg bw /day).

Examinations

Parental animals: Observations and examinations:

Clinical Observations
General clinical observations were made at least once a day, preferably at the same time each day. The health condition of the animals was recorded. Twice daily all animals were observed for morbidity and mortality except on weekends and public holidays when observations were made once daily.
Clinical observations included spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoea, asphyxia, vocalisation, diarrhoea, changes in the skin and fur, eyes and mucous membranes (salivation, discharge), piloerection and pupil size. Changes in gait, posture, response to handling as well as the presence of clonic or tonic movements, stereotypes, difficult or prolonged parturition or bizarre behaviour were recorded.


Body Weight and Food Consumption
The animals were weighed once before the assignment to the experimental groups, on the first day of dosing and weekly thereafter as well as at the end of the study. During pregnancy, females were weighed on gestation days (GD) 0, 7, 14 and 20 and within
24 hours of parturition (day 0 post-partum), on PND 4 and PND 13 along with pups. All animals were weighed at termination.
Food consumption was measured on the corresponding days of the body weight measurements after the beginning of the dose administration. Food consumption was not measured during the mating period in males and females and the post-mating period in males.

Oestrous cyclicity (parental animals):

Estrous cycles were monitored before treatment initiation to select for the study females with regular estrous cyclicity. Vaginal smears were also examined daily from the beginning of the treatment period until evidence of mating. Vaginal smears were examined on the day of necropsy to determine the stage of estrous cycle.

Litter observations:

The duration of the gestation was recorded and was calculated from day 0 of the pregnancy. Each litter was examined as soon as possible after delivery of the dam to establish the number and sex of pups, stillbirths, live births, runts and the presence of gross abnormalities.
Live pups were counted and sexed and litters weighed within 24 hours of littering
(PND 0) and on PND 4 and PND 13. Live pups were identified by tattooing. In addition to the observations of the parent animals, any abnormal behaviour of the offspring was recorded.
The anogenital distance (AGD) of each pup was measured on PND 0. Pup body weight measured on PND 0 was converted to cube root and used for the calculation of relative AGD (Relative AGD = AGD/Cube root of pup weight). The number of nipples/areolae in male pups was counted on PND 12.

Blood samples were collected from the defined site in serum separator tubes from 2 female pups /litter (wherever possible) on day 4 after birth from all dams, 2 pups/litter at termination on day 13 and from all adults males at termination. All blood samples were stored under appropriate conditions. Blood samples from the day 13 pups and the adult males were assessed for serum levels for thyroid hormones (T4). Further assessment of T4 in blood samples from the dams and day 4 pups were not done. Pup blood was pooled by litter for thyroid hormone analysis.

Postmortem examinations (parental animals):

Pathology
The males were sacrificed any time after the completion of the mating period (Day 29-30 after a minimum dosing period of 28 days) and females were sacrificed on the respective PND 13 by using an anesthesia (ketamine/xylazin). All surviving pups were killed by cervical dislocation/decapitation on PND 13. Vaginal smears were examined on the day of necropsy to determine the stage of estrous cycle.
Dead pups and pups sacrificed on PND 13 were carefully examined externally for gross abnormalities.
Non-pregnant females were sacrificed on study day 26 from the day of mating or from the last day of mating period.
All animals were subjected to a detailed gross necropsy which included careful examination of the external surface of the body, all orifices and the cranial, thoracic and abdominal cavities and their contents.
Special attention was paid to the organs of the reproductive system. The ovaries, uterus with cervix, oviducts, vagina, testes, epididymides, accessory sex organs (prostate, seminal vesicles with coagulating glands as a whole), the thyroid gland and all organs showing macroscopic lesions of all adult animals were preserved in 4% neutral-buffered formaldehyde, except for testes and epididymides which were preserved in modified Davidson’s Solution for 24 hours and then transferred to 70% ethanol.
The number of implantation sites and corpora lutea was recorded for each parental female at necropsy. The number of corpora lutea and implantation sites was recorded for any females sacrificed 26 days after the end of the mating period with no evidence of mating and for any females sacrificed on day 26 post-coitum due to non-delivery.


Organ Weights
The wet weight of the reproductive organs of all sacrificed adult males and females (only lactating females were evaluated) from each group were recorded as soon as possible. Paired organs were weighed together.
Testes, levator ani + bulbocavernosus muscle complex, epididymides, glans penis, prostate, seminal vesicles and coagulating glands uterus with cervix, Cowper’s gland, ovaries.
Thyroid gland from 1 pup/sex/litter/ group and from all adult males and females was preserved. The thyroid gland weight was measured after fixation.
The following tissues from all male and female animals were preserved in 4% neutral-buffered formaldehyde except for eyes, testes and epididymides which were fixed in Modified Davidson’s fixative for approximately 24 hours before they were transferred to 70% ethanol:
all gross lesions, epididymides, ovaries, oviducts, adrenal glands, prostate and seminal vesicles with coagulating glands as a whole, testes, uterus with cervix and vagina, thyroid gland.
The following tissues were preserved in case of intercurrent death:
adrenal glands, all gross lesions, brain (incl. medulla/pons, cerebellar and cerebral cortex), caecum, colon, duodenum, epididymides, heart, ileum (including Peyer´s patches), jejunum, kidneys, liver, lungs, lymph nodes (mesenteric and axillary), ovaries, oviducts, prostate and seminal vesicles with coagulating glands as a whole, rectum, spleen, stomach, testes, thymus, thyroid gland, trachea, urinary bladder, uterus with cervix and vagina. Thyroid gland from 1 pup/sex/litter/ group sacrificed on PND 13 and adult animals was preserved for the potential histopathological examination.


Histopathology
A full histopathology was carried out on the preserved organs and tissues of all animals of the control and high dose groups which were sacrificed at the end of the treatment period. Thyroid gland from pups and from the adult animals was not evaluated as it was not considered necessary.
Testes, epididymides, ovaries, uterus with cervix, vagina, accessory sex organs (prostate, seminal vesicle with coagulating gland) were trimmed, embedded into paraffin, cut at an approximated thickness of 2-4 µm and stained with hematoxylin and eosin and were examined in control and HD animals and in non pregnant female animals of the LD and MD animals. Testes, epididymides and accessory sex organs (prostate, seminal vesicle with coagulating gland) were also examined in the mating partners of the non pregnant female LD and MD animals.
These examinations were not extended to animals of all other dosage groups as no treatment-related changes were observed in the HD group.
Any gross lesion macroscopically identified was examined microscopically in all animals. Discoloration possibly due to the test item was evaluated in the organs of all dose groups.
For the testes, a detailed qualitative examination was made; taking into account the tubular stages of the spermatogenic cycle at evaluation of additional hematoxylin-PAS (Periodic Acid Schiff) stained slides.
Histological processing of tissues to microscope slides was performed at the GLP-certified contract laboratory AnaPath GmbH, AnaPath Services, Hammerstrasse 49, 4410 Liestal, Switzerland (test site for tissue processing). Histopathological evaluation was performed at the GLP-certified contract laboratory AnaPath GmbH, Buchsweg 56, 4625 Oberbuchsiten, Switzerland (test site for histopathology). The study phases from test site 1 and 2 was performed in compliance with the Swiss Ordinance relating to Good Laboratory Practice adopted 18 May 2005 [SR 813.112.1] (Status as of 01 December 2012). Blocking, embedding, cutting, H&E staining and scientific slide evaluation were performed according to the corresponding SOP’s of the test sites.
The principal investigator for histopathological tissue processing sent all raw data (including blocks, slides, paper raw data, statement of compliance and quality assurance statement) to the study director.
The principal investigator for histopathological evaluation provided the histopathology results to the study director by e-mail and sent a pathology phase report to the study director upon the completion of the study.

Peer Review:
As mentioned in the dose selection justification section of the study plan, dose levels for this study were selected based on two 28 day studies. In one 28 day toxicity study, the test item was orally administered to male and female animals for 28 days at the dose levels of 100, 300 and 1000 mg/kg bw/day. Histopathological examination of the dose groups revealed cytoplasmic vacuolation of the adrenal cortex, especially in female rats, combined with slight parenchymal degeneration and single cell necrosis. Cytoplasmic vacuolation was also observed in interstitial cells of the ovary. All investigated dose groups were affected in a comparable manner. No signs of reversibility were discernable after the 2 week treatment free period.
In another 28 day repeated dose toxicity study with this test item at dose levels of 3, 10 and 30 mg/kg bw/day, histopathology evaluation of the female rats of all dose groups exhibited a cytoplasmic vacuolation in the zona fasciculata of the adrenal gland. Incidence and severity of this observation was clearly dose-dependent. The males were not affected in this study. 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. The histopathology findings were not reversible after 2 weeks recovery period.
The aim of the present study was to provide initial information on the effects on reproduction and / or development, at doses that caused the histopathological findings described above (i.e. 1, 3 and 10 mg/kg bw /day).
Histopathological evaluation of organs from animals dosed up to 10 mg/kg bw /day revealed no treatment related changes in adrenal glands and reproductive organs of both male and females.
As no histopathological findings were observed in this study up to highest dose tested, the sponsor had decided to perform a peer review for the histopathological evaluation of adrenals and ovaries in all female animals to confirm the findings from this study.
A peer review was performed by a second pathologist:
Dr. Anja Knippel
Merck KGaA
Biopharma
Non-Clinical Safety
HPC: U009/101
64271 Darmstadt
Germany
Email: [e-mail]
Initially, the peer reviewing pathologist performed the evaluation for internal confirmation and it was decided that in case of no discrepancies, a formal peer review consultation with the principal investigator for histopathology evaluation will not be made and no peer review statement will be issued. In case of marked discrepancies, the peer review pathologist and the principal investigator was supposed to discuss the findings and discrepancies. Based on the outcome of this discussion, the peer review pathologist and the principal investigator was supposed to prepare a signed peer review statement, which was to be included as an annexure to the study report.

Postmortem examinations (offspring):

All surviving pups were sacrificed by decapitation on post-natal day 13.
Dead pups and pups sacrificed on day 13 post-partum were carefully examined externally for gross abnormalities.

Statistics:

A statistical assessment of the results of the body weight, food consumption and absolute and relative organ weights were performed for each gender by
comparing values of dosed with control animals of the main groups using a one-way ANOVA and a post-hoc Dunnett Test. These statistics were performed with GraphPad Prism V.6.01 software (p<0.05 was considered as statistically significant).

Reproductive indices:

see Results F1 section

Offspring viability indices:

see Results F1 section

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

see Details on results P0

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

see Details on results P0

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

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

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Other effects:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

effects observed, non-treatment-related

Description (incidence and severity):

Test item had no biologically significant effect on the estrous cycle analyzed during 2 weeks premating period after the first administration. There were no considerable differences in the length or sequence of cycle stages between the dose groups and the control group. Deviations from the physiological 4 or 5 day cycle in the rat were observed occasionally in few animals of all treatment groups including control. As this effect was also observed in control animals, it was considered as biological variation and not related to the treatment with the test item. There was higher incidence (7/10) of number of abnormal cycles observed in HD group. However, majoritiy of those females with abnormal cycle had followed normal proestrus i.e. stage before estrous. During transition from proestrus to estrous, estrous stage was already passed at the time of vaginal smear observation was made and next stage of cycle metestrous or diestrous was recorded. Since this estrous cyclicity data is based on actual smear stages observed at the time of observation and one cycle duration is considered as days between 2 estrous stages, it appears abnormal cycles but as such it was not and therefore considered to be toxicologically irrelevant.

Reproductive function: sperm measures:

not examined

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

Reproductive Indices
There were no test item related effects on the reproductive indices (copulation, fertility, delivery and viability indices) in the dose groups when compared to the control group. However, a slightly reduced fertility index (number of females pregnant / No. of females copulated X 100) of 90 % in the LD and MD group compared to 100 % in control group. In the absence of dose response dependency, the finding was not considered to be of toxicological relevance.

Details on results (P0)

Mortality
No mortalities/moribund sacrifice were observed during the entire period of this study.


Clinical Observations
In males, no clinical signs were observed in any treatment group or control animals during the entire study period.
In females, Isolated incidences of alopecia in one control and one MD group female and slight piloerection in one MD and one HD female were observed for very few days and therefore were considered to be incidental.
None of the females showed signs of abortion or premature delivery.



Body Weight Development
IIn both males and females, there was no test item treatment related effect on body weight and body weight gain observed in the dose groups during the study period. There were no statistically significant differences between the dose groups and the control group. There was marginally lower body weight gain observed during gestation in HD and during lactation in MD and HD group. As this difference was marginal and within the biological variation, it was not considered to be adverse effect due to treatment with test item.



Food Consumption
In correlation to the body weight and body weight gain, the food consumption in both males and females tended to increase with the progress of the study in the control, the LD, the MD and the HD group.
No test item related or statistically significant effect on food consumption was observed in males and females during the whole study period.

Precoital Interval and Duration of Gestation
There were no effects on the duration of precoital interval and the duration of gestation in the dose groups when compared to the control group.

Pathology- Macroscopic Findings
Few specific macroscopic changes were recorded for the male and female animals, which based on microscopic examination were not considered to be of test item treatment relevance.
The macroscopic changes observed were soft mass at tail of epididymis (male no. 32 of HD), bilateral dilation of ureters, solid abnormal content in urinary bladder (Female no. 58 of LD) and cyst on ovaries (Female No. 67 from MD).
These changes were within the range of normal background alterations which may be recorded in animals of this strain and age.



Organ Weight
In males and female, there were no statistically significant differences in the absolute and relative organ weights of the dose groups when compared to the control group.



Histopathology
Under the conditions of this study, the test item caused no histological changes indicating toxicity in adrenal glands and reproductive organs.
Gross lesions were unremarkable and not related to treatment with the test item.
There were two non-mating males (19 and 29) and two non-mating females (59 and 69). The evaluation of the reproductive organs did not reveal a morphological injury.
Histopathology and sperm staging did not reveal any abnormality that could be related to treatment with the test item.
The peer reviewing pathologist performed the evaluation for internal confirmation and no discrepancies were observed with the principal investigator for histopathology evaluation, therefore no peer review statement has been issued and included as an annexure to the study report.





Dose Formulation Analysis
Concentration analysis of formulation samples was determined at three concentrations, 0.2 mg/mL, 0.6 mg/mL and 2.0 mg/mL in study weeks 1, 3, 5 and the last week of the study. The mean recoveries observed in the low dose (LD), medium dose (MD) and high dose (HD) groups were 98.5%, 94.5% and 97.8% of the nominal concentration, respectively. Nominal concentrations were confirmed for all dose groups, as measured concentrations were within acceptance criterion of 15%. However for the single samples no. 15 (MD), recovery lower than 15% was measured and verified by reanalysis.
Homogeneity of formulation samples was determined at two concentrations, 0.2 mg/mL and 2.0 mg/mL, in study weeks 1 and 5. The mean recoveries observed for the LD dose group was 103.4% and 96.7% of the nominal value and 102.9% and 97.4% of the nominal value for HD dose group. The coefficients of variation of the different sampling locations (top, middle, bottom) were 2.7% and 3.7% in LD dose group, 1.0% and 3.5% in HD dose group.

Effect levels (P0)

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 10 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

gross pathology

histopathology: non-neoplastic

Remarks on result:

other: Generation: P + F1 (migrated information)

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

see Details on results F1

Body weight and weight changes:

no effects observed

Description (incidence and severity):

see Details on results F1

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Details on results (F1)

Litter Data
There were no test item treatment related effects on litter data including total number of pups born, number of male pups, number of female pups, sex ratio, number of live pups, still births and runts on PND 0 as well as number of male pups, number of female pups, number of live pups and sex ratio on PND 4 and PND 13. There were no statistically significant changes noted for these litter data and all litter data parameters were within the range of biological variation and historical control data.


Litter Weight Data
There were no effects on pup mean weight, total litter weight, male and female litter weight on PND 0, PND 4 and PND 13. There was no statistically significant change in dose groups compared to corresponding controls.
 

Pre- and Post-Natal Data
There were no test item treatment related effects on the number of corpora lutea, number of implantation sites, number of live pups (PND 0, PND 4 and PND 13) and percentage of pre- and post-implantation loss in the dose groups when compared to the control group. All pre and post-natal data parameters including post-implantation loss were within the range of biological variation and historical control data.



Pup Survival Data
There were no effects on the survival of the pups from PND 1 through PND 13 in the dose groups when compared to the control group.
A marginally higher mean mortality of pups between PND 0 and PND 4 was observed in the HD group (2.50%) compared to the control group (0.00%). This outcome did not achieve statistical significance and was attributed to missing one single pup of one single dam (pup no. 4 of dam no. 78) on PND 1. Thus, it was considered as incidental and not related to the treatment with the test item.


Anogenital Distance and Nipple Retention
No test item related effect of toxicological relevance was observed on anogenital distance and nipple retention in the pups of any of the groups.


Thyroid Hormone (T4) Analysis
No test item related effect of toxicological relevance was observed on pup thyroid weight, male and PND 13 pup thyroxine hormone (T4) in the treatment groups when compared to the controls.


Pup External Findings on PND 0 and at Death
No test item related gross external abnormalities of toxicological relevance on PND 0 were observed in the pups of any of the groups. Few specific findings like dark snout (pup no. 1 from control dam 43), dark tail tip (pup no. 1 from control dam 46), cold and pale (Pup no. 14 from LD dam no. 56) and stiffness of hindlegs (pup No. 4 from HD dam no. 78) were observed.
The external findings hair coat absent (pup 1-11, 13 from control dam 46, pup 1-7, 10-12 from MD dam 62, pup 1-5 from MD dam 65) at death were considered to be spontaneous and not related to test item treatment.

Effect levels (F1)

Dose descriptor:

NOEL

Generation:

F1

Effect level:

10 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

sexual maturation

clinical signs

mortality

body weight and weight gain

organ weights and organ / body weight ratios

gross pathology

histopathology: non-neoplastic

Overall reproductive toxicity

Reproductive effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

On the basis of this reproduction/ developmental toxicity screening test with 4-Ethyl-2',3'-difluor-4''-propyl-1,1':4',1''-terphenyl in male and female Wistar rats with dose levels of
1, 3, and 10 mg/kg body weight day the following conclusion can be made:
The NO(A)EL of 4-Ethyl-2',3'-difluor-4''-propyl-1,1':4',1''-terphenyl in this study for general and reproductive toxicity screening is considered to be 10 mg/kg body weight/day.

Executive summary:

Summary

The aim of this study was to assess the possible effects of 4-Ethyl-2',3'-difluor-4''-propyl-1,1':4',1''-terphenylo n male and female fertility and development after repeated dose administrationinWistarrats.

The test item was administered daily in graduated doses to 3 groups of test animals, one dose level per group for a treatment period up to 63 days, i.e. during 14 days of pre-mating and maximum 14 days of mating in both males and females, during the gestation period and up to post-natal day 12 in females. Males were dosed after the mating period until the minimum total dosing period of 28 days was completed. Animals of an additional control group were handled identically as the dose groups but received0.25 % aqueous hydroxypropyl methylcellulose, the vehicle used in this study. The 4 groups comprised 10 male and 10 femaleWistarrats.Before dosing all females were screened for two weeks for regular estrous cyclicity and animals (10 females/ group) with regular estorus cycle (4-5 day cycle) were used in the study.

The following doses were evaluated:

Control:                 0 mg/kg bw /day

Low Dose:             1 mg/kg bw/day

Medium Dose:       3 mg/kg bw/day

High Dose:            10 mg/kg bw/day

The test item formulation was prepared once a week based results obtained from stability investigations (study160287). The test item formulation was stored at approximately2 to 8 °C in refrigerator.

The test item was suspended in0.25 % aqueous hydroxypropyl methylcelluloseand administered daily during 14 days of pre-mating and maximum 14 days of mating in both males and females, during the gestation period and up to post-natal day 12 in females. Males were dosed for 28-30 days. Dose volumes were adjusted individually based on weekly body weight measurements. Theadministration volume was 5 mL/kg body weight.

During the period of administration, the animals were observed each day for signs of toxicity. At the conclusion of the test, all animals were sacrificed and observed macroscopically.

Body weight and food consumption were measured weekly, except for food consumption measurements which were not taken during the mating period in female animals and the mating and post-mating period in male animals.

After 14 days of treatment to both male and female, animals were mated (1:1) for a maximum of 14 days. The subsequent morning onwards the vaginal smears of females were checked to confirm the evidence of mating. After the confirmation of the mating, females were separated and housed individually. Each litter was examined as soon as possible after delivery of the dam to establish the number and sex of pups, stillbirths, live births, runts and the presence of gross abnormalities. Live pups were counted, sexed and litters weighed within 24 hours of parturition, on day 4 and day 13 post-partum. The anogenital distance (AGD) of each pup was measured on PND 0. The number of nipples/areolae in male pups was counted on PND 12. Blood samples from the day 13 pups and the adult males were assessed for serum levels for thyroid hormones (T4).

The males were sacrificed after completion of the mating period on treatment days 29 and 30 and the females along with their pups were sacrificed on post natal day 13. Two female pups wherever possible were sacrificed for blood collection on post natal day 4. Non-pregnant females were sacrificed on day 26.

The number of implantation sites and corpora lutea was recorded for each parental female at necropsy.

Pups sacrificed on post-natalday 4 or 13 and those found dead, were carefully examined for gross external abnormalities.

A full histopathological evaluation of the preserved tissues (adrenal glands, testes, epididymides, ovaries, oviducts, uterus with cervix, vagina, prostate and seminal vesicle with coagulating gland) was performed on high dose and control animals, in non pregnant female animals and male mating partners of the LD and MD animals. These examinations were not extended to animals of all other dosage groups as treatment-related changes were not observed in the high dose group. For the testes, a detailed qualitative examination was made; taking into account the tubular stages of the spermatogenic cycle at evaluation ofadditional hematoxylin-PAS (Periodic Acid Schiff)stained slides.All gross lesions macroscopically identified were examined microscopically in all animals.

The peer reviewing pathologistperformedtheevaluationfor internal confirmation.

Summary Results

No mortalities/moribund sacrifice were observed during the entire period of this study.

In males, no clinical signs were observed in any treatment group or control animals during the entire study period.In females, Isolated incidences of alopecia in one control and one MD group female and slight piloerection in one MD and one HD female were observed for very few days and therefore were considered to be incidental.None of the females showed signs of abortion or premature delivery.

In both males and females, there was no test item treatment related effect on body weight and body weight gain observed in the dose groups during the study period. There were no statistically significant differences between the dose groups and the control group.

No test item related or statistically significant effect on food consumption was observed in males and females during the whole study period.

Test item had no biologically significant effect on the estrous cycle analyzed during 2 weeks premating period after the first administration.

There were no test item treatment related effects on litter data including total number of pups born, number of male pups, number of female pups, sex ratio, number of live pups, still births and runts on PND 0 as well as number of male pups, number of female pups, number of live pups and sex ratio on PND 4 and PND 13. There were no statistically significant changes noted for these litter data.

There were no effects on pup mean weight, total litter weight, male and female litter weight on PND 0, PND 4 and PND 13. There was no statistically significant change in dose groups compared to corresponding controls.

There were no effects on the duration of precoital interval and the duration of gestation in the dose groups when compared to the control group.

There were no test item treatment related effects on the number of corpora lutea, number of implantation sites, number of live pups (PND 0, PND 4 and PND 13) and percentage of pre- and post-implantation loss in the dose groups when compared to the control group.

There were no test item related effects on the reproductive indices (copulation, fertility, delivery and viability indices) in the dose groups when compared to the control group. However, a slightly reduced fertility index of 90 % in the LD and MD group compared to 100 % in controlgroup.

There were no effects on the survival of the pups from PND 1 through PND 13 in the dose groups when compared to the control group.No test item related effect of toxicological relevance was observed on anogenital distance and nipple retention in the pups of any of the groups.

No test item related effect of toxicological relevance was observed on pup thyroid weight, male and PND 13 pup thyroxine hormone (T4) in the treatment groups when compared to the controls.

No test item related gross external abnormalities of toxicological relevance on PND 0 and at death were observed in the pups of any of the groups.

Few specific macroscopic changes were recorded for the male and female animals, which based on microscopic examination were not considered to be of test item treatment relevance.

In males and female, there were no statistically significant differences in the absolute and relative organ weights of the dose groups when compared to the control group.

Under the conditions of this study, the test item caused no histological changes indicating toxicity in adrenal glands and reproductive organs and therefore based on the pathology evaluation, the NOEL may be established at 10 mg/kg/day.

The peer review evaluation revealed no discrepancies with the principal investigator for histopathology evaluation.

Dose formulation analysis for nominal concentration and homogeneity revealed that nominal concentrations for all formulations were confirmed and all dose formulations were homogenous at the time of application throughout the study period.

Conclusion

On the basis of this reproduction/ developmental toxicity screening test with 4-Ethyl-2',3'-difluor-4''-propyl-1,1':4',1''-terphenyli n male and femaleWistarrats with dose levels of 1, 3, and 10 mg/kg body weight day the following conclusion can be made:

The NO(A)EL of 4-Ethyl-2',3'-difluor-4''-propyl-1,1':4',1''-terphenylin this study for general and reproductive toxicity screening is considered to be 10 mg/kg body weight/day.