2,2''-dihydroxy-4,4''-(2-hydroxy-propane-1,3-diyldioxy)dibenzophenone

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

201502-19 to 2015-07-30

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3500 (Preliminary Developmental Toxicity Screen)

Principles of method if other than guideline:

NA

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

424-210-0

EC Name:

-

Cas Number:

23911-85-5

Molecular formula:

C29 H24 O7

IUPAC Name:

2-benzoyl-5-[3-(4-benzoyl-3-hydroxyphenoxy)-2-hydroxypropoxy]phenol

Test material form:

other: solid

Details on test material:

- Name of test material (as cited in study report): Fadex HE 1819 PK
- Chemical name: Methanone, 1,1’-[(2-hydroxy-1,3-propanediyl)bis[oxy(2-hydroxy-4,1-phenylene]]bis[phenyl-
- Molecular formula (if other than submission substance): C29H24O7
- Molecular weight (if other than submission substance): 484.50 g/mol
- Physical state: solid
- Color: yellow
- Analytical purity: 96.6 %
- Purity test date: 2014-11-28
- Lot/batch No.: #CHA0123335
- Expiration date of the lot/batch: 2015-07-19
- Stability under test conditions:
- Storage condition of test material: Keep containers tightly closed in a cool, well-ventilated place.

Test animals

Species:

rat

Strain:

other: Hsd.Brl.Han: of Wistar origin

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Toxi-Coop Zrt., Cserkesz u. 90., H-1103 Budapest, Hungary
- Age at study initiation: (P): 79 – 82 d (Males), 72 – 74 d (Females)
- Weight at study initiation: (P) Males: 314 – 369 g; Females: 177 – 208 g
- Housing: Before mating: 2 animals of the same sex/cage; mating: 1 male and 1 female/cage; mated females were housed individually; males after mating: 2 animals/cage
- Diet: ssniff® SM R/M-Z+H "Autoclavable complete feed for rats and mice – breeding and maintenance", ad libitum
- Water: Tap water, ad libitum
- Acclimation period: 19 d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 30 – 70 %
- Air changes (per hr): 10 – 15 air exchanges/hour by central air-condition system.
- Photoperiod (hrs dark / hrs light): 12 hours daily, from 6.00 a.m. to 6.00 p.m.

IN-LIFE DATES: From: 20015-02-24 To: 2015-04-24

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: 1 % aqueous methylcellulose

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The test item was formulated in the vehicle in concentrations of 200, 60 and 20 mg/mL. Formulations were prepared in the formulation laboratory of the Test Facility beforehand not longer than for three days before the application and were stored in a refrigerator (at 5±3 oC).

VEHICLE
- Justification for use and choice of vehicle: The test item was not soluble in water therefore 1 % aqueous methylcellulose was used for preparing formulations appropriate for oral administration. 1 % aqueous methylcellulose was a suitable vehicle to facilitate formulation analysis for the test item.

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: One female and one male of the same dose group were placed in a single cage (1:1 mating). Females were cohabited with the same male until copulation occurred. The mating period was elongated for one female animal at 100 mg/kg bw/day (up to 19 days) and for one female animal at 1000 mg/kg bw/day (up to 20 days) in the lack vaginal plug or sperm in the vaginal smear (as evidence of copulation). Mating pairs were changed for these animals on mating days 15 within the same groups. Duration of the mating period was determined according to the last day of mating (i.e. when the last positive vaginal smear was observed).
- Proof of pregnancy: The presence of vaginal plug or sperm in the vaginal smear was considered as evidence of copulation (day 0 of pregnancy as defined by OECD 421). Sperm positive females were caged individually.
- Examination of Placental Sign: All sperm positive animals were examined for vaginal bleeding (placental sign of gestation) on day 13 of gestation. If the test was negative on day 13 the examination was repeated on day 14 of gestation.
- Observation of the Delivery Process: Females were allowed to litter and rear their offspring. Delivery process was monitored whilst keeping possible interferences at a minimum. Observations were reported individually for each animal. The duration of gestation was recorded and was calculated from day 0 of pregnancy.
Dams were observed whether they made a nest from the bedding material and cover their newborns or not. The sucking success was monitored by the presence of milk in the pups' stomach. All observations were recorded individually for each dam.
Each litter was examined as soon as possible after delivery to establish the number and sex of pups, stillbirths, live births, runts (pups that are significantly smaller than normal pups), and the presence of gross abnormalities.
Live pups were counted, sexed and litters were weighed within 24 hours of parturition (on the day when parturition was complete) and on day 4 post-partum with an accuracy of 0.1 g.
In addition to the observations on parent animals, any abnormal behavior of the offspring was monitored.
All the litters were checked and recorded daily for the number of viable and dead pups. The pups found dead were subjected to necropsy by a macroscopic examination. On the day of birth, a lung flotation test was performed on all pups found dead to separate stillborn from those that died after delivery (dead pups). The lung flotation test is negative for stillborn (pups that died intrauterine) but positive for pups that died after delivery.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analysis of formulations (checking of concentration and homogeneity) was performed in the Analytical Laboratory of Test Facility. Five samples (5 mL, each) were taken from different places from each concentration (Groups 2, 3 and 4) and all samples were measured. Similarly, five samples (5 mL, each) were taken from the vehicle, from different places, (Group 1) and analyzed.
Date of sampling: 2015-02-24 and 2015-04-07.
Date of analysis: 2015-02-26 and 2015-04-08.
Concentration of the test item in the dosing formulations varied in the range of 96 and 107 % of the nominal values at both analytical occasions.

Duration of treatment / exposure:

Dosing of both sexes began two weeks before mating and was continued up to and including the day before the necropsy.

Male animals were dosed for 42 days.

Female animals were dosed for 14 days pre-mating, during mating period, through gestation and up to lactation days 3 - 6 (altogether for 42 – 59 days depending on day of mating). The day of delivery (viz. when parturition was complete) was defined as day 0 post-partum.

Non-pregnant female animals were treated up to and including the day before necropsy (altogether for 42 days).

Frequency of treatment:

once daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

100 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

0 mg/kg bw/day (nominal)

No. of animals per sex per dose:

12 animals per sex per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Justification of the dose selection: The dose setting with 1000, 300 and 100 mg/kg bw/day based on findings obtained in a previous 28-day oral gavage toxicity study with Fadex HE 1819 PK in rats. The high dose was chosen with the aim of inducing toxic effects but no mortality or severe suffering of animals. The low dose was chosen to induce no toxic effect.

Positive control:

No positive control was used.

Examinations

Parental animals: Observations and examinations:

- All animals were observed twice daily, once in the morning and once in the afternoon, for mortality and moribundity.
- General clinical observations were made once a day, after treatment at approximately the same time.
- Detailed physical examinations were were made at the times of weekly weighing, prior to and during the mating and until necropsy. Observations were performed on the skin, fur, eyes and mucous membranes, autonomic activity (lachrymation, piloerection, pupil size, respiratory pattern, occurrence of secretions and excretions), circulatory and central nervous system, somatomotor activity and behavior pattern, changes in gait, posture and response to handling.
-Parent male animals were weighed on the first day of dosing (Day 0), weekly thereafter and on the day of necropsy.
Parent females were weighed on the first day of dosing (Day 0) then weekly, on gestation days 0, 7, 14 and 21 and on post-partal day 0 (lactation day 0; within 24 hours after parturition) and post-partal day 4 (lactation day 4).
- The food consumption was determined weekly by reweighing the non-consumed diet with an accuracy of 1 g during the treatment period except mating days (pre-mating and post mating for male animals, during pre-mating, gestation days 0, 7, 14 and 21, lactation days 0 and 4 for dams).
- A complete necropsy was conducted on all F0 parental animals found dead, euthanized in extremis or at termination.

The observations and examinations performed in parental males are detailed in the following:
− Clinical observations
− Body weight
− Body weight gain
− Food consumption
− Percentage of pairings
− Percentage of fertile pairings
− Percentage of infertile males
− Male copulatory index
− Male fertility index
− Necropsy findings
− Organ weights (absolute and relative to the body and brain weights)
− Histopathology findings

The observations and examinations performed in parental fermales are detailed in the following:
− Clinical observations
− Body weight
− Body weight gain
− Food consumption
− Percentage of pairings
− Percentage of pregnant females
− Percentage of sperm positive, but non-pregnant females
− Female copulatory index
− Female fertility index
− Gestation index
− Duration of pregnancy (days)
− Number of Corpora lutea / dams
− Number of implantations / dams
− Percentage of dams with live pups on post-partal days 0 and 4
− Pre-implantation mortality
− Post-implantation mortality
− Intra uterine mortality
− Necropsy findings
− Histopathology findings

Oestrous cyclicity (parental animals):

The estrous cycle was not investigated.

Sperm parameters (parental animals):

No sperm parameters were investigated.

Litter observations:

− Litter weight on postnatal days 0 and 4.
− Mean body weight gain per litter between postnatal days 0-4.
− Number of live births per litter, and number of viable pups per litter on postnatal days 0 and 4.
− Survival Index of pups on postnatal day 4.
− Sex ratio % (on postnatal days 0 and 4).

Postmortem examinations (parental animals):

GROSS NECROPSY
- Gross necropsy was performed on each animal one day after the last treatment. Animals were anesthetized by Isofluran CP® and then were exsanguinated.
After examination of the external appearance the cranial, thoracic and abdominal cavities were opened and the appearance of the tissues and organs was observed, and any abnormality was recorded with details of the location, color, shape and size.
Special attention was paid to the organs of the reproductive system. The number of implantation sites and of corpora lutea was recorded.
The testes, epididymides and brain of all adult male animals were weighed.
The uterus with cervix, vagina, testes, epididymides (total and cauda), prostate, and seminal vesicles with coagulating glands, ovaries, pituitary of all adult animals were preserved. A sample of the skin under the left ear of one male animal at 100 mg/kg bw/day was also fixed due to the macroscopic finding. Testes and epididymides were fixed in modified Davidson solution, all other organs in 4 % buffered formaldehyde solution.


HISTOPATHOLOGY / ORGAN WEIGHTS
Detailed histological examinations were performed on the testes and epididymides of all animals in the control and high dose group. For the testes and epididymides, examinations were performed with special emphasis on stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure.

Detailed histological examinations were performed on the ovaries of all animals in the control and high dose group. Detailed histological examination of the ovaries covered the follicular, luteal, and interstitial compartments of the ovary, as well as the epithelial capsule and ovarian stroma.

Postmortem examinations (offspring):

SACRIFICE
- Offspring was carefully examined for external abnormalities and euthanized on postnatal day 4.

GROSS NECROPSY
- Each litter was examined as soon as possible after delivery to establish the number and sex of pups, stillbirths, live births, runts (pups that are significantly smaller than normal pups), and the presence of gross abnormalities.

Statistics:

The statistical evaluation were performed with the statistical program package SPSS PC+4.0.
The homogeneity of variance between groups was checked by Bartlett’s homogeneity of variance test.
Where no significant heterogeneity was detected a one-way analysis of variance (ANOVA) was carried out. If the obtained result was significant Duncan Multiple Range test was used to access the significance of inter-group differences. Getting significant result at Bartlett’s test the Kruskal-Wallis analysis of variance was used and the inter-group comparisons were performed using Mann-Whitney U-test. Chi2 test was performed if feasible.
The frequency of clinical signs, pathology and histopathology findings were calculated.
Results were evaluated in comparison with values of control group (i.e. control value).

Reproductive indices:

The following reproductive indices were calculated: Male and female copulatory index, male and female fertility index, gestation index.
The formulas for calculation can be found below in "Any other information on materials and methods incl. tables".

Offspring viability indices:

The following pup mortality and sex ratio indices were calculated: pre-implantation mortality index, post-implantation mortality index, intra uterine mortality index, post-natal mortality index, sex ratio index (male and female), survival index.
The formulas for calculation can be found below in "Any other information on materials and methods incl. tables".

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
There was no mortality in any group (control, 1000, 300 or 100 mg/kg bw/day) during the course of study.
Adverse signs of systemic toxicity related to the test item were not detected at any dose level, neither at the daily nor at the detailed weekly clinical observations.
Alopecia and scab were detected under the left ear in one male animal at 100 mg/kg bw/day (1/12) from Day 28 up to the termination of the study. Alopecia and scab are common spontaneous findings in this strain of experimental rats and is seen also in untreated rats. As these dermal changes were only present in one low dose treated animal, it was not considered to be relevant. Brownish-red contamination on the hairs around the left eye was seen in one male of the low dose group (1/12 at 100 mg/kg bw/day) transiently between Days 7 and 15. Brownish-red contamination on the hairs around the eyes is considered to be originated from tears rich in porphyrin and which is also a common findings in rats.
The behavior and physical condition of animals was not affected by the test item at any dose level (1000, 300 or 100 mg/kg bw/day) based on the weekly detailed clinical observations during the entire treatment period.
Observations on the fur and skin as described above were also detected in single male animals at 100 mg/kg bw/day at the detailed weekly observation (brownish-red contamination, alopecia and scab).

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
The body weight development was unaffected in any treatment group (male and female, 1000, 300 and 100 mg/kg bw/day).
The mean body weight and body weight gain were similar to that of the control group in male and female animals of 1000, 300 and 100 mg/kg bw/day groups during the course of entire treatment period (pre-mating, mating and post mating period in male animals and pre-mating, mating, gestation and lactation periods in female animals).
Statistical significance was only noted with respect to the control for the slightly higher mean body weight gain of female animals in 300 mg/kg bw/day group between Days 0 and 7. However, this slight difference was not associated with relevant or significant differences in the mean body weight value in female animals of the mid dose group and. Moreover, the body weight gain of the mid dose group was within the historical control range and similar finding was not detected at the high dose treated animals. Therefore this slight change in the body weight gain was judged to be toxicologically not significant but indicative of biological variation.
There were no test item or treatment related changes in the food consumption of male and female animals at any dose level (1000, 300 and 100 mg/kg bw/day).
Compared to the control animals, statistical or biological significances were not detected in the mean food consumption of male or female animals at 1000, 300 or 100 mg/kg bw/day during the course of the pre-mating, mating and post mating period in male animals and pre-mating, mating, gestation and lactation periods in female animals.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
The examined parameters of reproductive performance were not affected by the treatment with the test item at 1000, 300 or 100 mg/kg bw/day in male or female animals.
Statistical significances with respect to the relevant control were noted for the lower percentage of mated male animals at 1000 and 100 mg/kg bw/day and for the higher percentage of fertile male animals at 300 and 100 mg/kg bw/day, which resulted in a lower copulatory index at 1000 and 100 mg/kg bw/day and a higher fertility index at 300 and 100 mg/kg bw/day.
Compared to the concurrent control groups, statistical significances were noted for the lower percentage of non-pregnant females and for the higher percentage of pregnant females in the 300 and 100 mg/kg bw/day groups as well as for the higher fertility index at 300 and 100 mg/kg bw/day.
These slight, but statistically significant differences in the parameters above were considered to be indicative of biological variation due to the lack of a clear and consistent dose-response-relations ship.

DELIVERY DATA OF DAMS (PARENTAL ANIMALS)
The delivery data of dams were not affected by the treatment with the test item at 1000, 300 or 100 mg/kg bw/day.
The mean number of corpora lutea, implantation sites, pre-implantation loss, post-implantation loss and total intrauterine mortality were similar in the control and all test item treated groups.
There were no significant differences between the control and test item treated groups in the mean duration of pregnancy, in the mean number of total births, live-borns, stillborns or viable pups on post-partal day 0, in the percentage of dams with viable offspring on postpartal days 0 and 4.

ORGAN WEIGHTS (PARENTAL ANIMALS)
The absolute and relative organ weights of brain, testes and epididymides did not demonstrate any test item related alterations.
There were no statistically or biologically significances between the control and Fadex HE 1819 PK treated groups (1000, 300 and 100 mg/kg bw/day) in the examined organ weights or body weight at the necropsy.

GROSS PATHOLOGY (PARENTAL ANIMALS)
Test item related alterations were not detected at the macroscopic observations of organs or tissues in male or female animals at any dose level (1000, 300 and 100 mg/kg bw/day) at necropsy.
Alopecia and scab were detected on the skin under the left ear of one male animal at 100 mg/kg bw/day (1/12). In non-pregnant female animals, slight hydrometra was observed in the control group (1/2) and in the 1000 mg/kg bw/day group (1/1).
These isolated findings of male and female animals were considered to be individual alterations, which occur also in not treated animals of this strain with similar age and these have no toxicological relevance in this study.

HISTOPATHOLOGY (PARENTAL ANIMALS)
Histological examination of male and female genital organs (testes, epididymides and ovaries) did not reveal any test item related alterations at the highest dose of 1000 mg/kg/bw/day.
The investigated organs of the reproductive system (testes, epididymides) were histopathologically normal in all animals of the control and 1000 mg/kg bw/day groups (12/12, both) even in those male (1/12 at 1000 mg/kg bw/day and 2/12 in the control group), which did not fertilize female animals. The various spermatogenic cells (the spermatogonia, the spermatocytes, the spermatids and spermatozoa), representing different phases in the development and differentiation of the spermatozoons and the interstitial cells were the same in quantity and morphologically for all dose groups. Compared to the concurrent control, epididymides were histopathologically not affected in all animals.
The ovaries had a normal structure characteristic for the species, age and phase of sexual cycle in all examined female animals of the control and 1000 mg/kg bw/day group (12/12, both) and non-pregnant animals of 1000 mg/kg bw/day (1/1) and control (2/2) group. The cortex contained primary, secondary and tertiary follicles and corpora lutea, indicating the active maturation of oocytes and ovulation. The epithelial capsule and ovarian stroma were normal in all cases as well.
In one male animals of 100 mg/kg bw/day group (1/1), focal exsudative dermatitis was observed. This finding was considered to be an individual disorder as it was only present in single animal of low dose group.

Effect levels (P0)

1

Key result

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no systemic toxicity effects

2

Key result

Dose descriptor:

NOAEL

Remarks:

reproductive performance

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no reproductive performance effects

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Details on results (F1)

MORTALITY (OFFSPRING)
There was no test item related effect on pup’s mortality.
The mean number of live pups per litter, the mean number of viable pups per litter on postnatal days 0 and 4 were similar in all groups. There were no differences between the control and test item treated (1000, 300 or 100 mg/kg bw/day) groups in the survival indices.

CLINICAL SIGNS (OFFSPRING)
Test item related clinical signs were not detected in the offspring.
The number and percentage of pups with findings (cold, pale) were low in the control or test item treated groups. The percentage of not suckled offspring (no milk in the stomach) was higher in all test item treated groups than in the control groups. In the lack of any dose relevancy and because of the transient occurrence (it was only detected on the day of birth) as well as there were no related changes in the litter/body weight parameters or in the mortality rate, it was not considered to be toxicologically relevant.

BODY WEIGHT (OFFSPRING)
A test item related effect on the body weight development of the offspring was not found.
The mean litter weights and mean pup weights were similar in the control and in all test item treated groups (1000, 300 and 100 mg/kg bw/day) on postnatal days 0 and 4.

SEX DISTRIBUTION (OFFSPRING)
There were no test item related differences between the control and test item treated groups in the ration or in the litter means of genders on postnatal days 0 or 4.

GROSS PATHOLOGY (OFFSPRING)
Test item related macroscopic alterations were not found in offspring subjected to gross pathological examination.

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Remarks:

development

Generation:

F1

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no developmental effects

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Any other information on results incl. tables

no remarks

Applicant's summary and conclusion

Conclusions:

Under the conditions of the present study, Fadex HE 1819 PK administered at 1000, 300 or 100 mg/kg bw/day did not cause signs of systemic toxicity and did not adversely influence the reproductive performance (gonad function, mating behavior, conception, pregnancy, parturition) in parental male and female Hsd.Brl.Han: Wistar rats. The development of the F1 offspring from conception to day 4 postpartum after repeated oral administration was not impaired at any dose level.

Based on these observations the No Observed Adverse Effect Levels (NOAEL) were determined as follows:
NOAEL for systemic toxicity of male/female rats: 1000 mg/kg bw/day
NOAEL for reproductive performance of male/female rats: 1000 mg/kg bw/day
NOAEL for F1 Offspring: 1000 mg/kg bw/day