3,20-bis(ethylenedioxy)pregna-5,7-diene

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08 January 2013 - 04 March 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: In accordance with GLP and OECD.

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:

other: OECD Guidelines for Testing of Chemicals, Guideline 422, Combined Repeated Dose Toxicity Study with Reproduction/Developmental Toxicity Screening Test, March 1996

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, July 2000

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

3,20-bis(ethylenedioxy)pregna-5,7-diene

EC Number:

243-175-0

EC Name:

3,20-bis(ethylenedioxy)pregna-5,7-diene

Cas Number:

19592-55-3

Molecular formula:

C25H36O4

IUPAC Name:

(1S,3aR,9aR,9bS,11aS)-9a,11a-dimethyl-1-(2-methyl-1,3-dioxolan-2-yl)-1,2,3,3a,6,8,9,9a,9b,10,11,11a-dodecahydrospiro[cyclopenta[a]phenanthrene-7,2'-[1,3]dioxolane]

Test material form:

solid: crystalline

Details on test material:

- Name of test material (as cited in study report): Proketal
- Molecular formula C25H36O4
- Molecular weight 400.5
- Structural formula attached as image file (if other than submission substance): see Fig.
- Description: Slightly yellow powder
- Analytical purity: 95.9%
- Purity test date:
- Lot/batch No.: 0385
- Expiration date of the lot/batch: 19 March 2013 (retest date)
- Stability under test conditions: stable
- Storage condition of test material: at room temperature (<25°C) in the dark

Test animals

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: 11 weeks old
- Weight at study initiation: all animals within ±20% of the sex mean.
- Fasting period before study: n/a
- Housing: during acclimatization and pre-mating, animals were housed in groups of a maximum of 5 animals/sex/cage in Macrolon plastic cages (MIV type, height 18cm) During mating, females were caged together with males on a one-to-one basis in Macrolon plastic cages (MIIItype, height 18cm) with a max of 5 animals/cage
- Diet: free access to pelleted roden diet (SM R/M-Z from SSNIFF Spezialdiaten GmbH)
- Water: ad libitum
- Acclimation period: 5 days prior to start of treatment

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-24
- Humidity (%): 40-70%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
VEHICLE
- Justification for use and choice of vehicle : The rationale for the used vehicle was based on trial formulations performed at WIL Research Europe B.V.
- Concentration in vehicle: The test item was administered at doses of 100, 300 or 1000 mg/kg bw/day (Groups 2, 3 and 4
respectively). Rats of the control group (Group 1) received the vehicle, propylene glycol, alone. The dose volume was 5 mL/kg body weight. Actual
dose volumes were calculated according to the latest body weight



VEHICLE
- Justification for use and choice of vehicle (if other than water):
- Concentration in vehicle:
- Amount of vehicle (if gavage):
- Lot/batch no. (if required):
- Purity:

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses were conducted on a single occasion during the treatment phase (31 January 2013), according to a validated method (Project 501713). Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations).
Stability in vehicle over 6 hours at room temperature was also determined (highest and lowest concentration).
The accuracy of preparation was considered acceptable if the mean measured concentrations were 85% - 115% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%. Formulations were considered stable if the relative difference before and after storage was maximally 10%.

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: Mating period followed after a minimum of 14 days of exposure for the males and females. Once mating occurred, the males and females were separated.
- Proof of pregnancy: Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the
appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum.
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility.

Duration of treatment / exposure:

The test item was administered at doses of 100, 300 or 1000 mg/kg bw/day (Groups 2, 3 and 4 respectively). Rats of the control group (Group 1) received the vehicle, propylene glycol, alone. The dose volume was 5 mL/kg body weight. Actual dose volumes were calculated according to
the latest body weight. Animals were treated by oral gavage, using a plastic feeding tube. Formulations were placed on a magnetic stirrer during dosing. The animals were dosed once daily for 7 days per week, approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose. Males were exposed for 29 days, i.e. 2 weeks prior to mating, during mating, and up to the day prior to scheduled necropsy. Females were exposed for 42-55 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of
lactation (up to the day prior to scheduled necropsy). Female no. 44 (Group 1) was not dosed during littering.
Pups were not dosed directly but could have been exposed to the test substance in utero, via maternal milk or from exposure to maternal
urine/faeces.

Frequency of treatment:

daily

Duration of test:

28 days

Doses / concentrations

Remarks:

Doses / Concentrations:

Basis:

No. of animals per sex per dose:

The F0 generation consisted of 40 males and 40 females (i.e. 10 animals/sex/group).

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels were selected based on the results of a 14-day dose range finding study (Project 501721; Abbott Study S102.7.006) . In that study, doses of 10, 100 or 1000 mg/kg bw/day were administered once daily by oral gavage to female Wistar Han rats. There
were no signs of toxicity up to 1000 mg/kg bw/day based on the measured parameters (mortality, clinical signs, body weight, food consumption, macroscopic appearance at necropsy, and liver and kidney organ weights). No toxicological relevant effects on the estrous cycle were noted up to
1000 mg/kg bw/day. Based on these data, the selected dose levels for this main study were 0, 100, 300 and 1000 mg/kg bw/day, respectively.

- Rationale for animal assignment (if not random):
In the current study Wistar Han rats Crl:WI(Han) (outbred, SPF-Quality) were used. This species and strain of rat has been recognized as appropriate
for general and reproduction toxicity studies. WIL Research Europe B.V. has general and reproduction/developmental historical data in this
species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were checked at least twice daily for mortality/viability

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once daily from start of treatment onwards, detailed clinical observations were made for all animals. Once prior to start of
treatment and at weekly intervals during the treatment period this was also performed outside the home cage in a standard arena. The time of onset, grade and duration of any observed sign were recorded. Signs were graded for severity and the maximum grade was predefined at 3 or 4. Grades were coded as slight (grade 1), moderate (grade 2), severe (grade 3) and very severe (grade 4). For certain signs, only its presence (grade 1) or absence
(grade 0) was scored.

BODY WEIGHT: Yes
- Time schedule for examinations: Males and females were weighed on the first day of exposure and weekly thereafter. Mated females were weighed
on Days 0,4 7, 11, 14, 17, and 20 post coitum and during lactation on Days 1 and 4.


POST-MORTEM EXAMINATIONS: Yes
SACRIFICE
-Necropsy was conducted on the following days:
Condition Day of necropsy
Females which delivered Lactation Days 5-7.
Females which failed to deliver(nos. 42, 43, 55, 67 and 80) Post-coitum Days 26-27 (females with evidence of mating).

- Organs examined:

Adrenal glands
Brain - cerebellum, mid-brain, cortex
Caecum
Cervix
Clitoral gland
Colon
Coagulation gland
Duodenum
Eyes (with optic nerve (if detectable) and Harderian gland)
Female mammary gland area
Femur including joint
Heart
Ileum
Jejunum
Kidneys (Lacrimal gland, exorbital)
Liver
Lung, infused with formalin Lymph nodes - mandibular, mesenteric
Ovaries
Peyer's patches [jejunum, ileum] if detectable
Pituitary gland
Preputial gland
Rectum
Sciatic nerve
Skeletal muscle
Spinal cord -cervical, midthoracic, lumbar
Spleen
Sternum with bone marrow
Stomach
Thymus
Thyroid including parathyroid if detectable
Trachea
Urinary bladder
Uterus
Vagina

All remaining females which failed to deliver:
Cervix
Preputial gland
Clitoral gland
Prostate gland
Coagulation gland
Ovaries
Uterus
Vagina
All gross lesions

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes

Fetal examinations:

- External examinations: Yes: [all per litter / half per litter / #? per litter ] / No / No data
- Soft tissue examinations: Yes: [all per litter / half per litter / #? per litter ] / No / No data
- Skeletal examinations: Yes: [all per litter / half per litter / #? per litter ] / No / No data
- Head examinations: Yes: [all per litter / half per litter / #? per litter ] / No / No data

Statistics:

The following statistical methods were used to analyze the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
- The Kruskal-Wallis nonparametric ANOVA test was applied to motor activity data to determine intergroup differences. In case intergroup
differences were seen, the Wilcoxon test was applied to compare the treated groups to the control group.

All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous
data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact
values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore,
two groups may display the same printed means for a given parameter, yet display different test statistics values.

Indices:

Mating index (%) (Number of females mated/Number of females paired) x 100

Fertility index (%) (Number of pregnant females/Number of females paired) x100

Conception index (%) (Number of pregnant females/Number of females paired) x100

Gestation index (%) (Number of females bearing live pups/Number of pregnant females) x100

Duration of gestation Number of days between confirmation of mating and the beginning of parturition

Percentage live males at First Litter Check: (Number of live male pups at First Litter Check/Number of live pups at First Litter Check) x 100

Percentage live females at First Litter Check: (Number of live female pups at First Litter Check/Number of live pups at First Litter Check) x 100

Historical control data:

Rats of the control group (Group 1) received the vehicle, propylene glycol, alone.
The dose volume was 5 mL/kg body weight. Actual dose volumes were calculated according to the latest body weight.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
No toxic effects

Effect levels (maternal animals)

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
No toxic effects

Effect levels (fetuses)

Remarks on result:

not determinable due to absence of adverse toxic effects

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

In conclusion, treatment with Proketal by oral gavage in male and female Wistar Han rats at dose levels of 100, 300 or 1000 mg/kg bw/day revealed no toxicologically relevant parental toxicity up to 1000 mg/kg bw/day. No reproduction and developmental toxicity was observed for
treatment up to 1000 mg/kg bw/day. Based on these results the No Observed Adverse Effect Level (NOAEL) was established at the highest tested dose of 1000 mg/kg bw/day.

Executive summary:

No developmental toxicity was observed up to the highest dose level tested (1000 mg/kg bw/day). No treatment-related changes were noted in any of the developmental parameters investigated in this study (i.e. gestation index and duration, parturition, maternal care and early postnatal pup development consisting of mortality, clinical signs, body weight and macroscopy).