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

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

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 OECD and GLP .

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

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
-- Stability under test conditions: stable
- Storage condition of test material: at room temperature (<25°C) in the dark

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

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 mazimum 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

Remarks:

specific gravity 1.036

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: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 wereadministered 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.

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

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%.

Duration of treatment / exposure:

The test item was administered at doses of 100, 300 or 1000 mg/kg bw/day. Rats of the control group received the vehicle, propylene glycol only. The dose volume was 5 mL/kg body weight. Actual dose volumes were calculated according to the latest body weight.
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.

Males were exposed for 29 days (2 weeks prior to mating during mating and up to the day prior to scheduled necropsy) Females were exposed for 42-55 days (2 weeks prior to mating, during mating, during port-coitum and during at least 4 days of lactation up to the day prior to scheduled necropsy.

Frequency of treatment:

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.

Doses / concentrations

Remarks:

Doses / Concentrations:
100, 300 or 1000 mg/kg bw/day.
Basis:
actual ingested

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 no treatment

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 :
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.

Positive control:

no

Examinations

Observations and examinations performed and frequency:

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. These clinical
observations will be conducted at least immediately after dosing, based on the absence of clinical signs after dosing in the 14-day dose range
finding study (Project 501721; Abbott Study S102.7.006).
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.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: between 7 and 10:30am on the day of necropsy
- Animals fasted: Yes - deprived of food overnight with a maximum of 24 hours before blood sampling but water available
- How many animals: 5 animals/sex/group
- Parameters checked in table table attached below were examined.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: between 7 and 10:30am on the day of necropsy
- Anaesthetic used for blood collection: Yes, isoflurane (Abbott B.V. Hoofddorp, The Netherlands)
- Animals fasted: Yes - deprived of food overnight with a maximum of 24 hours before blood sampling but water available
- How many animals: 5 animals/sex/group
- Parameters checked in table attached below were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: The selected males will be tested during Week 4 of treatment and the selected females will be tested towards the end of the scheduled lactation period (from lactation Day 4 onwards). These tests will be performed after observation for clinical signs (incl. arena observation, if applicable).
- Dose groups that were examined: selected 5 animals/group/sex
- Battery of functions tested: hearing ability, pupillary reflex, static righting reflex, grip strength

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see attached Pathology Report)
HISTOPATHOLOGY: Yes (see attached Pathology Report)

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.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

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

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
No mortality occurred during the study period. No clinical signs of tozicity were noted during the observation period.
Incidental findings noted included alopecia, scabbing and wounds on different parts of the body and salivation (all at a slight degree)

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
No toxicologically relevant changes in body weights and body weight gain were noted. The slightly lower mean body weight recorded for females at 1000 mg/kg bw/day on the first day of lactation was considered not to be toxicologically relevant as changes as compared to controls were relatively slight (5%) and remained within the normal range of biological variation.
No toxicologically relevant changes in food consumption before or after allowance for body weight were noted.

ORGAN WEIGHTS (PARENTAL ANIMALS)
No toxicologically relevant changes were noted in organ weights and organ to body weight
ratios.

GROSS PATHOLOGY (PARENTAL ANIMALS)
Macroscopic observations at necropsy did not reveal any alterations that were considered to have arisen as a result of treatment.

HISTOPATHOLOGY (PARENTAL ANIMALS)
There were no treatment-related microscopic findings.

HAEMATOLOGY
There were no differences noted in haematological parameters between control and treated rats that were considered to be related to treatment with Proketal.

CLINICAL CHEMISTRY
Clinical biochemistry parameters of treated rats were considered not to have been affected by treatment.

Effect levels

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: highest tested dose of 1000 mg/kg bw/day.

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Formulation analysis

Accuracy of preparation The concentrations analysed in the formulations of Group 2, Group 3 and Group 4 were in agreement with target concentrations (i.e. mean accuracies between 85% and 115%). No test substance was detected in the Group 1 formulation.

Homogeneity

The formulation of Group 4 was determined to be homogeneous (i.e. a coefficient of variation (CV) of ≤ 10%). The coefficient of variation obtained for the Group 2 formulation was 16% when taken all samples into account and 7% when excluding the samples taken at 50% height. As the No Observed Adverse Effect Levels (NOAEL) in this study was 1000 mg/kg bw/day (Group 4) and analysis of this group was within acceptance criteria, the deviating result of the Group 2 formulation did not adversely affect the outcome of the study.

Stability

Formulations at the entire range were stable when stored at room temperature under normal laboratory light conditions for at least 6 hours.

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 Adeverse Effect Level (NOAEL) was established at the highest tested dose of 1000 mg/kg bw/day.

Executive summary:

Proketal was administered by daily oral gavage to male and female Wistar Han rats at dose levels of100, 300 or 1000 mg/kg bw/day (Groups 2, 3 and 4, respectively). The rats of the control group (Group 1)received the vehicle, propylene glycol, alone. 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). Formulation analysis showed that mean concentrations of Group 2-4 dose preparations were within target, and no test substance was detected in the Group 1 formulation. The formulation of Group 4 was determined to be homogeneous (i.e. a coefficient of variation (CV) of ≤ 10%). The coefficient of variation obtained for the Group 2 formulation was 16% when taken all samples into account and 7% when excluding the samples taken at 50% height. As the No Observed Adverse Effect Levels (NOAEL) in this study was 1000 mg/kg bw/day (Group 4) and analysis of this group was within acceptance criteria, the deviating result of the Group 2 formulation did not adversely affect the outcome of the study. Formulations were found to be stable for at least 6 hours at room temperature.

Developmental results:

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).

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).