Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
12 Oct 2017 - 07 Feb 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Currently studies in laboratory animals provide the best available basis for extrapolation to humans and are required to support regulatory submissions. Acceptable models which do not use live animals currently do not exist.
Cross-reference
Reason / purpose:
reference to same study
Reference
Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
12 Oct 2017 - 07 Feb 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Currently studies in laboratory animals provide the best available basis for extrapolation to humans and are required to support regulatory submissions. Acceptable models which do not use live animals currently do not exist.
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
July 2016
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA, Health Effects Test Guidelines OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test (July 2000)
Version / remarks:
July 2000
Deviations:
no
Qualifier:
according to
Guideline:
other: other guidance as listed under Principles of method if other than guideline
Principles of method if other than guideline:
- OECD Guidelines for Testing of Chemicals, Guideline 421, Reproduction/Developmental Toxicity Screening Test (July 2016)
- The United States EPA Health Effects Test Guidelines, OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test (July 2000)
- Commission regulation (EC) No 440/2008 Part B: Methods for the Determination of Toxicity and other Health Effects; B.7: "Repeated Dose (28 days) Toxicity (oral)". Official Journal of the European Union No. L142 (May 2008)
- OECD Guidelines for Testing of Chemicals, Guideline 407, Repeated Dose 28-day Oral Toxicity Study in Rodents (October 2008)
- The United States EPA Health Effects Test Guidelines, OPPTS 870.3050, Repeated dose 28-day oral toxicity study in rodents (July 2000)
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
STABILITY OF TEST MATERIAL
- Stability for at least 7 days in the refrigerator is confirmed over the concentration range 1.0 to 200 mg/mL
- Stability for at least 6 hours at room temperature under normal laboratory light conditions is confirmed over the concentration range 1.0 to 200 mg/mL

CORRECTION FACTOR
Purity/Composition correction factor: Yes, correction factor is 2.554 based on solid content.

No specific handling conditions required
Species:
rat
Strain:
other: Crl:WI(Han)
Details on species / strain selection:
The Wistar Han rat was chosen as the animal model for this study as it is an accepted rodent species for toxicity testing by regulatory agencies. Charles River Den Bosch 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. The total number of animals used in this study was considered to be the minimum required to properly characterize the effects of the test item. This study has been designed as such that it does not require an unnecessary number of animals to accomplish its objectives.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Age at study initiation: Approximately 11 weeks (males); 13 weeks (females).
- Weight at study initiation: 276-312 gr (males); 205-242 gr (females).
- Fasting period before study: no
- Housing:
Pre-mating: Animals were housed in groups of 5 animals/sex/cage in Macrolon cages.
Mating: Females were caged together with males on a one-to-one-basis in Macrolon cages.
Post-mating: Males were housed in their home cage with a maximum of 5 animals/cage. Females were individually housed in Macrolon cages.
Lactation phase: females were housed in Macrolon plastic cages (MIII type, height 18 cm). Pups were housed with the dam, except during locomotor activity monitoring of the dams, when the pups were kept warm in their home cage using bottles filled with warm water. In order to avoid hypothermia of pups, pups were not left without their dam or a bottle filled with warm water for longer than 30-40 minutes.
Locomotor activity monitoring: animals were housed individually in a Hi-temp polycarbonate cage without cageenrichment, bedding material, food and water.
General: Sterilised sawdust as bedding material and paper as cage enrichment were supplied.
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water: Free access to tap water.
- Acclimation period: 6 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 22.
- Humidity (%): 37 - 59.
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 15-Aug-2017 to 07 Dec 2017
Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS
Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared at least every 7 days as a solution, formulated in daily portions and stored in the refrigerator. The dosing formulations were removed from the refrigerator and stirred for at least 30 minutes before dosing. Test item dosing formulations were kept at room temperature until dosing. If practically possible, the dosing formulations and vehicle were continuously stirred until and during dosing. Adjustment was made for specific gravity of the test item.

DOSE VOLUME
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 performed using a validated analytical procedure.
Duplicate sets of samples (approximately 500 mg) were sent to the analytical laboratory:
- Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 10% for solutions of target concentration.
- Homogeneity results were considered acceptable if the coefficient of variation (CV) of concentrations was ≤ 10%.
- Stability analyses performed previously in conjunction with the method development and validation study demonstrated that the test item is stable stable in the vehicle when prepared and stored under the same conditions at concentrations bracketing those used in the present study.
Duration of treatment / exposure:
Males were treated for 29 days, i.e. 2 weeks prior to mating, during mating and up to and including the day before scheduled necropsy.
Females that delivered were treated for 50-56 days, i.e. 14 days prior to mating (with the objective to cover at least two complete estrous cycles), the variable time to conception, the duration of pregnancy and 13 or 15 days after delivery, up to and including the day before scheduled necropsy. Females without offspring were treated for 53 days (no evidence of mating) or 42-43 days (not pregnant or implantation site only).

Frequency of treatment:
Once daily, 7 days per week.
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
Low dose group
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Remarks:
Mid dose group
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
High dose group
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
Dose selection rationale: The dose levels were selected based on the results of an 11-day dose range finder with oral administration of Miranol Ultra C32 in rats. No guidelines were applicable as this study was intended for dose level selection purposes only.

Selection of animals for selected measurements: 5 animals/sex/group were randomly selected at allocation for functional observations, clinical pathology, macroscopic examination, organ weights and histopathology. Only females with live offspring were selected.
Positive control:
No.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS:
Yes
- Time schedule: Throughout the study, animals were observed for general health/mortality and moribundity twice daily, in the morning and at the end of the working day. Animals were not removed from cage during observation, unless necessary for identification or confirmation of possible findings.

DETAILED CLINICAL OBSERVATIONS:
Yes
- Time schedule: Clinical observations were performed at least twice daily, beginning during the first administration of the test item and lasting throughout the dosing periods up to the day prior to necropsy.
- During the dosing period, these observations were performed immediately after dosing and 1 hour (± 15 minutes) after dosing.

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 PND 1, 4, 7 and 13.

FOOD CONSUMPTION:
Yes. Quantitatively measured weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13.

FOOD EFFICIENCY:No

WATER CONSUMPTION : No.
Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY:
- Time schedule for collection of blood: on the day of scheduled necropsy.
- Anaesthetic used for blood collection: Yes (isoflurane).
- Animals fasted: Males (with a maximum of 24 hours). Females were not fasted. Water was provided.
- How many animals: 5 animals/sex/group.
- Parameters checked were: According to test guidelines.

CLINICAL CHEMISTRY:
- Time schedule for collection of blood: on the day of scheduled necropsy.
- Animals fasted: Yes (with a maximum of 20 hours). Water was provided.
- How many animals: 5 animals/sex/group.
- Parameters checked were: According to test guidelines.
- Blood samples were processed for serum, and serum was analyzed for total Thyroxine (T4).

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION:
Yes
- Time schedule for examinations: The selected males were tested during Week 4 of treatment and the selected females were tested during lactation (LND 6-13).
- Tests were performed after completion of clinical observations (including arena observation).
- Dose groups that were examined: all (5 animals/sex/group)
- Battery of functions tested: According to test guidelines. Hearing ability, pupillary reflex, static righting reflex , fore- and hindlimb grip strength (recorded as the mean of three measurements per animal) and locomotor activity (recording period: 1-hour under normal laboratory light conditions, using a computerized monitoring system). Total movements and ambulations were reported. Ambulations represent movements characterized by a relocation of the entire body position like walking, whereas total movements represent all movements made by the animals, including ambulations but also smaller or more fine movements like grooming, weaving or movements of the head.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
- All animals were subjected to a full post mortem examination, with special attention being paid to the reproductive organs.

ORGAN WEIGHTS
- The organs were weighed at necropsy for all scheduled euthanasia animals. Organ weights were not recorded for animals found dead or euthanized in extremis. Paired organs were weighed together. In the event of gross abnormalities, in addition to the combined weight, the weight of an organ of a pair was determined. Organ to body weight ratios (using the terminal body weight) were calculated.

HISTOPATHOLOGY: Yes
All tissues as defined in the guidance were examined histopathologically. For the testes of all selected males of the control and high dose groups, all males that failed to sire, and one male that died before mating, a detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle.
Statistics:
All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% and or 5% levels.
Numerical data collected on scheduled occasions for the listed variables were analyzed as indicated according to sex and occasion. Descriptive statistics number, mean and standard deviation (or %CV or SE when deemed appropriate) were reported whenever possible. Inferential statistics were performed according to the comparison matrix below when possible, but excluded semi-quantitative data, and any group with less than 3 observations. The following pairwise comparisons were made: low dose group vs. control group; mid dose group vs. control group; high dose group vs. control group. Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test). Datasets with at least 3 groups was compared using a Steel-test (many-to-one rank test). The motor activity data set was compared using an overall Kruskal-Wallis. The Wilcoxon Rank-Sum test was applied to compare the treated groups to the control group. An overall Fisher’s exact test was used to compare all groups at the 5% significance level. The above pairwise comparisons were conducted using Fisher’s exact test whenever the overall test is significant.

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Animals of the 1000 mg/kg bw/day group and, on only a few occasions, in animals of the 300 mg/kg bw/day group: Observed salivation was considered to be a physiological response rather than a sign of systemic toxicity considering its slight severity and the time of occurrence (i.e. after dosing).
Rales were noted in several males and females of the 1000 mg/kg bw/day group (and in a single male and female of the 100 mg/kg bw/day group). As these animals showed rales on only one or a few days, this finding was considered not to be toxicologically relevant (it was likely related to the dosing technique).
Any other clinical signs noted incidentally occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and showed no dose-related trend. At the incidence observed, these were considered to be unrelated to treatment.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One male and two females died in the high dose group. Mortality was considered to be associated to the dosing technique and not test-item related.
The male was euthanized in extremis on day 17 of the treatment period (day 3 of mating period). Body weight gain was normal up to day 15. Necropsy showed macroscopic findings such as mucous contents in the trachea and gas distended parts of the gastrointestinal tract. Microscopic findings showed acute inflammation of the trachea.
One female was found dead on day 16 of the treatment (day 1 of the post-coitum period). The weight gain during the whole period was poor. Macroscopic findings were swollen lungs; microscopic findings were alveolar content and congestion of the lungs, marked bronchial mucosal erosion and erosion/ulceration of the trachea.
One female was euthanized in extremis on day 40 of the treatment (day 1 of lactation period). Food consumption and weight gain was reduced since day 17-20 of the gestation period. Macroscopic findings were pale liver and greenish kidneys. Microscopic findings were marked ulceration in the forestomach and lymphogranulocytic inflammation.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Mean body weights and body weight gain were considered not to be adversely affected by treatment. A few findings at 1000 mg/kg bw/day were regarded as not toxicologically relevant as explained below.
Two males in the dose groups 1000 mg/kg bw/day showed considerably reduced body weight gain over the 4-week treatment period whereas the other males of this dose group grew normally. There were no associated signs of toxicity and mean body weights of 1000 mg/kg bw/day males remained close to control values (4% difference at the end of the treatment period, not statistically significant).
Findings of note in 1000 mg/kg bw/day females consisted of slightly lower mean body weight gain in the last week of the gestation period and reduced weight gain or slight weight loss in three females during the lactation period. Mean body weights of 1000 mg/kg bw/day females did not differ statistically significantly from those of controls (5% difference at the end of the post-coitum and lactation periods). Except for the female which was euthanized in extremis on Day 1 of the lactation period), the lower weight gain was not associated with signs of toxicity.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Females in the high dose group showed reduced food consumption (before and after correction for body weight) in two periods: Periods between days 14-20 of the post-coitum period (about 10%, statistically significant) and during the lactation period (statistically significant between Days 7-13; mean absolute food consumption in this interval was 20% lower than the control value).These findings were considered not to be toxicologically relevant as they were not associated with an adverse effect on body weight gain.
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
There were no treatment-related changes in red blood cell parameters, white blood cell parameters or number of platelets.
Males treated at 1000 mg/kg bw/day had slightly lower activated partial thromboplastin time (APTT) values than concurrent controls. As all values at 1000 mg/kg bw/day remained within the historical control range, this change was regarded as non-adverse.
Female rats showed no treatment-related changes in coagulation parameters.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Clinical chemistry parameters showed no differences between control and treated rats that were considered to be toxicologically significant.
Mean values for alanine aminotransferase (ALAT) and bile acids in males treated at 1000 mg/kg bw/day were higher compared to the control group means (40 and 77%, respectively). These differences were not statistically significant, mean values at dose group 1000 mg/kg bw/day remained in the historical control ranges, and there were no associated anatomic pathology alterations. As such, these clinical chemistry findings were regarded as non-adverse.
Isolated, statistically significant variations noted in clinical chemistry parameters (higher creatinine and sodium in males at 300 mg/kg bw/day) were considered to be unrelated to treatment due to the lack of a dose-related trend and/or small magnitude of the difference from controls.
Serum levels of T4 in males were not affected by treatment.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals.
Grip strength was not affected by treatment. A statistically significantly higher forelimb grip strength noted in males at 300 mg/kg bw/day was judged to be unrelated to treatment due to the lack of a dose-related trend.
The variation in motor activity did not indicate a relation with treatment. All groups showed a similar habituation profile with a decreasing trend in activity over the duration of the test period. In the absence of a dose-related trend, the higher values for total movements and ambulations noted in 100 mg/kg bw/day females, particularly two of them, were regarded as unrelated to treatment.
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
In females at 1000 mg/kg, a statistically significantly lower mean absolute brain weight (relative difference 7%) was regarded as unrelated to treatment. Brain weights of 1000 mg/kg females were close to the historical control mean (except for one female whose brain weight was at the lower end of the historical range), whereas most concurrent control values were at the higher end of the historical range. Moreover, the brain to body weight ratios did not differ statistically significantly. For these reasons, the intergroup difference in absolute brain weight was considered to reflect normal background variation rather than an effect of the test item.
Gross pathological findings:
no effects observed
Description (incidence and severity):
All of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain, and were therefore considered to be unrelated to treatment.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
All of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.
Histopathological findings: neoplastic:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No toxicity was observed up to the highest dose level tested.
Key result
Critical effects observed:
not specified

Analysis of dose preparations: The concentrations analyzed in the test item formulations were in agreement with target concentrations (i.e. mean accuracies between 93% and 100%). A small response noted at the retention time of the test item in the chromatograms of the control group formulation was considered to derive from carry-over in the analytical system. This had no significant effect on the results of the other study samples because of the minor magnitude (maximally 0.0092% relative to low dose group samples). The formulations of the low and the high dose group were homogeneous (i.e. coefficient of variation ≤5.8%).

Summary results dose range finding study: Mortality was not observed. At 500 and 1000 mg/kg bw/day, slight salivation was noted after dosing. At 250 mg/kg bw/day, reduced weight gain in one animal was noted. At 500 mg/kg bw/day, weight loss was found in one animal between Days 5-11, in the other two reduced weight gain was found. At 1000 mg/kg bw/day, weight loss was seen in one animal, the other two were normal. No effects were seen on food consumption and at macroscopic examination. Kidney weights were normal in all rats, liver weights were slightly higher in one animal in each group at 250 and 1000 mg/kg bw/day each.

Conclusions:
An oral Repeated Dose Toxicity Study combined with a Reproduction/Developmental Toxicity Screening was performed according to OECD/EC guidelines and GLP principles. No adverse effects were observed for Miranol Ultra C32. Therefore, the NOAEL was established to be 1000 mg/kg bw/day.
Executive summary:

A combined oral repeated dose study with screening for reproductive and/ or developmental effects was performed according to OECD/EC guidelines and GLP principles. Miranol Ultra C32 was administered by daily oral gavage to male and female rats at dose levels of 100, 300 and 1000 mg/kg bw/day. Males were treated for 29 days, up to and including the day of the scheduled necropsy. Females that delivered were treated for 50-56 days, i.e. 14 days prior to mating (with the objective to cover at least two complete estrous cycles), the variable time to conception, the duration of pregnancy and 13 or 15 days after delivery, up to and including the day before scheduled necropsy. Females without offspring were treated for 53 days (no evidence of mating) or 42-43 days (not pregnant or implantation site only). Treatment with Miranol Ultra C32 was associated with a few non-adverse changes at the highest dose group i.e. slight salivation in both sexes, lower food consumption in females in the last week of gestation and during lactation, and lower activated partial thromboplastin time in males. No treatment-related or toxicologically relevant changes were noted in the other parameters investigated in this study.

Based on the absence of adverse effects up to 1000 mg/kg bw/day, a parental No Observed Adverse Effect Level (NOAEL) for Miranol Ultra C32 of 1000 mg/kg bw/day was established.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
July 2016
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA, Health Effects Test Guidelines OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test (July 2000)
Version / remarks:
July 2000
Deviations:
no
Qualifier:
according to
Guideline:
other:
Principles of method if other than guideline:
In addition, the procedures described in this report essentially conform to the following guidelines:
- OECD Guidelines for Testing of Chemicals, Guideline 421, Reproduction/Developmental Toxicity Screening Test (July 2016)
- The United States EPA Health Effects Test Guidelines, OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test (July 2000)
- Commission regulation (EC) No 440/2008 Part B: Methods for the Determination of Toxicity and other Health Effects; B.7: "Repeated Dose (28 days) Toxicity (oral)". Official Journal of the European Union No. L142 (May 2008)
- OECD Guidelines for Testing of Chemicals, Guideline 407, Repeated Dose 28-day Oral Toxicity Study in Rodents (October 2008)
- The United States EPA Health Effects Test Guidelines, OPPTS 870.3050, Repeated dose 28-day oral toxicity study in rodents (July 2000)
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
- Appearance: Clear amber viscous liquid
- Storage conditions: At room temperature

Specific details on test material used for the study:
STABILITY OF TEST MATERIAL
- Stability for at least 7 days in the refrigerator is confirmed over the concentration range 1.0 to 200
mg/mL
- Stability for at least 6 hours at room temperature under normal laboratory light conditions is conf
irmed over the concentration range 1.0 to 200 mg/mL
CORRECTION FACTOR
Purity/Composition correction factor: Yes, correction factor is 2.554 based on solid content.
No specific handling conditions required

Test animals

Species:
rat
Strain:
other: Crl:WI(Han).
Details on species / strain selection:
The Wistar Han rat was chosen as the animal model for this study as it is an accepted rodent species for toxicity testing by regulatory agencies. Charles River Den Bosch 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. The total number of animals used in this study was considered to be the minimum required to properly characterize the effects of the test item. This study has been designed such that it does not require an unnecessary number of animals to accomplish its objectives.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Age at study initiation: Approximately 11 weeks (males); 13 weeks (females).
- Weight at study initiation: 276-312 gr (males); 205-242 gr (females).
- Fasting period before study: no
- Housing:
Pre-mating: Animals were housed in groups of 5 animals/sex/cage in Macrolon cages.
Mating: Females were caged together with males on a one-to-one-basis in Macrolon cages.
Post-mating: Males were housed in their home cage with a maximum of 5 animals/cage. Females were individually housed in Macrolon cages.
Lactation phase: females were housed in Macrolon plastic cages (MIII type, height 18 cm). Pups were housed with the dam, except during locomotor activity monitoring of the dams, when the pups were kept warm in their home cage using bottles filled with warm water. In order to avoid hypothermia of pups, pups were not left without their dam or a bottle filled with warm water for longer than 30-40 minutes.
Locomotor activity monitoring: animals were housed individually in a Hi-temp polycarbonate cage without cageenrichment, bedding material, food and water.
General: Sterilised sawdust as bedding material and paper as cage enrichment were supplied.
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water: Free access to tap water.
- Acclimation period: 6 days.

ENVIRONMENTAL CONDITIONS (set conditions)
- Temperature (°C): 20 - 22.
- Humidity (%): 37 - 59.
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 15-Aug-2017 to 07 Dec 2017

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS
Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared at least every 7 days as a solution, formulated in daily portions and stored in the refrigerator. The dosing formulations were removed from the refrigerator and stirred for at least 30 minutes before dosing. Test item dosing formulations were kept at room temperature until dosing. If practically possible, the dosing formulations and vehicle were continuously stirred until and during dosing. Adjustment was made for specific gravity of the test item.

DOSE VOLUME
5 mL/kg body weight. Actual dose volumes were calculated according to the latest body weight.
Details on mating procedure:
- M/F ratio per cage:1/1 (one female was cohabitated with one male of the same treatment group, avoiding sibling mating (Charles River supplied non-litter mates).
- Length of cohabitation: A maximum of 14 days was allowed for mating.
- Proof of pregnancy: Detection of mating was confirmed by evidence of sperm in the vaginal lavage, by staging of the estrous cycle and/or or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating had occurred, the males and females were separated.
- After 14 days of unsuccessful pairing one female who at 100 mg/kg bw/day had not shown evidence of mating was separated from her male.
- After successful mating each pregnant female was caged individually in Macrolon cages (MIII type, height 18 cm).
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were performed using a validated analytical procedure.
Duplicate sets of samples (approximately 500 mg) were sent to the analytical laboratory:
- Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 10% for solutions of target concentration.
- Homogeneity results were considered acceptable if the coefficient of variation (CV) of concentrations was ≤ 10%.
- Stability analyses performed previously in conjunction with the method development and validation study demonstrated that the test item is stable stable in the vehicle when prepared and stored under the same conditions at concentrations bracketing those used in the present study.
Duration of treatment / exposure:
Males were treated for 29 days, i.e. 2 weeks prior to mating, during mating and up to and including the day before scheduled necropsy.
Females that delivered were treated for 50-56 days, i.e. 14 days prior to mating (with the objective to cover at least two complete estrous cycles), the variable time to conception, the duration of pregnancy and 13 or 15 days after delivery, up to and including the day before scheduled necropsy. Females without offspring were treated for 53 days (no evidence of mating) or 42-43 days (not pregnant or implantation site only). Pups were not treated directly, but were potentially exposed to the test substance in utero and through lactational transfer.
Frequency of treatment:
Once daily, 7 days per week.
Details on study schedule:
- Age at mating of the mated animals in the study: Approximately 13 weeks
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
Low dose group
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Remarks:
Mid dose group
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
High dose group
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
Dose selection rationale: The dose levels were selected based on the results of a 11-day dose range finder with oral administration of Miranol Ultra C32 in rats. No guidelines were applicable as this study was intended for dose level selection purposes only.

Selection of animals for selected measurements: 5 animals/sex/group were randomly selected at allocation for functional observations, clinical pathology, macroscopic examination, organ weights and histopathology. Only females with live offspring were selected.
Positive control:
no

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS:
Yes
- Time schedule: Throughout the study, animals were observed for general health/mortality and moribundity twice daily, in the morning and at the end of the working day. Animals were not removed from cage during observation, unless necessary for identification or confirmation of possible findings.

DETAILED CLINICAL OBSERVATIONS:
Yes
- Time schedule: Clinical observations were performed at least twice daily, beginning during the first administration of the test item and lasting throughout the dosing periods up to the day prior to necropsy.
- During the dosing period, these observations were performed immediately after dosing and 1 hour (± 15 minutes) after dosing.

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 PND 1, 4, 7 and 13.

FOOD CONSUMPTION:
Yes. Quantitatively measured weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13.

FOOD EFFICIENCY:No

WATER CONSUMPTION : No.
Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY:
- Time schedule for collection of blood: on the day of scheduled necropsy.
- Anaesthetic used for blood collection: Yes (isoflurane).
- Animals fasted: Males (with a maximum of 24 hours). Females were not fasted. Water was provided.
- How many animals: 5 animals/sex/group.
- Parameters checked were: According to test guidelines.

CLINICAL CHEMISTRY:
- Time schedule for collection of blood: on the day of scheduled necropsy.
- Animals fasted: Yes (with a maximum of 20 hours). Water was provided.
- How many animals: 5 animals/sex/group.
- Parameters checked were: According to test guidelines.
- Blood samples were processed for serum, and serum was analyzed for total Thyroxine (T4).

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION:
Yes
- Time schedule for examinations: The selected males were tested during Week 4 of treatment and the selected females were tested during lactation (LND 6-13).
- Tests were performed after completion of clinical observations (including arena observation).
- Dose groups that were examined: all (5 animals/sex/group)
- Battery of functions tested: According to test guidelines. Hearing ability, pupillary reflex, static righting reflex , fore- and hindlimb grip strength (recorded as the mean of three measurements per animal) and locomotor activity (recording period: 1-hour under normal laboratory light conditions, using a computerized monitoring system). Total movements and ambulations were reported. Ambulations represent movements characterized by a relocation of the entire body position like walking, whereas total movements represent all movements made by the animals, including ambulations but also smaller or more fine movements like grooming, weaving or movements of the head.
Oestrous cyclicity (parental animals):
Estrous cycles were evaluated by examining the vaginal cytology of samples obtained by vaginal lavage. Daily vaginal lavage was performed for all females beginning 14 days prior to treatment (pretest period), the first 14 days of treatment and during mating until evidence of copulation was observed. Vaginal lavage was continued for those females with no evidence of copulation until termination of the mating period.
On the day of necropsy, a vaginal lavage was also taken to determine the stage of estrous. This was done for all females, except for females that had to be euthanized in extremis or died spontaneously.
Sperm parameters (parental animals):
Slides of the testes were prepared for histopathological staging of spermatogenesis (5 males of the control and high dose group, the male that failed to sire and the male that died at 1000 mg/kg bw/day before termination of the experiment).
Litter observations:
STANDARDISATION OF LITTERS
- To reduce variability among the litters, on PND 4 eight pups from each litter of equal sex distribution (if possible) were selected. Blood samples were collected from two of the surplus pups (if possible from one male and one female pup). Selective elimination of pups was not done. Whenever the number of male or female pups prevented having four of each sex per litter, partial adjustment (for example, five males and three females) was acceptable.
- Maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded. Blood samples were collected from two of the surplus pups for the thyroid hormone analysis.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross abnomalies, weight gain, anogenital distance, areola/nipple retentionphysical or behavioural abnormalities.

- Mortality: The numbers of live and dead pups on Day 1 of lactation and daily thereafter were determined. If possible, defects or cause of death were evaluated.
- Clinical signs: At least once daily, detailed clinical observations were made in all animals.
- Body weights: Live pups were weighed on Days 1, 4, 7 and 13 of lactation.
- Sex: Sex was determined for all pups on Days 1 and 4 (externally) and at PND 14-16 (both externally and internally).

GROSS EXAMINATION OF DEAD PUPS
- Yes, if possible, defects or cause of death were evaluated.
- The stomach of pups not surviving to the scheduled necropsy date was examined for the presence of milk, if possible.
- If possible, defects or cause of death were evaluated.
Postmortem examinations (parental animals):
GROSS PATHOLOGY: Yes
- All animals were subjected to a full post mortem examination, with special attention being paid to the reproductive organs.

ORGAN WEIGHTS
- The organs were weighed at necropsy for all scheduled euthanasia animals. Organ weights were not recorded for animals found dead or euthanized in extremis. Paired organs were weighed together. In the event of gross abnormalities, in addition to the combined weight, the weight of an organ of a pair was determined. Organ to body weight ratios (using the terminal body weight) were calculated.

HISTOPATHOLOGY: Yes
All tissues as defined in the guidance were examined histopathologically. For the testes of all selected males of the control and high dose groups, all males that failed to sire, and one male that died before mating, a detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle.
Postmortem examinations (offspring):
SACRIFICE
- On PND 4, the surplus pups (> 8 pups per litter) were euthanized by decapitation. From two surplus pups per litter, blood was collected, if possible.
- All remaining pups were euthanized on PND 14-16.

GROSS NECROPSY
- Descriptions of all external abnormalities were recorded.
- Pups that died or were euthanized before scheduled termination were examined externally and sexed (both externally and internally).Particular attention was paid to the external reproductive genitals to examine signs of altered development.


HISTOPATHOLOGY / ORGAN WEIGTHS
No.
Statistics:
All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% and or 5% levels.
Numerical data collected on scheduled occasions for the listed variables were analyzed as indicated according to sex and occasion. Descriptive statistics number, mean and standard deviation (or %CV or SE when deemed appropriate) were reported whenever possible. Inferential statistics were performed according to the comparison matrix below when possible, but excluded semi-quantitative data, and any group with less than 3 observations. The following pairwise comparisons were made: low dose group vs. control group; mid dose group vs. control group; high dose group vs. control group. Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test). Datasets with at least 3 groups was compared using a Steel-test (many-to-one rank test). The motor activity data set was compared using an overall Kruskal-Wallis. The Wilcoxon Rank-Sum test was applied to compare the treated groups to the control group. An overall Fisher’s exact test was used to compare all groups at the 5% significance level. The above pairwise comparisons were conducted using Fisher’s exact test whenever the overall test is significant.
Reproductive indices:
For each group, the following calculations were performed:
- Mating index: (Number of females mated/Number of females paired) x 100
- Precoital time: Number of days between initiation of cohabitation and confirmation of mating
- Fertility index: (Number of pregnant females/Number of females paired) x 100
- Conception index: (Number of pregnant females/Number of females mated) x 100
- Gestation index: (Number of females bearing live pups/Number of pregnant females) x 100
- Duration of gestation: Number of days between confirmation of mating and the beginning of parturition
- Post implantation survival index: (Total number of offspring born/Total number of uterine implantation sites) x 100
Offspring viability indices:
- Live birth index: (Number of live offspring on day 1 after littering/Total number of offspring born) x 100
- 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
- Viability index: (Number of live offspring on day 4 before culling/Number live offspring on day 1 after littering) x 100
- Lactation index: (Number of live offspring on Day 13 after littering/Number live offspring on day 4 (after culling)) x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Animals of the 1000 mg/kg group and, on only a few occasions, in animals of the 300 mg/kg group:
Observed salivation was considered to be a physiological response rather than a sign of systemic toxicity considering its slight severity and the time of occurrence (i.e. after dosing).
Rales were noted in several males and females of the 1000 mg/kg group (and in a single male and female of the 100 mg/kg group). As these animals showed rales on only one or a few days, this finding was considered not to be toxicologically relevant (it was likely related to the dosing technique). Any other clinical signs noted incidentally occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and showed no dose-related trend. At the incidence observed, these were considered to be unrelated to treatment.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One male and two females died in the highest dose group. The male was euthanized in extremis on day 17 of the treatment period (day 3 of mating period). Body weight gain was normal up to day 15. Necropsy showed macroscopic findings such as mucous contents in the trachea and gas distended parts of the gastrointestinal tract. Microscopic findings showed acute inflammation of the trachea. One female was found dead on day 16 of the treatment (day 1 of the post-coitum period). The weight gain during the whole period was low. Macroscopic findings were swollen lungs; microscopic findings were alveolar content and congestion of the lungs, marked bronchial mucosal erosion and erosion/ulceration of the trachea. One female was euthanized in extremis on day 40 of the treatment (day 1 of lactation period). Food consumption and weight gain was reduced since day 17-20 of the gestation period. Macroscopic findings were pale liver and greenish kidneys. Microscopic findings were marked ulceration in the forestomach and lymphogranulocytic inflammation. Mortality was considered to be associated to the dosing technique and not test-item related.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Mean body weights and body weight gain were considered not to be adversely affected by treatment.
A few findings at 1000 mg/kg bw/day were regarded as not toxicologically relevant as explained below.
Two males in the dose groups 1000 mg/kg bw/day showed considerably reduced body weight gain over the 4-week treatment period whereas the other males of this dose group grew normally. There were no associated signs of toxicity and mean body weights of 1000 mg/kg bw/day males remained close to control values (4% difference at the end of the treatment period, not statistically significant).
Findings of note in 1000 mg/kg females consisted of slightly lower mean body weight gain in the last week of the gestation period and reduced weight gain or slight weight loss in three females during the lactation period. Mean body weights of 1000 mg/kg bw/day females did not differ statistically significantly from those of controls (5% difference at the end of the post-coitum and lactation periods). Except for the female which was euthanized in extremis on Day 1 of the lactation period, the lower weight gain was not associated with signs of toxicity.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Females in the dosing groups of 1000 mg/kg showed reduced food consumption (before and after correction for body weight) in two periods: Periods between days 14-20 of the post-coitum period (about 10%, statistically significant) and during the lactation period (statistically significant between days 7-13; mean absolute food consumption in this interval was 20% lower than the control value).These findings were considered not to be toxicologically relevant as they were not associated with an adverse effect on body weight gain.
Food efficiency:
not specified
Description (incidence and severity):
Relative Food Consumption calculated against the body weight for scheduled intervals
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment-related changes in red blood cell parameters, white blood cell parameters or
number of platelets.
Males treated at 1000 mg/kg had slightly lower activated partial thromboplastin time (APTT) values than concurrent controls. As all values at 1000 mg/kg remained within the historical control range, this change was regarded as non-adverse.
Female rats showed no treatment-related changes in coagulation parameters.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Clinical chemistry parameters showed no differences between control and treated rats that were considered to be toxicologically significant.
Mean values for alanine aminotransferase (ALAT) and bile acids in males treated at 1000 mg/kg were higher compared to the control group means (40 and 77%, respectively). These differences were not statistically significant, mean values at dose group 1000 mg/kg remained in the historical control ranges, and there were no associated anatomic pathology alterations. As such, these clinical chemistry findings were regarded as non-adverse.
Isolated, statistically significant variations noted in clinical chemistry parameters (higher creatinine and sodium in males at 300 mg/kg) were considered to be unrelated to treatment due to the lack of a dose-related trend and/or small magnitude of the difference from controls.
Serum levels of T4 in males were not affected by treatment.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals.
Grip strength was not affected by treatment. A statistically significantly higher forelimb grip strength noted in males at 300 mg/kg was judged to be unrelated to treatment due to the lack of a dose-related trend.
The variation in motor activity did not indicate a relation with treatment. All groups showed a similar habituation profile with a decreasing trend in activity over the duration of the test period. In the absence of a dose-related trend, the higher values for total movements and ambulations noted in 100 mg/kg females, particularly two of them, were regarded as unrelated to treatment.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
All of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.
Histopathological findings: neoplastic:
no effects observed
Other effects:
effects observed, non-treatment-related

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
effects observed, non-treatment-related
Description (incidence and severity):
Length and regularity of the estrous cycle were not affected by treatment. During the treatment (premating) period, all females had regular cycles of four days.
Extended di-estrus during pairing occurred in one female of the 100 mg/kg group which showed no evidence of mating. The irregular cycle noted in one female of the 1000 mg/kg group was not test item-related as it occurred prior to initiation of treatment.
Reproductive function: sperm measures:
effects observed, treatment-related
Description (incidence and severity):
One male at 300 mg/kg bw/day showed tubular atrophy in the testes and reduced luminal sperm with luminal cell debris in the epididymides which accounted for the lack of offspring. This male had no normal spermatogenic staging profile. For the other evaluated testes no indications for abnormal spermatogenesis were seen.
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
Seven couples had no offspring: 2/10 control couples (both females not pregnant); 2/10 couples at 100 mg/kg bw/day (one female without evidence of mating; one female not pregnant); 2/10 couples at 300 mg/kg bw/day (one female not pregnant; one female implantation sites only); 1/10 couples at 1000 mg/kg bw/day (one female not pregnant).

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Remarks:
Parental/ Reproduction
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No toxicity was observed up to the highest dose level tested.

Target system / organ toxicity (P0)

Key result
Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Clinical signs were seen only in pups of the 1000 mg/kg bw/day group that did not survive until scheduled sacrifice. The findings in the pups of one litter (cold, no milk in the stomach) were related to the moribundity of the dam, which was not test item-related.
The nature of the findings in a few other pups (pale appearance, cold, no milk in the stomach) remained within the range seen normally in pups that die within a few days after birth. These findings were therefore regarded as unrelated to treatment.
Treated pups that survived until scheduled sacrifice showed no clinical signs and the incidental findings in pups of the control group (alopecia, pale appearance) remained within the range considered normal for pups of this age.
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
The number of live offspring in day 1 after littering compared with the total number of offspring born was considered not to be affected by treatment. At 1000 mg/kg bw/day, seven pups (from 4 litters) were found dead at first litter check. The pup mortality in one of the litters was considered to be related to the moribundity of the dam (euthanized in extremis at PND 1; her moribundity was not test item-related. The number of dead pups in the other 1000 mg/kg bw/day litters was within normal limits and judged to be unrelated to treatment.
The same was true for the incidental mortality at 100 mg/kg bw/day (one pup from 1 litter).The number of live offspring on PND 4 compared with the number of live offspring on PND 1 was considered not to be affected by treatment. At 1000 mg/kg bw/day, a total of 15 pups (out of 4 litters) died at PND 1-2 (versus none in the control group; the difference was statistically significant). These pups went missing, presumably cannibalized, died spontaneously, or were euthanized in extremis (10 pups of one dam that were euthanized at PND 1). The moribundity of the dam, resulting in poor health of her pups, was not test item-related. Pup mortality in the other 1000 mg/kg bw/day litters remained within the range considered normal for pups of this age and was therefore considered to be unrelated to treatment. For the same reason the incidental pup mortality at the lower dose levels was regarded as unrelated to treatment (one pup out of one litter at dose 100 mg/kg bw/day, two pups out of two litters at dose 300 mg/kg bw/day).
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Body weights of pups were not affected by treatment.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
Serum T4 levels in male and female PND 14-16 pups were not affected by treatment. Assessment of T4 for PND 4 pups and TSH for PND 14-16 pups was considered not relevant because no treatment-related changes in T4 were noted in pups at PND 14-16.
Sexual maturation:
not examined
Anogenital distance (AGD):
no effects observed
Description (incidence and severity):
Anogenital distance (absolute and normalized for body weight) in male and female pups was not affected by treatment.
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
Treatment up to 1000 mg/kg be/day had no effect on areola/nipple retention. For none of the examined male pups nipples were observed at PND 13.
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
No macroscopic findings were noted among pups that were considered to be test item-related. Absence of milk in the stomach was noted in most of the pups found dead at first litter check. As absence of milk in the stomach is a normal finding in pups that die prematurely and the incidence among pups of surviving dams remained in the range considered normal for pups of this age, this macroscopic finding was regarded as unrelated to treatment. No other macroscopic findings were noted in pups that died prematurely. Pups that survived until scheduled sacrifice showed no abnormalities at macroscopic examination.
Histopathological findings:
not examined
Other effects:
no effects observed

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Remarks:
Repro/ development
Generation:
F1
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effects seen up to the highest dose level tested (1000 mg/kg bw/day)

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

Analysis of dose preparations: The concentrations analyzed in the test item formulations were in agreement with target concentrations (i.e. mean accuracies between 93% and 100%). A small response noted at the retention time of the test item in the chromatograms of the control group formulation was considered to derive from carry-over in the analytical system. This had no significant effect on the results of the other study samples because of the minor magnitude (maximally 0.0092% relative to low dose group samples). The formulations of the low and the high dose group were homogeneous (i.e. coefficient of variation≤5.8%).

Applicant's summary and conclusion

Conclusions:
In an oral OECD 422 screening study, the parental and reproductive NOAEL were derived to be 1000 mg/kg bw/day. No parental, reproduction and developmental toxicity was observed up to the highest dose level tested (1000 mg/kg bw/day).
Executive summary:

A combined oral repeated dose study with screening for reproductive and/ or developmental effects was performed according to OECD/EC guidelines and GLP principles. Miranol Ultra C32 was administered by daily oral gavage to male and female rats at dose levels of 100, 300 and 1000 mg/kg bw/day. Males were exposed for 2 weeks prior to mating, during mating, and up to termination (for 29 days). The females were exposed for 2 weeks prior to mating, during mating, during post-coitum, and at least 4 days of lactation (for 50-56 days). Treatment with Miranol Ultra C32 was associated with a few non-adverse changes at the highest dose group i.e. slight salivation in both sexes, lower food consumption in females in the last week of gestation and during lactation, and lower activated partial thromboplastin time in males. No treatment-related or toxicologically relevant changes were noted in the other parameters investigated in this study.

Based on the absence of adverse effects up to 1000 mg/ kg bw/day, a parental No Observed Adverse Effect Level (NOAEL) for Miranol Ultra C32 of 1000 mg/kg bw/day was established. The NOAEL for reproduction was established to be 1000 mg/ kg bw/day.