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Diss Factsheets

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental start date: 27 September 2018. Experimental completion: 28 February 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report date:
2019

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
N,N-bis(2-hydroxyethyl)-3-[(C16-18)alkoxy]-1-propanamine
EC Number:
930-859-5
Molecular formula:
C23-25H49-53NO3
IUPAC Name:
N,N-bis(2-hydroxyethyl)-3-[(C16-18)alkoxy]-1-propanamine
Test material form:
solid
Remarks:
Amber solid
Specific details on test material used for the study:
Test item: EXP1505486
Appearance: Amber gelatinous solid
Storage conditions: At ambient temperature (15 to 25°C) in the dark (may be used/formulated in light)
Supplier: Sponsor
Batch number: E00268-015-141
Stability/expiry date: 10 June 2019
Purity: 100%

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
The rat was chosen as the test species because of the requirement for a rodent species by regulatory agencies. The RccHanTM;WIST was used because of the historical control data available at this laboratory.
Sex:
male/female
Details on test animals or test system and environmental conditions:
Animals
Strain/Species RccHanTM;WIST rat.
Supplier Envigo RMS Limited.
Number of animals ordered 52 males and 56 females. Spare animals were removed from the study room after treatment commenced.
Duration of acclimatization Males: 7 days before commencement of treatment. Females: 6days before commencement ofestrus cycle evaluation.
Age of the animals at the start of the study Males 10 to 11 weeks old. Females 13 to 14 weeks old.
Weight range of the animals at the start of the study Males 260 to 306g. Females 198 to 236g.

Animal Replacement
Before the commencement of treatment, study allocation was revised to ensure all animals had regular estrous cycles, and to reduce inter/intra group body weight variation by replacement of animals with spares of suitable weight. Sevenfemales with irregular estrus cycles were replaced before dosing.

Animal Care and Husbandry
Environmental Control
Rodent facility: Limited access - to minimise entry of external biological and chemical agents and to minimise the transference of such agents between rooms.
Air supply: Filtered fresh air which was passed to atmosphere and not recirculated.
Temperature and relative humidity: Monitored and maintained within the range of 20-24ºC and 40-70%. Although conditions were occasionally outside the indicated ranges, these deviations were minor and/or of short duration and were not considered to have influenced the health of the animals and/or the outcome of the study.
Lighting: Artificial lighting, 12 hours light: 12 hours dark.
Electricity supply: Public supply with automatic stand-by generators.

Animal Accommodation
Cages: Cages comprised of a polycarbonate body with a stainless steel mesh lid; changed at appropriate intervals. Solid (polycarbonate) bottom cages were used during the acclimatization, pre-pairing, gestation, litteringand lactation periods. Grid bottomed polypropylene cages were used during pairing. These were suspended above absorbent paper which was changed daily during pairing.
Cage distribution: The cages were distributed on the racking to equalise, as far as possible, environmental influences amongst the groups.
Bedding: Solid bottom cages contained softwood based bark-free fiber bedding, which was changed at appropriate intervals each week.
Nesting material: Paper shavings, which was changed at the same frequency as bedding. Provided during gestation and lactation phases.
Number of animals per cage: Pre-pairing up to four animals of one sex. Pairing one male and one female. Males after mating up to four animals. Gestation one female. Lactation one female + litter

Environmental Enrichment
Aspen chew block: A soft white untreated wood block;provided to each cage throughout the study (except during pairing and lactation) and replaced when necessary.
Plastic shelter: Provided to each cage throughout the study (except during pairingand lactation) and replaced at the same time as the cages.

Diet Supply
Diet: SDS VRF1 Certified pelleted. The diet contained no added antibiotic or other chemotherapeutic or prophylactic agent.
Availability: Non-restricted.

Water Supply
Supply: Potable water from the public supply via polycarbonate bottles with sipper tubes. Bottles were changed at appropriate intervals.
Availability: Non-restricted.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on exposure:
Formulation
Method of preparation: The required amount of test item was warmed and mixed with some warmed vehicle to form a paste. Further amounts of vehicle were gradually added and mixed to produce a smooth, pourable formulation which was diluted to volume. A series of formulations at the required concentrations were prepared by dilution of individual weighings of the test item.
Frequency of preparation: Weekly.
Storage of formulation: Refrigerated (2 to 8°C). Stable for 21 days.
Test item accounting: Detailed records of compound usage were maintained. The amount of test item necessary to prepare the formulations and the amount actually used were determined on each occasion. The difference between these amounts was checked before the formulations were dispensed.

Administration
Dosing was restricted to the F0 generation. Animals of the F1 generation were not dosed directly.
Route: Oral gavage using a suitably graduated syringe and a rubber catheter inserted via the mouth. If female was in parturition, dosing was not performed on that day.
Treated at: Constant doses in mg/kg/day.
Volume: dose 6mL/kg body weight.
Individual dose volume: Calculated from the most recently recorded scheduled body weight.
Control (Group 1): Vehicle at the same volume dose as treated groups.
Formulation: A daily record of the usage of formulation was maintained based on weights. This balance was compared with the expected usage as a check of correct administration. No significant discrepancy was found. Formulations were stirred using a magnetic stirrer before and throughout the dosing procedure.
Details on mating procedure:
Pairing commenced After a minimum of two weeks of treatment.
Male/female ratio 1:1 from within the same treatment groups.
Duration of pairing Up to two weeks.
Daily checks for evidence of mating Ejected copulation plugs in cage tray and sperm in the vaginal smear.
Day 0 of gestation When positive evidence of mating was detected.
Male/female separation Day when mating evidence was detected.
Pre-coital interval Calculated for each female as the time between first pairing and evidence of mating.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Stability and homogeneity
Before commencement of treatment, the suitability of the proposed mixing procedures was determined and specimen formulations at 3.75 and 250mg/mL were analyzed to assess the stability and homogeneity of the test itemin the liquid matrix. The homogeneity and stability offormulations during storage were confirmed as part of another study.
Achieved concentration Samples of each formulation prepared for administration in Weeks 1, 4 and 6 of treatment were analyzed for achieved concentration of the test item.

The analytical procedure was successfully validated with respect to specificity of chromatographic analysis, limit of detection and quantification, linearity of detector response, repeatability, method accuracy and precision.
Homogeneity was confirmed during distribution between the bottles, during magnetic stirring for 2 hours, and on re-suspension following storage at ambient temperature for 1 day. At each time-point, the mean analysed concentration for the three samples remained within 7% of the initial time zero value and the coefficient of variation was less than 3% with the exception of 3.75 mg/mL 2-hour time point which had a coefficient of variation of 5.50%.
Analysis of dose formulations prepared for Week 1, 4 and 6 were all within 10% of nominal concentrations, confirming accurate formulation.
Control dose formulation samples prepared for Week 4 showed a response for the test item but this was believed to be contamination arisen during the analytical process and not direct contamination of the control dose formulations administered to the animals. Control dose formulation samples prepared for Week 6 showed a positive response for the test item but this was below the limit of quantification.
Duration of treatment / exposure:
Males were treated daily for two weeks before pairing, up to necropsy after a minimum of four consecutive weeks. Females were treated daily for two weeks before pairing, throughout pairing, gestation and until Day 13 of lactation.
Frequency of treatment:
Once daily at approximately the same time each day.
Details on study schedule:
Duration of Treatment
Males: Two weeks pre-pairing up to necropsy after minimum of fourweeks.
Females: Two weeks before pairing, then throughout pairing and gestation until Day 13 of lactation. Animals of the F1 generation were not dosed.

Time Schedule
Treatment commenced: 18 October 2018
F0 pairing commenced: 01 November 2018
F0 necropsy: Males 22 November 2018. Females 06 to 17 December 2018
Experimental completion date (pathology): 28 February 2019
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
30 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
250 mg/kg bw/day (nominal)
No. of animals per sex per dose:
12 males and females per dose
Control animals:
yes, concurrent vehicle
Details on study design:
Rationale for Dose Level Selection
The doses used in this study (0, 30, 100and 250mg/kg/day) were selected in conjunction with the Sponsor. The dose levels selected, were based on ongoing data from a 28-day oral gavage toxicity study in the Wistar Han rat (CovanceStudy Number BB44ST), at doses of 30, 100 and 250 mg/kg/day. In that study dosing over 4 weeks was well tolerated. Body weight gain was lower than the control with the response more evident for males, food consumption was lower for males and salivation was noted after dosing. Small prostate and seminal vesicles was noted in males given 250 mg/kg/day. Dose related higher kidney weights were noted in males at all doses and higher liver weights noted in females given 100mg/kg/day and both sexes given 250mg/kg/day, however these findings were not considered adverse. As such the same dose sequence, 0, 30, 100 and 250mg/kg/day was used for this study.


Allocation and Identification
Allocation: On arrival and non-selective allocation to cages. On Day 1 of study all animals were weighed andestrous cycles and body weights were reviewed before dosing commenced by Study Management. Body weight of animals did not exceed ±20% of the mean for each sex.
Identification of animals: Each adult animal was assigned a number and identified uniquely within the study by a microchip before Day 1 of treatment. The offspring were numbered individually within each litter on Day 1 of age, using a toe tattoo.
Identification of cages: Each cage label was color-coded according to group and was numbered uniquely with cage and study number, as well as the identity of the occupants.

Examinations

Parental animals: Observations and examinations:
Mortality
A viability check was performed near the start and end of each working day. Animals were killed for reasons of animal welfare where necessary or if they exhibited pregnancy loss. A complete necropsy was performed in all cases

Clinical Observations
Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of animal ill-health amongst the occupant(s). Any deviation from normal was recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as appropriate. During the acclimatization period, observations of the animals and their cages were recorded at least once per day.
Signs Associated with Dosing
Daily during the first week of treatment, once weekly from Week 2 on wards and on Days 0, 7, 14 and 20 of gestation and Days 1, 6 and 12 of lactation for F0 females, detailed observations were recorded at the following times in relation to dose administration:
Pre-dose observation. 1 to 2 hours after completion of dosing. As late as possible in the working day
Clinical Signs
A detailed weekly physical examination was performed on each animal to monitor general health. F0 females were examined on Days 0, 7, 14 and 20 of gestation and Days 1, 7 and 13 of lactation.

Body Weight
The weight of animals was recorded as follows:
F0 males Weekly during acclimatisation. Before dosing on the day that treatment commenced (Week 0) and weekly thereafter. On the day of necropsy.
F0 females Weekly during acclimatisation. Before dosing on the day that treatment commenced (Week 0) and weekly before pairing. Days 0, 7, 14and 20 after mating. Day 1, 6, and 13of lactation. On the day of necropsy.

Food Consumption
The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded as follows:
F0 animals Weekly, from the day that treatment commenced. Food consumption was not recorded for males and females during the period when paired for mating (Week 3), but recommenced for males in Week 4. For females after mating food consumption was performed to match the body weight recording: Days 0-5, 6-12 and 13-19 after mating Days 1-3, 4-6 and 7-12 of lactation.
From these records the mean weekly or daily consumption per animal (g/animal/week or g/animal/day) was calculated for each phase.

Water Consumption
During Weeks 1 and 2 of treatment, water consumption was recorded by weight for each cage of animals, using water bottles fitted with sipper tubes.

Parturition Observations and Gestation Length
Duration of gestation Time elapsing between the detection of mating and commencement of parturition.
Parturition observations From Day 20 after mating, females were inspected three times daily for evidence of parturition. The progress and completion of parturition was monitored, numbers of live and dead offspring were recorded and any difficulties observed were recorded.

Thyroid Hormone Analysis
Blood samples were collected from animals at the following occasions:
Ocasion Animals
Day 4 of age: Offspring: 2 females per litter (where possible)
One for T4 (serum) (Single blood sample taken for both analytes, priority given to T4 sample)
One for TSH (serum)
No pups were allocated to these procedures on Day 4 of age if the resultant live litter size fell below 8 pups, or the resultant number of female pups fell below 3 pups. If only 4 female pups were available within a litter but the overall litter size is >8,one female may be selected with priority given to the serum sample.
Day 13 of age Offspring: 2 males and 2 females per litter (where possible)
Two for T4 (serum); where possible one male and one female (Single blood sample taken for both analytes, priority given to T4 sample)
Two for TSH (serum); where possible one male and one female
At termination All adults

Sequence of blood sampling: On each occasion, in order to minimise any potential confounding effect of the time of day of blood sampling, the order of blood sampling wascontrolled to allow satisfactory inter-group comparisons.
Blood sample site: Offspring: Decapitation. Adults: Sublingual vein.
Anesthetic: Offspring: None adults: Isoflurane.
Anticoagulant: None.
Blood volume: Adults: 1.0 mL. Offspring#: Maximum possible # Thyroxine (T4) is the more sensitive biomarker and in the event of insufficient offspring collection of serum for T4 took priority over collection of serum for TSH.
Treatment of samples: Samples were kept ambient temperature for a minimum of 30 minutes prior to centrifugation.
Centrifugation conditions: At 2000 g for 10minutes at 4°C.
Number of aliquots per sample: Adults: Two aliquots per animal (one for T4 and one for TSH) Offspring: One aliquot per animal (For T4 and TSH)
Temporary storage conditions: Serum was placed on dry ice following collection.
Final storage conditions: Deep frozen (approximately -60°Cto -90ºC).
Fate of plasma samples: Dispatched to a Principal Investigator.
Bioanalysis: Performed by a Principal Investigator. Initially analysis of T4 on adult males and Day 13 offspring was required, with analysis of T4 for adult females and Day 4 offspring, or analysis of T3 to be triggered depending on results obtained. Analysis of T4 on adult females was analysed in error, in addition to study plan requirements.
Oestrous cyclicity (parental animals):
Estrous Cycle
Dry and wet smears were taken as follows:
Dry smears For 16 days before treatment (all females including spares) For 15 days before pairing using cotton swabs For four days before scheduled termination (nominally Days10 to 13 of lactation).
Wet smears After pairing until mating, using pipette lavage.
Litter observations:
Clinical observations Examined at approximately 24 hours after birth (Day 1 of age) and then daily thereafter for evidence of ill health or reaction to maternal treatment; these were on an individual offspring basis or for the litter as a whole, as appropriate.
Litter size Daily records were maintained of mortality and consequent changes in litter size from Days 1-13 of age.
Sex ratio of each litter Recorded on Days 1, 4, 7 and 13 of age.
Individual offspring body weights Days 1, 4, 7 and 13 of age.
Ano-genital distance Recorded on Day 1 for all offspring.
Nipple/areolae count Recorded on Day 13 of age for surviving male offspring only.
Postmortem examinations (parental animals):
Method of Kill
All adult animals: Carbon dioxide asphyxiation with subsequent exsanguination.
Offspring: For offspring selected for thyroid hormone sampling, decapitation. For all other offspring, intraperitoneal injection of sodium pentobarbitone.
Sequence: To allow satisfactory inter-group comparison.

Necropsy
All adult animals were subject to a detailed necropsy. After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative.
Time of Necropsy
F0 males: After Week 5 investigations completed.
F0 females failing to mate: Day 25 after last day of pairing.
F0 females failing to produce a viable litter: Day 25 after mating.
F0 females whose litter died before Day 13: On or after day the last offspring died.
F0 females: Day 13 of lactation.
F1 offspring: Selected offspring for thyroid hormone analysis – Day 4 of age. Scheduled kill –Day 13 of age.
Females
The following were recorded:
Each uterine horn: Number of implantation sites was counted and confirmed if none were visible at visual inspection.
Female whose litter died before Day 13 of lactation: Mammary tissue appearance.

Organ Weights
For bilateral organs, left and right organs were weighed together, unless specified above. Requisite organs were weighed for animals killed at scheduled intervals.

Fixation
Tissues were routinely preserved in 10% Neutral Buffered Formalin with the exception of those detailed below: Testes In modified Davidson’s fluid. Eyes In Davidson’s fluid.

Histology
Processing: Tissue samples were dehydrated, embedded in paraffin wax and sectioned at a nominal four to five micron thickness. For bilateral organs, sections of both organs were prepared. A single section was prepared from each of the remaining tissues required.
Full List: All animals killed or dying prematurely. All animals in Groups 1 and 4 at scheduled termination.
Abnormalities only: All animals.
Mesenteric lymph nodes, duodenum, jejunum, prostate and ovary: All animals in Groups 2 and 3 at scheduled termination.
Prostate: All animals in Groups 1 and 4 at scheduled termination.
Routine staining: Sections were stained with hematoxylinand eosin; in addition samples of the testes were stained using a standard periodic acid/Schiff (PAS) method.

Light Microscopy
For list of tissue preserved for examination please see "any other information on methods and materials section".
Findings were either reported as "present" or assigned a severity grade. In the latter case one of the following five grades was used - minimal, slight, moderate, marked or severe. A reviewing pathologist undertook a peer review of the microscopic findings. For the assessment of the testes, a detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment related effects such as missing germ cell layers or types, retained spermatids, multinucleate or apoptotic germ cells and sloughing of spermatogenic cells in the lumen. Any cell- or stage-specificity of testicular findings was noted. For the assessment of the ovaries a qualitative evaluation of one section from each ovary was made.
Postmortem examinations (offspring):
Premature deaths: Where possible, a fresh macroscopic examination (external and internal) with an assessment of stomach for milk content was performed.
Offspring at scheduled termination (excluding offspring for thyroid retention): Examined externally, if found to be normal offspring were discarded without further examination. Any externally abnormal offspring were also examined internally. Abnormal tissues were retained in an appropriate fixative.
Statistics:
Please see any other information on materials and methods.
Reproductive indices:
Mating Performance and Fertility
Individual data was tabulated. Group values were calculated for males and females separately for the following:
Percentage mating (%) = (Number of animals mating/ Animals paired) x 100
Conception rate (%) = (Number of animals achieving pregnancy/ Animals mated) x 100
Fertility index (%) = (Number of animals achieving pregnancy/ Aminals paired) x 100

Gestation Length and Index
Gestation length was calculated as the number of gestation days up to and including the day on which offspring were first observed, with Day 1 = day of mating for calculation purposes. Where parturition had started overnight, this value was adjusted by subtracting half of one day. Gestation index was calculated for each group as:
Gestation index (%) = (Number of live litters born/ Number pregnant) x 100

Offspring viability indices:
Survival Indices
The following were calculated for each litter:
Post-implantation survival index (%) = (Total number of offspring born/ Total number of uterine implantation sites) x 100
Post-implantation survival index was expressed as 100% where the number of offspring exceeded the number of implantation sites recorded.
Live birth index (%) = (Number of live offspring on Day 1 after littering/ Total number of offspring born) x 100
Viability index (%) = (Number of live offspring on Day 4/ Number live offspring on Day 1 after littering) x 100
Group mean values were calculated from individual litter values.

Sex Ratio
The percentage of male offspring in each litter was calculated at Day 1, and for live offspring on Days 1, 4 (before and after selection for thyroid hormone bleed) and 13 of age.
Percentage males = (Number of males in litter/ Total number of offspring in litter) x 100
Group mean values were calculated from individual litter values.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Excessive salivation was seen in one male given 100 mg/kg/day, and several males or females given 250 mg/kg/day, from Week 2 or 4 respectively. Female number 147 given 250 mg/kg/day had decreased activity and piloerection at the end of the working day on Lactation Day 1, these signs had resolved by the next day. Signs associated with the administration procedure included wet fur and red staining on the head/eyes/nose/muzzle following dosing. These signs were seen in animals from all groups, including controls, and are considered to be due to the method and duration of restraint and not test item related. There were no further test item related signs noted at the weekly physical examination or in association with dose administration.
Mortality:
mortality observed, treatment-related
Description (incidence):
There were 5 decedent females, all given 250 mg/kg/day.
One female (no 141) was found dead on Lactation Day 1 during parturition. Necropsy findings included a distended gastrointestinal tract with watery contents. Histopathological findings included marked decreased cellularity associated with aggregates of foamy macrophages within the mesenteric lymph node, marked aggregates of foamy macrophages in the duodenum and jejunumand minimal hypertrophy of corpora lutea in the ovary. The cause of death was undetermined.
One female (no 138) was euthanized for welfare reasons on Gestation Day 21due to apparent problems during parturition. Necropsy findings included thin build, a distended gastrointestinal tract with darkcontents, red stomach contentsand bilateral enlargement of the adrenals (not confirmed histologically). Histopathological findings included minimal to slight aggregates of foamy macrophages in the duodenum and jejunum and minimal hypertrophy of the corpora lutea in the ovary. The cause of death was undetermined.
One female (no 144) was euthanized for welfare reasons including neglecting litter on Lactation Day 3. Necropsy findings included thickened duodenum and abnormal (white) colour of the mesenteric lymph node. Histopathological findings included marked decreased cellularity associated with aggregates of foamy macrophages within the mesenteric lymph node and slightto moderate aggregates of foamy macrophages in the duodenum and jejunum. The causeof death was undetermined.
One female (no 146) was euthanized for welfare reasons, pup and maternal neglect,on Lactation Day 1. Necropsy findings included distended gastrointestinal tract with watery contents, red fluid and material within the stomach, raised area within the stomach (confirmed as moderate ulceration of the nonglandular region) and abnormal (white) colour of the mesenteric lymph node. Histopathological findings included moderate decreased cellularity associated with aggregates of foamy macrophages within the mesenteric lymph node and moderate aggregates of foamy macrophageswithin the duodenum and jejunum and minimal hypertrophy of the corpora lutea in the ovary. The cause of death was undetermined.
One female (no 148) was euthanized for welfare reasons including piloerection, unsteadiness and decreased activityand thin buildon Lactation Day 1. Necropsy findings included thickened duodenum, ileum and jejunum, small spleen (confirmed as lymphoid atrophy) and abnormal (white) colour of the mesenteric lymph node. Histopathological findings included marked decreased cellularity associated with aggregates of foamy macrophages within the mesenteric lymph node, and minimal to moderate aggregates of foamy macrophages in the duodenum and jejunum. The cause of death was undetermined.
There were no decedents amongst the males.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Group mean body weight gain over Weeks 0-5 for males given 100 or 250 mg/kg/day was lower than the control group (0.77X and 0.38X control respectively). There were no test item related effects on body weight over Weeks 0-5 in males given 30 mg/kg/day.
Group mean body weight gain over Weeks 0-2, prior to pairing was lower than the control group for females given 250 mg/kg/day (0.46X control) and remained lower during gestation (0.55X control). In the three females in this group that retained a litter to Lactation Day 13, group mean body weight gain during lactation was higher than the control group (1.77Xcontrol).
There were no test item related effects on group mean body weight gain prior to pairing or during gestation of females given 30or 100 mg/kg/day. Group mean body weight gain during lactation was higher than the control group for females given 250 mg/kg/day (1.27Xcontrol)
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Group mean food consumption over Weeks 1-5 for males given 100 or 250 mg/kg/day was lower than the control group (0.90 and 0.75X control respectively). There were no test item related effects on food consumption over Weeks 1-5 in males given 30 mg/kg/day.
Group mean food consumption over Weeks 1-2 prior to pairing, during gestationand over Days 1-4 of lactation was lower for females given 250 mg/kg/day (0.77X, 0.76X and 0.68X control respectively). There were no test item related effects on group mean food consumption of females given 250 mg/kg/day after Day 4 of lactation. There were no test item related effects on group mean food consumptionof females prior to pairing or during gestationor lactationgiven 30or 100 mg/kg/day
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
Group mean water consumption over the first two weeks of treatment was higher than controls in males given 250 mg/kg/day (1.34X control).
There were no overt effects on group mean water consumption of males or females given 30 or 100 mg/kg/day, or females given 250 mg/kg/day, over the first two weeks of treatment.
Haematological findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Animals Killed after 4 Weeks of Treatment for males and on Lactation Day 13 for females
Treatment Related Findings Changes related to treatment with EXP1505486 were seen in the duodenum, jejunum, mesenteric lymph nodes in males and females, and in the ovaries in females.
Duodenum
Minimal to slight aggregates of foamy macrophages located in the lamina propria of the villi wereseen in a few males given100 and 250 mg/kg/day. Minimal to slight aggregates of foamy macrophages at the same location were seen in a few females given100 and 250mg/kg/day.
Jejunum
Minimal to moderatefoamy macrophages located in lamina propria of the villi was seen in all males given250 mg/kg/day and in few males given 100 mg/kg/day. Minimal to moderate foamy macrophages at the same location were seen in some females given30, 100 and 250mg/kg/daywith a higher severity at higher doses.
Mesenteric lymph nodes
Slight to marked diffuse foamy macrophages were seen in all males and seven females given 250 mg/kg/day and minimal to moderate diffuse foamy macrophages were seen in some males and females given30 or 100 mg/kg/day. This finding was associated with decreased cellularity in all males given 250 mg/kg/day and some females given100 or 250 mg/kg/day.
Ovary
Minimal hypertrophy of corpora lutea was seen in onefemale given100 mg/kg/dayand one given 250 mg/kg/day. Minimal hypertrophy of corpora lutea was seen three decedent females given 250 mg/kg/day. This finding was not associated with increased organ weight and is considered to be non-adverse.
Incidental Findings
The incidence and distribution of all other findings were considered to be unrelated to the test item.
Histopathological findings: neoplastic:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
effects observed, treatment-related
Description (incidence and severity):
Prior to the study start all females allocated to dosing showed regular estrous cycles of 4, 4/5 or 5 days. Females that did not show regular estrous cycles were not allocated to dosing and were replaced.
In the first two weeks of treatment, before pairing, there was a test item related effect on estrous cycles in females given 250 mg/kg/day. Only 25% of these females showed regular estrous cycles (4, 4/5 or 5 days). The remaining females were either acyclic (25% of females) or had irregular cycles (50% of females), classified as 2, 3 or 6-10 days without estrous. There were no effects on estrous cycles in females of the control group or given 30 or 100 mg/kg/day. At termination, all females on Day 13 of lactation were in diestrus.
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
At 250 mg/kg/day, there was a test item related effect on mating performance with only six of the 12 pairings showing evidence of mating within the first 4 days after pairing. Four pairings showed evidence of mating between 5 to 8 days after pairing. The conception rate and the fertility index were all lower than the concurrent controls, with one male:female pairing not showing evidence of mating, and three females not achieving pregnancy.
Mating performance was good in the Control group and in animals given 30 or 100 mg/kg/day, with all male:female pairings showing evidence of mating, with 12, 12 and 11 females respectively appearing to mate within the first 4 days of pairing (within the first oestrus opportunity). In each of these three groups, a single female failed to litter, the female in the control group and given 30 mg/kg/day was non-pregnant and the female given 100 mg/kg/day was pregnant with a single implantation. Isolated incidences of male:female pairings failing to produce a litter is not unusual for this study design.

At 250 mg/kg/day, group mean implantation counts in the three females retaining live young to Lactation Day 13 was lower than the concurrent control group (0.79X control). Of interest, including all decedent females the group mean implantation count was only 0.90X control; the female decedents generally had a slightly higher number of implantations.

Litter size (total and live) at birth was consequently lower than the controls in this group, although there was no further test item related effects on survival. There were no test item related effects on pre-implantation survival index, live birth index or viability index, or sex ratio. There were no test item related effects related to parental treatment at 30 or 100 mg/kg/day on the number of implantations, litter size or sex ratio, or survival indices for post implantation, live birth and viability, or sex ratio.

There were no test item related effects on the gestation length or gestation index.

Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
30 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
mortality
body weight and weight gain
food consumption and compound intake
water consumption and compound intake
gross pathology
histopathology: non-neoplastic
reproductive function (oestrous cycle)
reproductive performance

Target system / organ toxicity (P0)

Critical effects observed:
yes
Lowest effective dose / conc.:
100 mg/kg bw/day (nominal)
System:
gastrointestinal tract
Organ:
duodenum
jejunum
mesenteric lymph node
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
There was a higher number of offspring from parents given 250 mg/kg/day with clinical observations related to maternal neglect or inability to thrive, such as cold to touch or little or no visible milk present. The majority of the affected offspring were from litters where the lactating dam was killed for welfare reasons or showed total litter loss post-partum.
There was also offspring in one litter from parents given 100 mg/kg/day with these same findings. The female these pups were from showed total litter loss post-partum.
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
Total litter loss post-partum has been seen in a single female given either 30, 100 or 250 mg/kg/day. The single incident of female with total litter loss during lactation was considered to have arisen by chance and not be directly test item related.
The numbers of females with live litters at Lactation Day 13 was 11, 10, 10 and 3 in the Control group and at 30, 100 or 250 mg/kg/day respectively.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Group mean body weight on Day 1 of age was slightly lower for male offspring (0.92X and 0.91X control) and female offspring (0.90X and 0.90X control) from the 100 and 250mg/kg/day group respectively. Group mean body weight gain from Lactation Day 1 to 13 of male offspring from the 100 and 250 mg/kg/day group was lower than the controls (0.86X control for both) and of female offspring from the 100 and 250mg/kg/day group was lower than the controls (0.88X and 0.85X control respectively).
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
There were no test item related effects on serum concentration of thyroxine (T4) in adult males or females or offspring on Day 13 of age.
Anogenital distance (AGD):
no effects observed
Description (incidence and severity):
There were no test item related effects related to the parental treatment on the ano-genital distance on Day 1 of age, corrected for the body weight
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
There were no nipples/areolae noted in any male offspring on Day 13
Gross pathological findings:
no effects observed
Description (incidence and severity):
With the exception of an increased observation of no milk present in the stomach, mainly in offspring dying between birth and Day 4 of age, there were no macroscopic changes noted in the offspring.

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
body weight and weight gain
Remarks on result:
other: A consequence of maternal neglect due to maternal toxicity

Target system / organ toxicity (F1)

Critical effects observed:
no

Overall reproductive toxicity

Reproductive effects observed:
yes
Lowest effective dose / conc.:
250 mg/kg bw/day (nominal)
Treatment related:
yes
Relation to other toxic effects:
reproductive effects as a secondary non-specific consequence of other toxic effects
Dose response relationship:
not specified
Relevant for humans:
not specified

Any other information on results incl. tables

Mortality

Timing

Number of decedents in Group/Sex/dose (mg/kg/day)

1M

2M

3M

4M

1F

2F

3F

4F

0

30

100

250

0

30

100

250

GD21

 

 

 

 

 

 

 

1

LD1

 

 

 

 

 

 

 

3

LD3

 

 

 

 

 

 

 

1

Category

Number of females or female identity in Groups

1

2

3

4

Group size

12

12

12

12

Failed to mate – non pregnant

 

 

 

1 (140)

Failed to litter

1 (102 NP)

1 (116 NP)

1 (156 P)

2 (145 NP, 139 NP )

Found dead LD1

 

 

 

1 (141)

Sacrificed following total litter loss

 

1 (118)

1 (126)

1 (137)

Sacrificed poor condition

 

 

 

4 (146 LD1; 138 GD21; 148 LD1; 144 LD3)

With live litter on LD13

11

10

10

3 (143,142,147)

LD Lactation Day  GD Gestation Day  NP Not pregnant   P Pregnant (single implantation)

Summary of findings in the small intestine for animals killed after 4 weeks of treatment for males and on Lactation Day 13 for females

Group/sex

1M

2M

3M

4M

1F

2F

3F

4F

Dose (mg/kg/day)

0

30

100

250

0

30

100

250

Duodenum thickened

0

0

0

8

0

2

3

5

Jejunum thickened

0

0

0

9

0

3

3

3

Ileum thickened

0

0

0

2

0

0

0

0

Number of animals examined

12

12

12

12

12

12

12

7

The females with total litter loss or failed to litter are included in table. Decedent females are not included in this table.

Summary of findings in the small intestine for Group 4 decedent females

 

Group/sex

4F

Dose (mg/kg/day)

250

Duodenum thickened

2

Jejunum thickened

1

Ileum thickened

1

Number of animals examined

5

Summary of findings in the mesenteric lymph nodes for animals killed after 4 weeks of treatment for males and on Lactation Day 13 for females

Group/sex

1M

2M

3M

4M

1F

2F

3F

4F

Dose (mg/kg/day)

0

30

100

250

0

30

100

250

Abnormal color (white)

0

0

4

10

1

1

8

4

Number of animals examined

12

12

12

12

12

12

12

7

The females with total litter loss or failed to litter are included in table. Decedent females are not included in this table.

 

Summary of findings in the mesenteric lymph nodes for Group 4 decedent females

Group/sex

4F

Dose (mg/kg/day)

250

Abnormal colour (white)

3

Number of animals examined

5

Summary of treatment related findings in the duodenum and jejunum for animals killed after 4 weeks of treatment for males and on Lactation Day 13 for females

The females with total litter loss or failed to litter are included in table. Decedent females are not included in this table.

 

Group/sex 1M 2M 3M 4M 1F 2F 3F 4F
Dose (mg/kg/day) 0 30 100 250 0 30 100 250
Duodenum                
Macrophages Aggregates, Foamy                
Minimal 0 0 1 7 0 0 2 3
Slight 0 0 0 1 0 0 0 2
Total 0 0 1 8 0 0 2 5
Number of tissues examined 12 12 12 12 12 12 12 7
Jejunum                
Macrophages aggregates, Foamy                
Minimal 0 0 6 1 0 3 8 0
Slight 0 0 6 2 0 0 1 4
Moderate 0 0 0 9 0 0 0 3
Total 0 0 6 12 0 3 9 7
Number of tissues examined 12 12 12 12 12 12 12 7

The females with total litter loss or failed to litter are included in table. Decedent females are not included in this table.

Summary of treatment related findings in the duodenum and jejunum for Group 4 decedent females

Group/sex

4F

Dose (mg/kg/day)

250

Duodenum

 

Macrophages Aggregates, Foamy

 

Minimal

2

Slight

1

Moderate

1

Marked

1

Total

5

Number of tissues examined

5

Jejunum

 

Macrophages aggregates, Foamy

 

Slight

1

Moderate

3

Marked

1

Total

5

Number of tissues examined

5

Summary of treatment related findings in the mesenteric lymph nodes for animals killed after 4 weeks of treatment for males and on Lactation Day 13 for females

Group/sex

1M

2M

3M

4M

1F

2F

3F

4F

Dose (mg/kg/day)

0

30

100

250

0

30

100

250

Macrophages Aggregates, Foamy

 

 

 

 

 

 

 

 

Minimal

0

12

3

0

0

6

0

0

Slight

0

0

6

0

0

0

0

2

Moderate

0

0

1

6

0

0

2

0

Marked

0

0

0

6

0

0

0

5

Total

0

12

10

12

0

6

2

7

Cellularity Decreased, General

 

 

 

 

 

 

 

 

Minimal

0

0

0

0

0

0

0

0

Slight

0

0

0

0

0

0

0

2

Moderate

0

0

0

5

0

0

2

0

Marked

0

0

0

7

0

0

0

5

Total

0

0

0

12

0

0

2

7

Number of tissues examined

12

12

12

12

12

12

2

7

The females with total litter loss or failed to litter are included in table. Decedent females are not included in this table.


Summary of treatment related findings in the mesenteric lymph nodes for Group 4 decedent females

Group/sex

4F

Dose (mg/kg/day)

250

Macrophages Aggregates, Foamy

 

Moderate

1

Marked

3

Total

4

Cellularity Decreased, General

 

Moderate

1

Marked

4

Total

4

Number of tissues examined

4*

*  Only 4 tissues examined as tissue was missing from Animal No. 138

Summary of treatment related findings in the ovary for females killed on Lactation Day 13

Group/sex

1F

2F

3F

4F

Dose (mg/kg/day)

0

30

100

250

Corpora Lutea, Hypertrophy

 

 

 

 

Minimal

0

0

1

1

Total

0

0

1

1

Number of tissues examined

12

12

12

7

The females with total litter loss or failed to litter are included in table. Decedent females are not included in this table.

 

Summary of treatment related findings in the ovary for Group 4 decedent females

Group/sex

4F

Dose (mg/kg/day)

250

Corpora Lutea, Hypertrophy

 

Minimal

3

Total

3

Number of tissues examined

5

Applicant's summary and conclusion

Conclusions:
It was concluded that the oral administration of the test item to parental Han Wistar rats at dose levels of 30, 100 or 250 mg/kg/day for two weeks before pairing, during pairing and then up to termination of the males after five weeks of treatment was associated with signs of toxicity at 100 and 250 mg/kg/day, including lower weight gain and food consumption and, at 250 mg/kg/day, higher water consumption. Oral administration of the same dose levels to female rats for two weeks before pairing, during pairing and during gestation and lactation and up to termination of females on Day 13 of lactation wasassociated with signs of toxicity at 250 mg/kg/day, including lower body weight and food consumption. It is possible these changes were secondary to the local toxic response seen in the gastrointestinal tract.
Pathological changes in the gastrointestinaltract and draining lymph nodes (foamy macrophage aggregates and, in the mesenteric lymph node, decreased cellularity) were seen at 100 and 250 mg/kg/day and were considered adverse. These changes are considered to have contributed in some way to the number of female decedents at 250 mg/kg/day.
Effects on reproductive/developmental performance were present at 250 mg/kg/day with an effect on estrous cycles and mating performance, minimalhypertrophy of the corpora lutea (although this was considered non-adverse in the context of this study), and slightly lower offspring body weight gain in utero and during lactation.
In the context of this study, the test item showed no evidence of being an endocrine disrupter. There were no effects on thyroxine levels in adults or offspring, organ weights, external examination of offspring, and nipple counts in male offspring.
Within the constraints of this screening study, the no-observed-adverse-effect level (NOAEL) of the test item for local/systemic toxicity was considered to be 30mg/kg/dayand the NOAEL for reproductive/developmental toxicity was considered to be 100 mg/kg/day.
Executive summary:

The purpose of this study was a screening test for reproductive/developmental effects, and assessment of endocrine disruptor relevant endpoints, with administration of test item by oral gavage administration for at least 4 weeks.

Three groups of twelve male and twelve female rats received test item at doses of 30, 100 or 250mg/kg/day by oral gavage administration.  Males were treated daily for two weeks before pairing, up to necropsy after a minimum of four consecutive weeks.  Females were treated daily for two weeks before pairing, throughout pairing, gestation and until Day 13 of lactation.  Females were allowed to litter, rear their offspring and were killed on Day 13 of lactation.  The F1 generation were killed on Day 13 of age and received no direct administration of the test item; any exposurewas in utero or via the milk. A similarly constituted Control group received the vehicle, arachis oil, at the same volume dose as treated groups.

During the study, clinical condition, body weight, food consumption, water consumption, thyroid hormone analysis, estrouscycles, pre-coital interval, mating performance, fertility, gestation length, organ weight and macroscopic pathology and histopathology investigations were undertaken.

The clinical condition, litter size and survival, sex ratio, body weight, ano-genital distance, nipple/areolae count (male only) and macropathology for all offspring were also assessed.  

Results

P0 responses

There were 5 treatment related decedents at 250 mg/kg/day. One female was sacrificed on Gestation Day 21, one female died on Lactation Day 1, and three females were sacrificed between Lactation Days 1 and 3. The animals were sacrificed due to poor condition, including pup neglect and showed the same small intestine and mesenteric lymph nodes changes described below.

Salivation was seen in one male given 100 mg/kg/day and several animals given 250mg/kg/day and higher water consumption was noted in males given 250 mg/kg/day.  Body weight gain and food consumption were lower in males and females given 250mg/kg/day and males given 100 mg/kg/day.

There were a lower number of females with regular estrous cycles and a higher number of females with irregular/acyclicestrous cyclesat 250 mg/kg/day.  Mating performance was impaired at 250 mg/kg/day with a longer pre-coital interval and a lower number offemales achieving pregnancy.

Prostate weight (body weight adjusted) was lower at 100 and 250 mg/kg/day but there were no associated pathological changes.

Foamy macrophage aggregates, noted macroscopically as thickening, were seen in the duodenum of males at 250 mg/kg/day and females at 100 and 250 mg/kg/day and in the jejunum of males at 250 mg/kg/day and females at 30, 100 and 250 mg/kg/day. Foamy macrophages, noted macroscopically as white colour, were also seen in the draining mesenteric lymph nodes in males at 250 mg/kg/day and females at 30, 100 and 250 mg/kg/day.

Minimal hypotrophy of corporal lutea was seen in one female at 100mg/kg/day and four females at 250mg/kg/day.

There was no effect oncirculating levels of thyroxine (T4) in adult males or females at termination.

F1 responses

In litters surviving to Day 13 of age, there was a slightly lower mean implantation rate at 250 mg/kg/day and, at 100and 250 mg/kg/day, a lower body weight on Day 1 of age and body weight gain to Day 13 of age.

At 250 mg/kg/day and in one litter at 100 mg/kg/day, no milk was visible in the stomach of some offspring between birth and day 4 of age.

There was no effect of parental treatment upon circulating levels of thyroxine (T4) in offspring on Day 13 of age.

The ano-genital distances of offspring were unaffected by paternal treatment and no nipples were seen on any male offspring on Day 13 of age.

No macroscopic findings considered to be related to paternal treatment were recorded.

There was no effect of parental treatment upon circulating levels of thyroxine (T4) in offspring on Day 13 of age.

Conclusion

It was concluded that the oral administration of the test item to parental Han Wistar rats at dose levels of 30, 100 or 250 mg/kg/day for two weeks before pairing, during pairing and then up to termination of the males after five weeks of treatment was associated with signs of toxicity at 100 and 250 mg/kg/day, including lower weight gain and food consumption and, at 250 mg/kg/day, higher water consumption.  Oral administration of the same dose levels to female rats for two weeks before pairing, during pairing and during gestation and lactation and up to termination of females on Day 13 of lactation was associated with signs of toxicity at 250 mg/kg/day, including lower body weight and food consumption. It is possible these changes were secondary to the local toxic response seen in the gastrointestinal tract.

Pathological changes in the gastrointestinal tract and draining lymph nodes (foamy macrophage aggregates and, in the mesenteric lymph node, decreased cellularity) were seen at 100 and 250 mg/kg/day and were considered adverse.  These changes are considered to have contributed in some way to the number of female decedents at 250 mg/kg/day.

Effects on reproductive/developmental performance were present at 250 mg/kg/day with an effect on estrous cycles and mating performance, minimalhypertrophy of the corpora lutea (although this was considered non-adverse in the context of this study), and slightly lower offspring body weight gain in utero and during lactation.

In the context of this study, the test item showed no evidence of being anendocrine disrupter.  There were no effects on thyroxine levels in adults or offspring, organ weights, external examination of offspring, and nipple counts in male offspring.

Within the constraints of this screening study, the no-observed-adverse-effect level (NOAEL) of the test item for local/systemic toxicity was considered to be 30mg/kg/day and the NOAEL for reproductive/developmental toxicity was considered to be 100 mg/kg/day.