2,2-bis[[(1-oxopentyl)oxy]methyl]propane-1,3-diyl divalerate

Toxicity to reproduction

Administrative data

Endpoint:

extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2020-02-12 to 2022-01-21

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)

Version / remarks:

Adopted 25 June 2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Justification for study design:

SPECIFICATION OF STUDY DESIGN FOR EXTENDED ONE-GENERATION REPRODUCTION TOXICITY STUDY WITH JUSTIFICATIONS

- Premating exposure duration for parental (P0) animals: 10 weeks.
- Basis for dose level selection: Based on results derived from a previously performed 90 day toxicity study, a deveopmental toxicity study and the preliminary reproductive performance study (Covance Study no. RQ21KL) dose levels of 100, 300 and 1000 mg/kg/day were selected.
- Exclusion of extension of Cohort 1B: No information indicate the genotoxicity, endocrine disruption related effect of this substance. No information indicate the substance leads to significant exposure or specific metabolism pattern that would require extended exposure. Therefore, extension of cohort 1B to include the F2 generation was not performed.
- Exclusion of developmental neurotoxicity Cohorts 2A/2B and Cohort 3: No indication of the developmental neurotoxicity or immunotoxicity of this substance was seen from existing information. Cohort 2A/2B and cohort 3 are considered not needed at this moment.
- Route of administration: Based on the information provided in the technical dossier and/or in the chemical safety report, the oral route - which is the preferred one as indicated in ECHA Guidance on information requirements and chemical safety assessment (version 4.1, October 2015) Chapter R.7a, section R.7.5.4.3 - was selected as most appropriate route of administration. Additionally, this mimics the most likely route of exposure to humans. Hence, the test was performed by the oral route.
- Other considerations: The Sprague Dawley rat was chosen as the animal model for this study as it is a rodent species accepted by regulatory agencies for reproductive toxicity testing. The total number of animals used in this study was considered to be the minimum required to properly characterise the effects of the test item. This study was designed such that it did not require an unnecessary number of animals to accomplish its objectives.

Test material

Test material information

Constituent 1

Reference substance name:

2,2-bis[[(1-oxopentyl)oxy]methyl]propane-1,3-diyl divalerate

EC Number:

239-937-7

EC Name:

2,2-bis[[(1-oxopentyl)oxy]methyl]propane-1,3-diyl divalerate

Cas Number:

15834-04-5

Molecular formula:

C25H44O8

IUPAC Name:

3-(pentanoyloxy)-2,2-bis[(pentanoyloxy)methyl]propyl pentanoate

Test material form:

liquid

Remarks:

light yellow clear liquid

Details on test material:

Batch Number: 8038T
Expiry date: 14 July 2021
Storage: at ambient temperature (15 to 25°C)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier): Perstorp Holding AB (Sponsor)
- Lot/Batch number: 8038T (exp. 14 July 2021)
- Purity: 96.95%


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient temperature (15 to 25°C)
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: Homogeneity and stability of the dose formulation at 1 and 250 mg/mL was established for 48 hours at ambient temperature (15 to 25°C) and 15 days when stored refrigerated (2 to 8°C).

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
Formulation fequency: weekly
Storage of formulation: refrigerated (2 to 8°C).
Preparation: The required amount of test item was weighed into a suitable container. Starting with the lowest concentration, approximately 50% of the final volume of vehicle was added to the test item and was magnetically stirred until uniformly mixed. A further amount of vehicle was added to make up to the required volume and further mixing, using a magnetic stirrer, was performed until the formulation was homogeneous. The remaining concentrations were then formulated in ascending order of concentration.
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.

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

The Sprague Dawley SD rat is the species and strain of choice because there is ample experience and background data on this species and strain. It is also the species and strain used in other toxicity studies of this substance which provide better results-comparison between studies.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain: Crl:CD (SD) rat
- Number of animals: 27 litters of four male siblings and 27 litters of four female siblings; no male/female sibling relationships
- Source: Charles River (UK) Ltd. Kent, UK
- Age at study initiation: (F0) 28-34 days, (F1) 21 days selection; 28 days allocation/formal start
- Weight at study initiation: (F0) Males: mean 95 g (76-114 g); Females: mean 87 g (70-104 g)
- Fasting period before study: none
- Housing: cages compromised of polycarbonate body with stainless steel mesh lid; solid bottom cages used except during pairing when grid bottom cages were used; softwood based bark-free fiber bedding; aspen wood based products, and plastic shelter and paper shavings were provided as environmental enrichment, number of animals per cage according to standards
- Diet (e.g. ad libitum): ad libitum SDS VRF1 Certified pelleted diet, no added antibiotic or other chemotherapeutic or prophylactic agent
- Water (e.g. ad libitum): potable water, ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 40-70%
- Air supply: Filtered fresh air which was passed to atmosphere and not recirculated
- Photoperiod (hrs dark / hrs light): Artificial lighting, 12 hours light: 12 hours dark

In-Life Dates:
19 Feb 2020 - 11 Sept 2020

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The dosing formulations were prepared at least weekly, stored in a refrigerator set to maintain 2 to 8ºC, or transferred immediately to the animal unit, and dispensed daily.

VEHICLE
- Justification for use and choice of vehicle (if other than water):TG recomends "where possible, the use of an aqueous solution/suspension is considered first,followed by consideration of a solution/suspension in oil". Corn oil was deemed most appropriate for species selection and substance properties by the Sponsor based on historical use of the vehicle.
- Concentration in vehicle (nominal): 0, 25, 75, 250 mg/ml (dose = 0, 100, 300, 1000 mg/kg/day)
- Amount of vehicle (if gavage): 4 ml/kg body weight

Details on mating procedure:

- F0 pairing commenced: After ten weeks of treatment
- M/F ratio per cage: 1/1(sibling pairing was not permitted)
- Length of cohabitation: max. 2 weeks (separation at detection of mating)
- Proof of pregnancy: The day of detection of an ejected copulation plug in cage tray and sperm in the vaginal smear was designated GD 0.
- After successful mating each pregnant female was caged: Mated females were transferred to individual solid bottomed cages. From Day 20 after mating and throughout lactation, approximately two handfuls of paper shavings were provided to each cage as nesting material; this nesting material was changed at the same frequency as the cage bedding.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The samples were analyzed in accordance with the validated Covance Analytical Procedure (DFA/M152/18). The analytical method involved extraction and dilution in acetone followed by gas chromatographic analysis with flame ionisation detection. Sample concentrations were
determined with reference to external standards prepared in the concentration range 5 µg/mL
to 100 µg/mL.

The homogeneity and stability of Pevalen in corn oil formulations was assessed with respect to the level of concentration at nominal concentrations of 1 mg/mL and 250 mg/mL. The original formulation preparation for 1 mg/mL formulations was outside of acceptance limits for nominal concentration and therefore the stability trial was repeated. No results are reported from the original stability trial for 1 mg/mL formulations. Homogeneity was confirmed during distribution between the bottles, during magnetic stirring for 2 hours, and on re-suspension following storage at ambient temperature (15 to 25ºC) for 2 days and refrigeration (2 to 8ºC) for up to 15 days. At each time-point, the mean analyzed concentration for the three samples remained within 8% of the initial time zero value and the coefficient of variation was within 3%. Recovery results during the trial remained within ±7.5% of the mean recovery found during validation showing the continued accuracy of the method (Table 4). On Day 15 of the stability trial for 1 mg/mL formulations, the recoveries were not analyzed in error. As the recoveries are not used to correct the formulation concentrations, this is considered to have no impact on the results.

Stability analyses performed previously demonstrated that the test item is stable in the vehicle when prepared and stored under the same conditions at concentrations bracketing those used in the present study.

Samples of each formulation prepared for administration in Week 1 (F0/F1 generations) and last week (F1 generation) of treatment were analyzed for achieved concentration of the test item.

The mean concentrations of formulation samples taken during the course of this study were within 6% of the nominal concentration, confirming the accuracy of formulation. The percent difference for all sample results from the mean concentrations and coefficient of variation values were within 3% confirming precise analysis and recovery results during the trial remained within ±7.5% of the mean recovery found during validation showing the continued accuracy of the method.

Duration of treatment / exposure:

The F0 animals were dosed for 10 weeks before pairing until termination after litters are weaned. The F1 animals were dosed from PND 21 (weaning) up to at least PND 90 (Cohort 1A 13 weeks, Cohort 1B 14 weeks)

Frequency of treatment:

Daily, approx. the same time each day

Details on study schedule:

- Age at mating of the mated animals in the study (F0): 14-15 weeks (after 10 weeks dosing)
-The F0 animals were dosed for 10 weeks before pairing until termination after litters are weaned. The F1 animals were dosed from PND 21 (weaning) up to at least PND 90 (Cohort 1A 13 weeks, Cohort 2A 14 weeks)

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw/day

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day

No. of animals per sex per dose:

25 males + 25 females (F0), 20 males + 20 females (F1 cohort 1A), 20 males + 20 females (F1 cohort 1B)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
Dose levels of 100, 300 and 1000 mg/kg/day were selected in conjunction with the Sponsor based on the findings from a preliminary reproductive study conducted at these laboratories . In the preliminary study dose levels of 300, 600 and 1000 mg/kg bw/day were well tolerated with no adverse effects of treatment on clinical condition of F0 or F1 animals, F0 body weight/food consumption, F1 offspring body weight and F1 selected body weight/food consumption. Possible effects of treatment were limited to a slight two day delay in attaining balano-preputial separation at 600 or 1000 mg/kg/day when compared with concurrent Controls but within the historical control range. Based on the results of the preliminary study and in the absence of any overt toxicity, there was nothing to preclude the use of 1000 mg/kg bw/day as the high dose in this OECD 443 study. Low and intermediate dose levels of 100 and 300 mg/kg bw/day were selected to provide an approximate 3-fold dose interval.

Examinations

Parental animals: Observations and examinations:

CLINICAL OBSERVATIONS: Yes
- Time schedule: 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.

Detailed observations of signs associated with dosing were recorded according to the following schedule:
F0 generation: Week 1 - daily, Week 2 to 4 - twice weekly (middle and end of week), Week 5 onward - once each week and for F0 females: Gestation phase - Days 0, 7, 14 and 20, during Lactation phase - Days 1, 7, 14 and 20
F1 generation: DWeek 1 - daily, Week 2 to 4 - twice weekly (middle and end of week), Week 5 onward - once each week

Observations were recorded at the following times during the day: Pre-dose, one to two hours after completion of dosing and as late as possible in the working day

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: A detailed physical examination was performed on each animal to monitor general health according to the following schedule:
Physical examination Once each week
After mating of F0 females: Days 0, 5, 12, 18 and 20 after mating and Days 1, 7, 14 and 21 of lactation.
Particular attention was paid to possible signs of neurotoxicity such as convulsions, tremor and abnormalities of gait or behavior.

BODY WEIGHT: Yes
- Time schedule for examinations: :
F0 males Day that treatment commenced.
Each week.
Before necropsy.

F0 females Day that treatment commenced.
Each week until mating detected.
Days 0, 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20 after mating.
Days 1, 4, 7, 11, 14, 18 and 21 of lactation.

F1 selected animals From nominal week 4 of age, twice during week 1 of the F1 generation and weekly thereafter.


FOOD CONSUMPTION AND COMPOUND INTAKE:
The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded as follows:
F0 males and females Weekly, from the day that treatment commenced until paired for mating.
For females after mating food consumption was performed to match the body weight recording:
Days 0-1, 2-3, 4-5, 6-7, 8-9, 10-11, 12-13, 14-15, 16-17 and 18-19 after mating
Days 1-3, 4-6, 7-13 and 14-20 of lactation.
From these records the mean weekly or daily consumption per animal (g/animal/week or g/animal/day) was calculated for each relevant phase.

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.

WATER CONSUMPTION AND COMPOUND INTAKE: No (ad libitum). Cohort 1A was deprived of water overnight during collection of urine.

Oestrous cyclicity (parental animals):

Dry and wet smears were taken as follows:
- Dry smears: For 15 days before pairing, using cotton swabs.

- Wet smears: After pairing until evidence of mating confirmed.For four days before scheduled termination (nominally Days 25 to 28 post partum). Females that failed to litter or mate were
retained and smeared for four days starting on the day on which the first batch of ‘true’ Day 25 post partum females started smearing, and were then killed with the first batch of females.

Sperm parameters (parental animals):

Motility:
A sample of sperm was expressed from the left vas deferens into prewarmed (target 37oC) medium M199, which contained 0.5% w/v bovine serum albumin (BSA Fraction V). A sample for assessment was taken into a 100 μm depth cannula by capillary action and, where possible, at least 200 sperm per animal analysed using the Hamilton Thorne IVOS II Computer Assisted Sperm Analyser (CASA)

Sperm morphology: Groups 1 and 4
A 200 µL aliquot of the sperm/medium mixture (described above) was diluted with 800 µL of 10% neutral buffered formalin. After staining with nigrosine and eosin an air-dried smear was prepared. Slides were examined by light microscopy for the assessment of sperm morphology. At least 200 sperm were assessed for each male, where possible.

For F0 Group 4M 78 insufficient sperm to be able to assess 200 sperm. Group 4M 83 only 199 sperm were assessed in error.

Sperm morphology: Groups 2 and 3
Fixed samples retained for possible future assessment.

Sperm count: Groups 1 and 4
The left cauda epididymis of each male was weighed and then the tunica was removed, then homogenised for at least 30 seconds in 10 mL of a mixture of 0.9% saline and 0.01% merthiolate (SM). An aliquot of this mixture was added to a pre-prepared IDENT stain tube before being assessed for sperm count using CASA.

Sperm count: Groups 2 and 3
Samples frozen for possible future assessment.

Homogenization-resistant spermatid counts: Groups 1 and 4
After removal of the tunica, the left testis of each male was then homogenised for at least 30 seconds in 25 mL of SM. An aliquot of this mixture was added to a pre-prepared IDENT stain tube before being assessed for homogenisation-resistant spermatid count using CASA.

Homogenization-resistant spermatid counts: Groups 2 and 3
Samples frozen for possible future assessment.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes, litters culled to 10 (where possible 5 males and 5 females).

PARAMETERS EXAMINED
The following parameters were examined in [F1] offspring: Clinical observations, body weight, food consumption and macropathology examinations were performed on all animals for signs of toxicity, with special emphasis on the integrity and performance of the male and female reproductive system and the health (including thyroid hormone analysis), growth, development and function of the offspring. At weaning, two cohorts of selected males and females were assigned for further investigations, including sexual maturation, reproductive organ integrity and function.

The numbers of live and dead pups born in each litter was recorded daily from Days 1-21 of age. The live pups were counted, sexed, weighed individually on PND 1 and examined daily for evidence of ill-health or reaction of treatment. The pups were weighed on days 1, 4 (before culling), 7, 14 and 21. On day 13, male offsprings nipple/aerolae were counted.

After being sexed directly before and after culling (PND 4), the pups were not sexed again until day 21 of age.

- Selection and weaning of F1 animals for Cohort 1A and 1B
The offspring with the lowest within-litter identification per sex from each selected litter were selected to form the F1 generation, after exclusion of grossly atypical animals.
Where possible, two male and two female were selected from each selected litter and were allocated to each of the two cohorts. If more were required, up to three males and three females were selected from each selected litter. Selected animals were microchipped on Day 18 to 21 of age and separated from littermates on Day 21 of age. Up to two male and two female F1 offspring per group were retained as spares, to provide potential replacement in the event of any mortality. These spares had body weights and clinical signs recorded weekly and were terminated after commencement of the F1 generation.

- Assessment of F1 Sexual Maturation (Cohorts 1A and 1B)
Males: Sexual maturation was assessed by daily examination from Day 38 of age until balano-preputial separation occurred. Body weight was recorded on the day of completion of separation.
Females: Sexual maturation was assessed by daily examination from Day 28 of age until vaginal opening occurred. Body weight was recorded on the day of vaginal opening. For Cohort 1A: a wet smear was taken daily from the day of
vaginal opening until first estrus was detected.

In-life procedures. observations and measurements F1 (Cohorts 1A and 1B)
- Mortality/Moribundity Checks
Observed approximately 24 hours after birth (Day 1 of age) and then twice daily for evidence of ill-health or reaction to treatment. A viability check was performed near the start and end of each working day. Animals were killed for reasons of animal welfare where necessary.

- Detailed Clinical Observations
A detailed physical examination will be performed at nominally the same time of day on each occasion by an observer. After removal from the home cage, animals will be assessed for physical condition and behavior during handling.
Particular attention will be paid to possible signs of neurotoxicity such as convulsions, tremor and abnormalities of gait or behavior. Any deviations from normal will be recorded with respect to nature, and, where appropriate, degree of
severity.
Once each week for all F0 and selected F1 generation animals.
Days 0, 5, 12, 18 and 20 after mating and Days 1, 7, 14 and 21 of lactation for F0 (and F1 females if mating triggered).

- Pre and Postdose Observations
Detailed observations were performed to establish and confirm a pattern of signs in association with dosing according to the following schedule:
Week 1 - Daily.
Weeks 2 to 4 - twice weekly (middle and end of the week)
Week 5 onward - once each week.
Detailed observations were recorded at the following times in relation to dose administration:
- Prior to dosing
- One to two hours after completion of dosing
- As late as possible in the working day

- Body weights
Recorded on Days 1, 4 (before and after culling), 7, 14 and 21 of age; Selected F1 generation: Days 23, 25, 27* and 29* of age.
* Only applicable before formal commencement of the F1 generation at nominal four weeks of age (Day 28 of age ± 2 days)
Before necropsy.

- Food consumption
From nominal Week 4 of age, twice during Week 1 of the F1 generation and weekly thereafter.

- Estrus Cycle Monitoring
For Cohort 1A: a wet smear was taken following onset of vaginal patency until first cornified (estrus) smear was recorded. For at least three days prior to the start of the necropsy phase and on the day of termination.
For cohort 1B: a wet smear was taken for at least three days prior to the start of the necropsy phase and on the day of termination.


- Haematology (Cohort 1A; 10 rats/sex/group):
At day of necropsy
Analysed parameters: Hematocrit, hemoglobin concentration, erythrocyte count, retuculocyte count, mean cell hemoglobin, mean cell hemoglobin concentration, mean cell volume, total leucocyte count, differential leucocyte count (Neutrophil count (absolute), Lymphocyte count (absolute), Monocyte count (absolute), Eosinophil count (absolute), Basophil count (absolute), Large unstained cells (absolute)) platelet count, prothrombin time, and activated partial thromboplastin time.

- Clinical Chemistry (Cohort 1A; 10 rats/sex/group):
At day of necropsy
Analysed parameters: Alanine aminotransferase, Aspartate aminotransferase, Alkaline phosphatase, Gamma-glutamyltransferase, bile acids, Total bilirubin, Urea, Creatinine, Calcium, Total protein, Albumin, Albumin/globulin ratio, Glucose, Total Cholesterol, Sodium, Potassium, Chloride, Inorganic phosphorus, total protein

- Urinalysis (Cohort 1A; 10 rats/sex/group):
Last week of dosing
Analysed parameters: Colour, Appearance/Clarity, Specific gravity, Volume, pH, Protein, Glucose, bile pigments, Ketones, Blood pigments, sodium, potassium, and chloride. Microscopic examination of the urine sediment was performed. An aliquot of the urine sample was centrifuged, stained with Kova stain and the resulting deposit spread on a microscope slide. The number of elements seen in nine high or low power fields (HPF or LPF) was recorded in the raw data and entered onto the database and the number seen /HPF or /LPF was derived from these data as described below.
-Epithelial cells (Epi)
-Leucocytes (WBC)
- Erythrocytes (RBC)
- Casts
- Other abnormal components (A)
The slide was also examined for abnormalities in spermatozoa and crystals


- Thyroid stimulating hormone, TSH and T4 (Conditions, blood sample site, and anaesthetic caried based on age)
F1 - culled offspring Ten litters per group - pooled litter sample on Day 4 of age.* Plus ten male and ten female animals per group on Day 22 of age from as many litters as possible
F1 adults- Cohort 1A - Ten male and ten female animals per group at termination (approximately 13 weeks of age)

Postmortem examinations (parental animals):

SACRIFICE
F0 animals:
- Male animals: After weaning of the F1 animals, after confirmation that no further mating was required
- Maternal animals: Day 28 post partum
- Females failing to mate: If an estrus smear was seen following completion of the pairing period animals were terminated as soon as logistically possible.
- Females failing to produce a viable litter and those with total litter loss: Terminated with first cohort of females with live litters.


Method:
-Animals 14 days and older: Carbon dioxide asphyxiation with subsequent exsanguination.
-Sequence: To allow satisfactory inter-group comparison.


GROSS NECROPSY
F0
All animals were subject to a complete macroscopic examination. Any abnormality in the appearance or size of any organ and tissue was recorded and the required tissue samples preserved in appropriate fixative.

For F0 females the implantation site count was recorded.

ORGAN WEIGHTS
F0:
adrenals, brain, L+R epididymides, heart (including auricular and ventricular regions), kidneys, liver (section from two lobes), optic nerves, pituitary, prostate- dorsolateral and ventral combined, seminal vesciles (with coagulating gland), spleen, L+ R testes, thymus, thyroid with parathyroids (weighed after partial fixation), uterus with cervix and oviducts. Paired organs were weighed together. Organ to body weight percentages (using the terminal body weight) were calculated.

MICROSCOPIC EVALUATION
F0:
abnormalities, adrenals, brain (cerebellum, cerebrum, midbrain), cecum, colon, duodenum, epididymides, esophagus, eyes, femurs, heart, ileum, jejunum, kidneys, liver (section from two lobes), lungs (section from two major lobes including bronchi), optic nerves, ovarie, pancreas, pituitary, prostate (dorsolateral and ventral combined), rectum, scieatic nerve, seminal vesicles, skeletal muscles, skin with mammary glands (inguinal area), spinal cord (transverse and longitudinal sections at the cervial thoracic and lumbar levels), spleen, sternum, stomach, testes, thymus, throid with parathyroids, trachea, urinary bladder, uterus with cervix and oviducts, vagina, and vas deferens.

Postmortem examinations (offspring):

SACRIFICE
F1 animals:
Unselected offspring On Day 4 and Day 22 of age.
Cohort 1A animals At approximately 13 weeks of age.
Cohort 1B animals At approximately 14 weeks of age.
- Method:
F1 adults: Carbon dioxide asphyxiation with subsequent exsanguination.
F1 offspring 14 days and older: Carbon dioxide asphyxiation with subsequent exsanguination.
F1 offspring less than 14 days of age: Intraperitoneal injection of sodium pentobarbitone.
F1 Day 4 offspring selected for blood sampling: decaptitation.


GROSS NECROPSY
F1 animals:
All animals, including surplus offspring culled on Day 4 of age were subject to a complete macroscopic examination. Any abnormality in the appearance or size of any organ and tissue was recorded and the required tissue samples preserved in appropriate fixative. Decedents offspring <21 days of age, (found dead or welfare kill), where possible, were examined and carcass retained.

ORGAN WEIGHTS
- F1 Cohort 1A
adrenals, brain, L+R epididymides, heart (including auricular and ventricular regions), kidneys, liver (section from two lobes), lymph nodes (mesenteric, left axillary), oovaries (L+R), pituitary, prostate- dorsolateral and ventral combined, seminal vesciles (with coagulating gland), spleen, L+ R testes, thymus, thyroid with parathyroids (weighed after partial fixation), uterus with cervix and oviducts. Paired organs were weighed together. Organ to body weight percentages (using the terminal body weight) were calculated.

- F1 Cohort 1B
epididymides, ovaries (L+R), pituitary, prostate (dorsolateral and ventral combined), seminal vesciles (with coagulation gland), testes, uterus with cervix and oviducts

- Unselected F1 offspring on Day 22 of age
brain (cerebellum, cerebrum, midbrain), spleen, thymus


MICROSCOPIC EVALUATION
- F1 Cohort 1A
abnormalities, adrenals, brain (cerebellum, cerebrum, midbrain), cecum, colon, duodenum, epididymides, esophagus, eyes, femurs, heart, ileum, jejunum, kidneys, liver (section from two lobes), lungs (section from two major lobes including bronchi), optic nerves, ovarie, pancreas, pituitary, prostate (dorsolateral and ventral combined), rectum, scieatic nerve, seminal vesicles, skeletal muscles, skin with mammary glands (inguinal area), spinal cord (transverse and longitudinal sections at the cervial thoracic and lumbar levels), spleen, sternum, stomach, testes, thymus, throid with parathyroids, trachea, urinary bladder, uterus with cervix and oviducts, vagina, and vas deferens

- F1 Cohort 1B
Abnormalities

Statistics:

All statistical analyses were carried out separately for males and females. Data relating to food consumption were analyzed on a cage basis. For all other adult parameters, the analyses were carried out using the individual animal as the basic experimental unit. For litter/fetal findings the litter was taken as the treated unit and the basis for statistical analysis and biological significance was assessed with relevance to the severity of the anomaly and the incidence of the finding within the background control population. The following data types were analyzed at each timepoint separately:
-Body weight, using absolute weights and gains over appropriate study periods
-Food consumption, over appropriate study periods
-Hematology
-Blood chemistry
-Urinalysis
-Estrous cycles
-Mating performance and fertility
-Gestation length
-Litter (implantations, litter size, sex ratio - percentage male, post implantation survival index, live birth index and viability index), for before cull study periods
-Ano-genital distance, adjusted for Day 1 offspring body weight
-Sexual maturation, age and body weight at completion
-Organ weights, absolute and relative to body weight
-Sperm analysis, motility, morphology and counts for F1 only
- Thyroid hormone analyses

The following comparisons were performed:
-Group 1 vs 2, 3 and 4
-Group 1 vs 4 for ovarian follicle counts and corpora lutea data

Significant differences between the groups compared were expressed at the 5% (p<0.05) or 1% (p<0.01) level.

Parametric/Non-Parametric
Tests include Bartlett's test, Willams' test, t-tests, Dunnett's tests, Shirley's test Kruskal-Wallis' tests, Fisher's exacttest, Wilcoxon rank sum tests, Cochran-Armitage tests, Chi-square tests, one- and two-tailed linear-by-linear tests

Reproductive indices:

Post implantation survival, live birth, viability, and lactation indices were calculate for F0.

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 offspring on Day 1 after littering)/(Total number of offspring born) x 100

Offspring viability indices:

Viability index (%) = (Number of live offspring on Day 4 before culling) / (Number of live offspring on Day 1 after littering) x 100

Lactation index (%) = (Number of live offspring on Day 21 after littering) / (Number of live offspring on Day 4 (after culling)) x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Description (incidence and severity):

There were no signs at routine physical examination that could be attributed to treatment and no signs were observed in association with dose administration.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Six animals died prior to scheduled termination:
Group 1 male no. 12 was found dead on Day 99 of treatment; no terminal signs were observed. There were no macroscopic or microscopic findings and the major factor contributing to death was recorded as ‘undetermined’.
Group 3 male no. 52 was found dead on Day 92 of treatment; no terminal signs were observed. Macroscopic examination revealed abnormal content in the thoracic cavity, pale frothy material in the trachea and a dark thymus. Microscopic examination revealed slight hemorrhage (correlating with the dark color) and minimal involution/atrophy of the thymus. The major factor contributing to death was recorded as ‘undetermined’.
Group 3 male no. 55 was found dead on Day 14 of treatment; no terminal signs were observed. Macroscopic examination did not reveal any abnormality and microscopic examination revealed slight increased extramedullary hemopoiesis in the spleen. The major factor contributing to death was recorded as ‘undetermined’.
Group 3 male no. 68 was found dead (partly cannibalized) on Day 98 of treatment; no terminal signs were observed. Macroscopic examination did not reveal any abnormality and microscopic examination revealed minimal tubular dilatation in the kidney. The major factor contributing to death was recorded as ‘undetermined’.
Group 2 female no. 241 was found dead on Day 19 of treatment; no terminal signs were observed. Macroscopic examination revealed a distended and perforated esophagus with abnormal fluid contents and adhesions in the thoracic cavity. Microscopic examination revealed marked inflammation in the pleura of the lungs, slight increased extramedullary hemopoiesis in the spleen, slight involution/atrophy in the thymus, slight neutrophilic inflammation of the esophagus, and minimal to slight increased cellularity of the bone marrow of the femur and sternum, respectively. The major factor contributing to death was recorded as ‘gavage injury’.
Group 4 female no. 281 was found dead on Day 20 of treatment; no terminal signs were observed. Macroscopic examination revealed a perforated esophagus with abnormal contents in the thoracic cavity. Microscopic examination revealed slight inflammation of the lungs and thymus, and minimally increased extramedullary hemopoiesis in the spleen. The major factor contributing to death was recorded as a ‘gavage injury’.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Body weight gain for males throughout treatment and for females before pairing, during gestation and lactation was unaffected by administration of Pevalen at all dose levels.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption for males and females before pairing and for females during gestation and lactation was unaffected by administration of Pevalen at all dose levels.

Food efficiency:

not examined

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):

Males that received Pevalen showed high white blood cell counts (lymphocytes, neutrophils, esonophils, monocytes and large unstrained cells); there was no evidence of a dose response and these differences were not apparent for females that received Pevalen.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Males that received Pevalen showed low plasma glucose levels (p<0.01) and low potassium levels (p<0.01) when compared with Controls; there was no dose response and these differences were not apparent in females. Males at 1000 mg/kg/day also showed slightly low phosphorous levels (p<0.05) and females at 1000 mg/kg/day showed marginally high calcium levels (p<0.05).

Endocrine findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no statistically significant differences on mean serum TSH concentrations in male or female F0 adult rats after administration of Pevalen at dose levels up to and including 1000 mg/kg/day.

There were no statistically significant differences on mean serum T4 concentrations in F0 adult females at dose levels up to and including 1000 mg/kg/day.

F0 males that received Pevalen showed statistically significantly high mean serum T4 concentrations when compared with Controls (p<0.01); however, there was no evidence for a dose response, the mean Control value for the F0 males was low when compared with the Control values of the F1 adult males and historical control data and a similar effect was not apparent in the F0 females. In the absence of any histopathological findings in the thyroid, these differences are not considered to be of any toxicological significance.

Urinalysis findings:

not examined

Behaviour (functional findings):

not specified

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 microscopic findings were considered spontaneous and/or incidental because they occurred at a low incidence, were randomly distributed across groups (including concurrent controls), and/or their severity was as expected for this animal strain and age; therefore, they were considered not test item related.

Histopathological findings: neoplastic:

not specified

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Estrous cycles showed no adverse effects of treatment, with the distribution of regular 4/5-day cycles, irregular cycles, females showing extended estrus or were acyclic similar across all groups including the Controls (refer to section 4).
The majority of females, with the exception of one female at 1000 mg/kg/day, showed an estrus smear prior to scheduled termination on Day 28 post partum.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

No adverse effects on sperm motility, motion parameters or morphology were observed following treatment with Pevalen at dose levels up to and including 1000 mg/kg/day.

Reproductive performance:

no effects observed

Description (incidence and severity):

Precoital interval:
The majority of animals showed positive evidence of mating within four days of pairing with no differences between the groups that could be associated to administration of Pevalen.

Mating performance & Fertility:
Mating performance and fertility was unaffected by administration of Pevalen at dose levels up to and including 1000 mg/kg/day.

Gestation Length & Gestation Index:
Gestation length and the gestation index for females receiving Pevalen were similar to Controls and considered unaffected by treatment.

Effect levels (P0)

Key result

Dose descriptor:

NOAEL

Effect level:

> 1 000 mg/kg bw/day

Sex:

male/female

Basis for effect level:

reproductive function (sperm measures)

reproductive performance

Target system / organ toxicity (P0)

Key result

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

The clinical condition of selected F1 animals allocated to Cohort 1A and 1B was unaffected by treatment with Pevalen. No signs were observed in association with dose administration

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

Two animals died prior to scheduled termination:
Group 4 male no. 556 was found dead on Day 63 of the F1 generation; there were no terminal signs. Macroscopic examination revealed pale areas on the lungs with abnormal dark content in the oral cavity. Microscopic examination was unremarkable although several tissues were too autolytic; the major factor contributing to death was recorded as ‘undetermined’.
Group 2 female no. 640 was killed for welfare reasons on Day 69 of the F1 generation. Terminal signs included labored breathing, thin build, piloerection and dark eyes. Macroscopic examination revealed a thickened stomach and cecum, a perforated esophagus, dark areas in the lungs, and abnormal fluid contents and adhesions in the thoracic cavity. Microscopic examination revealed a slight inflammatory cell infiltrate in the lungs, slight decreased glycogen in the liver, moderate atrophy of the thymus, perforation of the esophagus, and adhesions and moderate inflammation in the thoracic cavity. The major factor contributing to death was recorded as a ‘gavage injury’.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Body weight at weaning on Day 21 of age and subsequent body weight gain up to Day 25 of age did not show any adverse effects of treatment.
On Day 1 of the F1 generation at 28 ± 2 days of age the mean body weight for males and females receiving 1000 mg/kg/day was slightly but statistically low when compared with Controls (p<0.01). This difference persisted up to Day 15 for male animals and Day 5 for female animals.
The overall body weight gain for selected F1 animals up to scheduled termination was unaffected by administration of Pevalen at dose levels up to and including 1000 mg/kg/day.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption for selected F1 animals from the start of the F1 generation up to scheduled termination was unaffected by treatment.

Food efficiency:

not examined

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):

Total white blood cell (p<0.01) and lymphocyte counts (p<0.05) for males that received 300 or 1000 mg/kg/day and for females at dose levels up to and including 1000 mg/kg/day (p<0.05) were high when compared with Controls; there was no evidence for a dose response.
There were no other differences that could be related to administration of Pevalen.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Females that received Pevalen showed slightly low levels of plasma creatinine (p<0.05) and high levels of calcium (p<0.05/0.01) and phosphorous (p<0.05/0.01) when compared with Controls. Females at 300 or 1000 mg/kg/day also showed high levels of potassium (p<0.05). These differences were not evident in the males and there was no evidence for a dose response.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

Females that received 1000 mg/kg/day had low total urinary chloride (p<0.05); this difference was not apparent in males.

Sexual maturation:

no effects observed

Description (incidence and severity):

Sexual maturation was unaffected by administration of Pevalen at dose levels up to and including 1000 mg/kg/day.

Anogenital distance (AGD):

effects observed, non-treatment-related

Description (incidence and severity):

On Day 1 of age the ano-genital distance for male offspring were essentially similar across the groups; females on Day 1 of age showed marginally but statistically significantly higher body weight adjusted ano-genital distances

Nipple retention in male pups:

effects observed, non-treatment-related

Description (incidence and severity):

On Day 13 post partum one male from one litter at 300 mg/kg/day and eight males from four litters at 1000 mg/kg/day, showed faint areas of darkening on the ventral surface when examined for nipples.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

All differences in organ weight parameters, statistically significant or not, were consistent with normal variation and considered incidental. These differences were characterized by one or more of the following: inconsistency between sexes; presence only in absolute weight or in relative (to body weight) but not both; lack of a dose relationship or correlative findings; and/or the magnitude was considered small.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

In Cohorts 1A and 1B all macroscopic findings were considered spontaneous and/or incidental because they occurred at a low incidence, were randomly distributed across groups (including concurrent controls), and/or were as expected for this animal strain and age; therefore, they were considered not test item related.
Dark areas and pale areas were seen in the lungs of numerous animals in all groups and were considered unlikely to be related to treatment as there was no clear dose response and/or the incidence was generally similar between control and treated animals.

Histopathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

All microscopic findings were considered spontaneous and/or incidental because they occurred at a low incidence, were randomly distributed across groups (including concurrent controls), and/or their severity was as expected for this animal strain and age; therefore, they were considered not test item related.
This included interstitial inflammation, which frequently correlated to dark and/or pale areas macroscopically and was seen in the lungs of numerous animals in all groups/cohorts and included Controls. The histopathological appearance of this finding was not consistent with a treatment effect (no consistent zonal distribution) and any apparent relationship to dose level is considered fortuitous. This finding was considered to be due to Pneumocystis as demonstrated by positive staining for Grocott’s methenamine silver in several animals. As is typical for this agent, the lung findings were not associated with any clinical signs and were not considered to have a functional impact. In addition, the presence of this finding did not impact the ability to detect any treatment related findings in the lungs as it only affected a small proportion of the parenchyma in the majority of affected animals.

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Endocrine:
There was no treatment related effect evident on serum TSH concentrations in F1 animals after doses up to and including 1000 mg/kg bw/day. F1 offspring male rats at 300 and 1000 mg/kg/day showed statistically significantly low mean serum TSH concentrations when compared with Controls (p<0.05 and p<0.01, respectively). However, there was no effect on the thyroxine levels for these animals and as such these differences are not considered to be of toxicological significance. There were no statistically significant differences on mean serum T4 concentrations in male F1 adult males, F1 adult females or F1 offspring at dose levels up to and including 1000 mg/kg/day.

Minor differences were observed in the immunophenotyping parameters between groups of F1 Cohort 1A animals; these differences were not related to the oral gavage administration of Pevalen as they were within the ranges observed in Group 1 (Vehicle control) animals.

Estrous cycles:
One female at 100 mg/kg/day, four females at 300 mg/kg/day and for four females at 1000 mg/kg/day were acyclic compared to none in the concurrent Controls. One Control female, one female at 100 mg/kg/day and one at 1000 mg/kg/day showed irregular estrous cycles.
However, the majority of females at all dose levels showed regular 4/5-day cycles.

Stage of estrous cycle at termination - Cohort 1A and 1B:
The majority of females exhibited at estrus smear before termination and there was no consistent differences between groups that could be related to treatment with Pevalen when comparing groups and Cohorts.

Ovarian Follicle counts & Corpea Lutea:
The mean ovarian follicle counts at 1000 mg/kg/day were slightly low when compared with Controls (approximately 79% of Controls), whilst the mean corpora lutea count was slightly high at 112 % of Controls; overall it was considered that there was no adverse effect of treatment

Developmental neurotoxicity (F1)

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Development of the F1 offspring up to weaning were unaffected by treatment at all dose levels.

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

effects observed, non-treatment-related

Description (incidence and severity):

Minor differences were observed in the immunophenotyping parameters between groups of F1 Cohort 1A animals; these differences were not related to the oral gavage administration of Pevalen as they were within the ranges observed in Group 1 (Vehicle control) animals.

Effect levels (F1)

1

Key result

Dose descriptor:

NOAEL

Generation:

F1 (cohort 1A)

Effect level:

> 1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse treatment related effects were seen

2

Key result

Dose descriptor:

NOAEL

Generation:

F1 (cohort 1B)

Effect level:

> 1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse treatment related effects were seen

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Lowest effective dose / conc.:

1 000 mg/kg bw/day

Treatment related:

no

Applicant's summary and conclusion

Conclusions:

Oral administration of Pevalen at dose levels up to and including 1000 mg/kg bw/day was well tolerated with no treatment-related mortality, no adverse effects on general condition, body weight, food consumption, thyroid hormones, seminology, organ weights or pathology on either the F0 or F1 generation. Reproductive performance for the F0 adults and spleen cell immunophenotyping and ovarian follicle counts for the F1 animals were unaffected by treatment at dose levels up to and including 1000 mg/kg bw/day. The clinical condition of offspring, litter size, offspring survival and development were unaffected by parental treatment at dose levels up to and including 1000 mg/kg bw/day. Based on the results of this study, it is concluded that the No-Observed-Adverse-Effect-Level (NOAEL) for both systemic and reproductive toxicity in the Sprague Dawley rat is 1000 mg/kg bw/day - the highest tested dose in this study.

Executive summary:

In an extended one-generation reproductive toxicity study (OECD TG 443) in rats, including Cohorts 1A and 1B, Pevalen was administered to groups of 25 animals per sex per dose level by gavage at dose levels of 100, 300, or 1000 mg/kg bw/day (F0) at a volume dose of 10 mL/kg bw/day. Additionally, Pevalen was administered to 40 animals per sex per dose level by gavage at dose levels 100, 300, or 1000mg/kg bw/day (F1, Cohorts 1A and 1B) at a volume dose of 4 mL/kg bw/day. A similarly constituted Control group received the vehicle (F0 & F1), corn oil, at the same volume dose as the treated groups throughout the same periods.

 

Males were treated for ten weeks before pairing, up to necropsy after litters were weaned. Females were treated for ten weeks before pairing, throughout pairing up to necropsy on Day 28 of lactation. All F1 animals were then dosed directly from postnatal day (PND) 21 to at least PND 90. At weaning, two cohorts of selected males and females were assigned for further investigations, including sexual maturation, urinalysis, reproductive organ integrity and function (Cohorts 1A and 1B). 

 

Throughout the study, 8 animals were either found dead or were killed for reasons of animal welfare, none were deemed treatment-related. Various effects were seen throughout the study, but differences and/or effects were concluded to be non-treatment-related since they were either minor, lacked a dose response or were inconsistent between the sexes.

 

Overall, oral administration of Pevalen at dose levels up to and including 1000 mg/kg bw/day was well tolerated with no treatment-related mortality, no adverse effects on general condition, body weight, food consumption, thyroid hormones, seminology, organ weights or pathology on either the F0 or F1 generation. Reproductive performance for the F0 adults and spleen cell immunophenotyping and ovarian follicle counts for the F1 animals were unaffected by treatment at dose levels up to and including 1000 mg/kg/day. The clinical condition of offspring, litter size, offspring survival and development were unaffected by parental treatment at dose levels up to and including 1000 mg/kg bw/day.

 

Based on the results of this study, it is concluded that the No-Observed-Adverse-Effect-Level (NOAEL) for both systemic and reproductive toxicity in the Sprague Dawley rat is 1000 mg Pevalen/kg bw/day - the highest tested dose in this study.

 

This study is acceptable and satisfies the guideline requirements for an extended one-generation reproductive study (OECD 443) in the rat.