1-[([[(3R,3aS,6aR)-hexahydrofuro[2,3-b]furan-3-yl]oxy]carbonyl)oxy]pyrrolidine-2,5-dione

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018-10-15 to 2019-11-04

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

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3650 (Combined Repeated Dose Toxicity Study with the Reproduction/Deve lopmental Toxicity Screening Test)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

Test guideline 4

Qualifier:

equivalent or similar to guideline

Guideline:

other: EPA OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test, July 2000

Deviations:

no

Test guideline 5

Qualifier:

equivalent or similar to guideline

Guideline:

other: EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral).

Deviations:

no

Test guideline 6

Qualifier:

equivalent or similar to guideline

Guideline:

other: OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)

Deviations:

no

Test guideline 7

Qualifier:

equivalent or similar to guideline

Guideline:

other: EPA OPPTS 870.3050 (Repeated dose 28-day oral toxicity study in rodents)

Deviations:

no

Principles of method if other than guideline:

No testing guidelines were applicable for the pilot phase, as this part of the study was intended for dose level selection purposes only

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

1-[([[(3R,3aS,6aR)-hexahydrofuro[2,3-b]furan-3-yl]oxy]carbonyl)oxy]pyrrolidine-2,5-dione

EC Number:

813-811-7

Cas Number:

253265-97-3

Molecular formula:

C11H13NO7

IUPAC Name:

1-[([[(3R,3aS,6aR)-hexahydrofuro[2,3-b]furan-3-yl]oxy]carbonyl)oxy]pyrrolidine-2,5-dione

Test material form:

solid: particulate/powder

Details on test material:

- Physical state: powder
- Appearance: white powder

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: I18IB2522
- Expiration date of the lot/batch: 2020-03-17 (retest date)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: In refrigerator (2-8°C)
- Solubility and stability of the test substance in the solvent/vehicle: in Propylene glycol, the stability is insured for at least 6 hours at room temperature under normal laboratory light conditions and is confirmed over the concentration range 1 to 200 mg/mL (Test Facility Study No. 20164353)

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
Formulations (w/w) were prepared daily within 6 hours prior to dosing and were homogenized to a visually acceptable level. No adjustment was made for specific gravity/density of the test item, vehicle, and/or formulation. Adjustment was made for specific gravity of the vehicle. A correction was made for the purity/composition of the test item. A correction factor of 1.00 was used

FORM AS APPLIED IN THE TEST (if different from that of starting material)
Suspension (Groups 2-4).

OTHER SPECIFICS: correction factor is 1

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

This species and strain of rat has been recognized as appropriate for general and reproduction toxicity studies. Charles River Den Bosch has general and reproduction/developmental historical data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: males approx. 10-12 weeks (at start F0-treatment); females approx. 12-14 weeks
- Weight at study initiation: 268-295 g (males) and 204-235 g (females)
- Fasting period before study: No
- Housing:
Pretest: Females were housed in groups of 5 females/cage in Macrolon plastic cages (MIV type, height 18 cm).
Pre-mating: Animals were housed in groups of 5 animals/sex/cage in Macrolon plastic cages (MIV type, height 18 cm).
Post-mating: Males were housed in their home cage (Macrolon plastic cages, MIV type, height 18 cm) with a maximum of 5 animals/cage. Females were individually housed in Macrolon plastic cages (MIII type, height 18 cm).
- Diet (e.g. ad libitum): Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany)
- Water (e.g. ad libitum): Free access to tap-water
- Acclimation period: At least 5 days prior to start of pretest (females) or treatment (males).

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 21°C
- Humidity (%): 46 to 62%
- Air changes (per hr): at least 10 room air changes/hour
- Photoperiod (hrs dark / hrs light): 12-hour light/12-hour dark

IN-LIFE DATES: From: 2018-10-15 to 2019-05-24

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Remarks:

specific gravity 1.036

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily within 6 hours prior to dosing and were homogenized to a visually acceptable level. No adjustment was made for specific gravity/density of the test item, vehicle, and/or formulation. Adjustment was made for specific gravity of the vehicle. A correction was made for the purity/composition of the test item. A correction factor of 1.00 was used.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on trial formulations performed at Charles River Den Bosch
- Amount of vehicle (if gavage): 10 mL/kg; Actual dose volumes were calculated according to the latest body weight

Details on mating procedure:

- M/F ratio per cage: 1 animal/sex/cage
- Length of cohabitation: Following a minimum of 14 days of treatment for the males and females, the animals were cohabitated on a 1:1 basis within the same treatment group, avoiding sibling mating until detection of mating was confirmed.
Proof of pregnancy: Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating was confirmed, the males and females were separated.
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): Females were individually housed in Macrolon plastic cages (MIII type, height 18 cm)
- Any other deviations from standard protocol: no

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses were conducted on a single occasion during the treatment phase (19 Mar 2019, Day 1 of treatment) according to a validated method (Test Facility Study No. 20164353). Sextuplicate samples (i.e. 3 sets of duplicate samples) Three sets of duplicate samples were collected. Two sets of duplicate samples were stored in the refrigerator as reserve samples.
Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in vehicle over 6 hours at room temperature. In addition to the criteria mentioned in the validated analytical method, each calibration curve was accepted if the average of the retention times and response factors of the data points used to construct the calibration line were within a range of ±10.00% compared to those obtained during the method validation. The accuracy of preparation was considered acceptable if the mean measured concentrations were 85.00-115.00% of the target concentration2. Homogeneity was demonstrated if the coefficient of variation was ≤ 10.00%. Formulations were considered stable if the relative difference before and after storage was maximally 10.00%. Once analytical results were approved in the raw data by the Principal Scientist, the reserve samples were destroyed.

Duration of treatment / exposure:

Males-29 days ; 50-64 days (females that delivered); 41-52 days (females with total litter loss). Pups were not dosed directly but were potentially exposed to the test item in utero, via maternal milk or from exposure to maternal urine/faeces.

Frequency of treatment:

Once daily for 7 days per week, approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Control

Doses / concentrations 2

Dose / conc.:

11 mg/kg bw/day (nominal)

Remarks:

Group 2

Doses / concentrations 3

Dose / conc.:

33 mg/kg bw/day (nominal)

Remarks:

Group 3

Doses / concentrations 4

Dose / conc.:

90 mg/kg bw/day (nominal)

Remarks:

Group 4

No. of animals per sex per dose:

10 animals/sex/dose level

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels were selected based on the results of a dose range finding study (Test Facility Study No. 20164353) in which animals were dosed for 10 days at 40, 90, 100 and 500 mg/kg/day.
- Rationale for animal assignment (if not random): randomized

Positive control:

No

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least twice daily (early in the morning and close to the end of the working day)

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once daily from start of treatment onwards up to the day prior to necrops y, detailed clinical observations were made for all animals after treatment. Once prior to start of treatment and at weekly intervals during the treatment period this was also performed outside the home cage in a standard arena.

BODY WEIGHT: Yes
- Time schedule for examinations: Males and females were weighed on the first day of treatment (prior to first dosing) and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on PND 1, 4, 7 and 13. Body weight gain was calculated and reported.
Body weight gain was calculated and reported.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
Weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on PND 1, 4, 7 and 13. Relative food consumption was calculated and reported.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No
- Time schedule for examinations: Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no treatment related effect was suspected.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood samples were collected at the end of the treatment period on the day of scheduled necropsy from the selected 5 animals/sex/group, between 7.00 and 1 0.30 am
- Anaesthetic used for blood collection: Yes, isoflurane (Abbott B.V., Hoofddorp, The Netherlands)
- Animals fasted: yes, the animals were deprived of food overnight (with a maximum of 24 hours) bef ore blood sampling, but water was available.
- How many animals: 5 animals/sex/group ; blood samples were drawn from the retro-orbital sinus a ns collected into tubes prepared with K3-EDTA for haematological parameters (0.5 mL) and with citrate for clotting tests (0.45 mL)
- Parameters examined: white blood cells, differential white blood counts (neutrophils, lymphocytes, monocytes, eosinophiles, basophils), red blood cells, reticulocytes, red blood cell distribution width, haemoglobin, haematocrit, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, platelets, coagulation parameters (prothrombin time, activated partial thromboplastin time)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood samples were collected at the end of the treatment period on the day of the scheduled necropsy from the selected 5 animals/sex/group
- Animals fasted: yes, the animals were deprived of food overnight (with a maximum of 24 hours) before blood sampling, but water was available.
- Anaesthetic used for blood collection: Yes, isoflurane (Abbott B.V., Hoofddorp, The Netherlands)
- How many animals: 5 animals/sex/group ; blood samples were drawn from the retro-orbital sinus and collected into tubes prepared with Li-heparin for clinical biochemistry parameters (0.5 mL). An additional blood sample (0.25 mL) was collected into serum tubes for determination of bile acids.
- Parameters checked: alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, gamma glutamyl transpeptidase, total protein, albumin, total bilirubin, bile acids, urea, creatinine, glucose, cholesterol, sodium, potassium, chloride, calcium, inorganic phosphate.
- Thyroid hormone analysis: T4: F0 males: after at least 28 days of treatment

FUNCTIONAL OBSERVATIONS:
The following functional observations tests were performed on each individual animal of the selected 5 animals/sex/group: hearing ability, pupillary reflex, and static righting reflex,fore- and hind-limb grip strength, locomotor activity. Total movements and ambulations are reported. The selected males were tested during Week 4 of treatment and the selected females were tested once during the last week of lactation (e.g. PND 6-13). These tests were performed after observation for clinical signs.

Oestrous cyclicity (parental animals):

Daily vaginal lavage was performed to determine the stage of estrous beginning 14 days prior to treatment (pretest), the first 14 days of treatment and during mating until evidence of copulation was observed. Vaginal lavage continued for those females with no evidence of copulation until termination of the mating period. During pretest, this was done for 48 females. On the day of scheduled necropsy, a vaginal lavage was taken to determine the stage of estrous.

Sperm parameters (parental animals):

Parameters examined in Fo male parental generations: additional slides of the testes to examine staging of spermatogenesis were stained with PAS/haematoxylin

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: No. All pups were randomized per litter and individually identified by means of subcutaneous injection of Indian ink on post-natal day 1 (= day the litter was found completed)
- Maximum of 8 [pups/litter ([4/sex/litter as nearly as possible) were selected for culling on PND4. Blood samples were collected from two of the surplus pups. Selective elimination of pups, e.g. based upon body weight, or anogenital distance was not done. Whenever the number of male or female pups prevented having four of each sex per litter, partial adjustment (for example, five males and three females) was acceptable.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
- Mortality/viability: The numbers of live and dead pups were determined on PND1 and daily thereafter. If possible, defects or cause of death were evaluated. Animals showing pain, distress or discomfort, which was considered not transient in nature or was likely to become more severe, were sacrificed for humane reason.
- Clinical signs: At least once daily, detailed clinical observations were made for all animals. Only days on which clinical signs were present between first and last litter check are presented in the respective table in the study report.
- Body weights: Live pups were weighed on PND 1, 4, 7 and 13.
- Sex: Sex was determined for all pups on PND1 and 4.
- Anogenital distance: Anogenital distance (AGD) was measured for all live pups on PND1. The AGD was normalized to the cube root of body weight.
- Areola/nipple retention: On PND13, all males in each litter were examined for the number of areola/nipples.

GROSS EXAMINATION OF DEAD PUPS:
Yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead if possible. Pups found dead during the weekend were necropsied on the same day.

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: No

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: No

Postmortem examinations (parental animals):

SACRIFICE
The animals were deprived of food overnight (with a maximum of 24 hours) before blood sampling, but water was available.
All animals surviving to the end of the observation period were deeply anaesthetized using isoflurane and subsequently exsanguinated and subjected to a full post mortem necropsy, with special attention being paid to the reproductive organs.

GROSS NECROPSY
Necropsy was conducted according to the following schedule:
- Males: following completion of the mating period (after 28 days of dose administration).
- Females which delivered: on PND 14-16
- Females which failed to deliver
With evidence of mating: Post-coitum Day 25 (Nos. 41, 76 and 80) or postcoitum Day26 (No. 51).
Without evidence of mating: 24 days after the last day of the mating period (No. 78).
- Females with total litter loss (Nos. 68, 69): within 24 hours of litter loss
- Spontaneous deaths (No. 42): as soon as possible after death, and always within 24 hours
- Euthanized in extremis (Nos. 40, 62): when pain, distress or discomfort was considered not transient in nature or was likely to become more severe

GROSS PATHOLOGY: Yes
Descriptions of all macroscopic abnormalities were recorded.
- Samples of the following tissues and organs of the selected 5 animals/sex/group were collected and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution): Adrenal glands (M/F), (Aorta) (M/F), Brain - cerebellum, mid-brain, cortex (7 levels) (M/F), Caecum (M/F), Cervix (F), Clitoral gland (F), Colon (M/F), Coagulation gland (M), Duodenum (M/F), Epididymides (M), Eyes (with optic nerve (if detectable) and Harderian gland) (M/F), Mammary gland area (M/F), Femur with bone marrow including joint (M/F), Heart (M/F), Ileum (M/F), Jejunum (M/F), Kidneys (M/ F), (Lacrimal gland, exorbital) (M/F), (Larynx) (M/F), Liver (M/F), Lung, infused with formalin (M/F), Lymph nodes - mandibular, mesenteric (M/F), (Nasopharynx) (M/F), (Esophagus) (M/F), Ovaries (F), (Pancreas) (M/F), Peyer's patches [jejunum, ileum] if detectable (M/F), Pituitary gland (M/F), Prepu tial gland (M), Prostate gland (M), Rectum (M/F), (Salivary glands - mandibular, sublingual) (M/F), Sciatic nerve ( M/F), Seminal vesicles (M), Skeletal muscle (M/F), (Skin) (M/F), Spinal cord -cervical, midthoracic, lumbar (M/F), Spleen (M/F), Sternum with bone marrow (M/F), Stomach (M/F), Testes (M), Thymus (M/F), Thyroid including parathyroid if detectable (M/F), (Tongue) (M/F), Trachea (M/F), Urinary bladder (M/F), Uterus (F), Vagina (F), All gross lesions (M/F)
Tissues/organs mentioned in parentheses were not examined by the pathologist, since no signs of toxicity were noted at macroscopic examination.
- Samples of the following tissues and organs of all remaining animals, males that fail to sire and females which fail to deliver, were collected and fixed in 10% buffered formalin: Cervix (F), Clitoral gland (F), Coagulation gland (M), Epididymides (M), Mammary gland area inguinal region with skin (M/F), Ovaries (F), Preputial gland (M), Prostate gland (M), Seminal vesicles (M), T estes (M), Thyroid including parathyroid if detectable (M/F), Uterus (F), Vagina (F), All gross lesions (M/F).

HISTOTECHNOLOGY
All organ and tissue samples as defined under 'Histopathology' were processed, embedded and cut a t a thickness of 2-4 micrometers. These slides were stained with haematoxylin and eosin. The additional slides of the testes (to examine staging of spermatogenesis) were stained with PAS/haematoxyin . In addition, the heads (without pituitary gland) of the selected males and females of all 4 groups were decalcified. From the decalcified heads of selected animals of group 4, the nasal cavity at different levels were processed to slides.

ORGAN WEIGHTS
- Absolute organ weights and organ to body weight ratios were reported.
- The following organ weights and terminal body weight were recorded from the selected 5 animals/ sex/ group on the scheduled day of necropsy: Adrenal glands, Brain, Epididymides, Heart, Kidneys, Liver, Ovaries, Prostate, Seminal vesicles including coagulating glands, Spleen, Testes, Thymus, Thyroid, including parathyroid if detectable, Uterus including cervix
- The following organ weights and terminal body weight were recorded from all remaining animals on the scheduled day of necropsy: Epididymides, Prostate, Seminal vesicles, including coagulation glands, Testes, Thyroid

HISTOPATHOLOGY
The following slides were examined by a pathologist:
- The preserved organs and tissues of the selected 5 animals/sex of Groups 1 and 4
- From selected animals of Groups 2 and 3, stomach and brain (level 1, including olfactory bulbs, only ) of males and female, cecum of females and bone marrow-femur and sternum of males.
- From selected animals of Group 4, nasal cavity/skull at levels 3, 4 and 8 to further investigate the microscopic findings in the brain/olfactory bulbs.
- The preserved organs and tissues of the animals of all dose groups which died spontaneously or were euthanized in extremis.
- The mammary gland of all females with total litter loss.
- All gross lesions of all animals (all dose groups).
- The reproductive organs of all males that failed to sire and all females that failed to deliver healthy pups.

Postmortem examinations (offspring):

SACRIFICE
Pups, younger than 7 days were euthanized by decapitation. All remaining pups (PND 7-16) were sacrificed using Euthasol® 20% by intraperitoneal (ip) injection.
On PND 4 (at culling), from 2 pups per litter, blood samples (0.4 mL in total) were collected by decapitation between 7.00 and 10.30 a.m. Blood samples from the 2 pups per litter were collected into one serum tube.
Pups found dead during the weekend were fixed in an identified container containing 70% ethanol as they were not necropsied on the same day. On PND 14-16, from 2 pups per litter (if possible from one male and one female) blood samples were collected at planned necropsy. As part of the necropsy procedure, blood samples (0.9 mL) were drawn by aorta puncture under anaesthesia using isoflurane , between 7.00 and 10.30 a.m., followed by exsanguination. Blood was collected into serum tubes.

GROSS NECROPSY
Necropsy was conducted on the following days:
- Culling: PND 4.
- Terminal sacrifice: PND 14-16.
- Spontaneous deaths: As soon as possible after death and always within 24 hours.
- Euthanized in extremis: When pain, distress or discomfort was considered not transient in nature or was likely to become more severe.

GROSS PATHOLOGY
All pups were sexed by both external as well as internal examination. Descriptions of all abnormalities were recorded.
At terminal sacrifice (PND14-16), the thyroid from 2 pups per litter, i.e. the same pups as selected for blood sampling were preserved in 10% buffered formalin.
The stomach of pups not surviving to the scheduled necropsy date was examined for the presence of milk, if possible. If possible, defects or cause of death were evaluated.

HISTOPATHOLOGY / ORGAN WEIGTHS
not examined

Statistics:

The following statistical methods were used to analyse the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one ttest) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
- For the motor activity data set (at least 3 groups) parametric (ANOVA) tests on group means were applied with Bonferroni correction for multiple testing. Mixed modelling techniques, comparing six dif ferent covariance structures, were used in orde to select the best fitting statistical model.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means an d pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.

Reproductive indices:

For each group, the following calculations were performed:
Mating index (%) = (Number of females mated/Number of females paired) x 100
Precoital time = Number of days between initiation of cohabitation and confirmation of mating
Fertility index (%) = (Number of pregnant females/Number of females mated) x 100
Gestation index (%) = (Number of females with living pups on Day 1/Number of pregnant females) x 100
Duration of gestation = Number of days between confirmation of mating and the beginning of parturition

Offspring viability indices:

Survival indices:
Post-implantation survival index (%) = (Total number of offspring born/Total number of uterine implantation sites) x 100
Live birth index (%) = (Number of live offspring on Day 1 after littering/Total number of offspring born) x 100
Percentage live males at First Litter Check (%) = (Number of live male pups at First Litter Check/Number of live pups at First Litter Check) x 100
Percentage live females at First Litter Check (%) = (Number of live female pups at First Litter Check/Number of live pups at First Litter Check) x 100
Viability index (%) = (Number of live offspring on Day 4 before culling/Number live offspring on Day 1 after littering) x 100
Lactation index (%) = Number of live offspring on Day 13 after littering/Number live offspring on Day 4 (after culling) x 100
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):

Piloerection was noted in 2 females at 33 mg/kg/day for 1 to 6 consecutive days, and up to 6 females at 90 mg/kg/day on most days from Day 22 of treatment onwards. Hunched posture was noted in 3 females at 90 mg/kg/day on several days. Salivation seen after dosing among animals of all dose groups during the treatment period was considered to be a physiological response rather than a sign of systemic toxicity considering the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). Incidental findings that were noted included rales, alopecia, uncoordinated movements and lean appearance. These findings occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study. At the incidence observed, these were not considered to be signs of toxicological relevance. No findings were noted during the arena observations in this study.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Male No. 40 (90 mg/kg/day) was euthanized for ethical reasons after 22 days of treatment. Sligt rales were noted 2 days before its death, and slight body weight loss compared to Day 15 (0.94x) was noted. Diffuse granulocytic inflammation of the meninges of brain (moderate) and spinal cord (mild) was regarded the cause of moribundity for this animal. Additional findings were present in the thymus as moderate decreased lymphoid cellularity, most likely resulting from the poor health condition of this animal. No reflux-related inflammatory findings were recorded for the additionally prepared levels of the nasal cavity which could have explained the pathogenesis of the meningitis. It is however unlikely the granulocytic inflammation of the meninges is related to the test item, but this cannot be fully excluded. Female No. 42 (control) was found dead after 47 days of treatment (Day 10 of lactation) without prior signs of bad condition. At necropsy, the lungs were not collapsed, indicative of an obstruction of the trachea and a dark red discoloration of the lungs was observed. Cause of death for this animal was the severe erosion/ulceration with inflammation of the trachea, which was most likely related to the gavage procedure. Female No. 62 (33 mg/kg/day) was euthanized for ethical reasons on Day 5 post-coitum. Slight lethargy, flat posture, piloerection and ptosis were noted on the day of preterm sacrifice and prior to that slight body weight loss on Day 3 post-coitum compared to Day 0 post-coitum (0.95x) was noted. Cause of moribundity for this animal was a malignant lymphoma. Infiltration of the lymphoma was present in a variety of organs (including adrenal glands, lung, spleen, liver, kidneys and bone marrow) and was the microscopic correlate for the enlarged liver and spleen recorded at necropsy. Malignant lymphomas can be seen as a spontaneous neoplasia in young Wistar rats and this single incidence in the mid dose was regarded to be unrelated to the treatment with the test substance.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

In males up to 33 mg/kg/day and in females at 11 mg/kg/day, body weight (gain) was similar between treated and control animals. In females at 33 mg/kg/day, decreased body weight (0.94x control, not statistically significant) and body weight gain (0.74x control) were noted on Day 20 post-coitum. Although this could partly be contributed to individual females with smaller litters or pups with lower body weight at birth, body weight gain for all females in this group was below the average body weight gain in concurrent controls. Subsequent body weight on Day 1 of lactation was within the same range as concurrent controls, whereas body weight (up to 0.93x control) and body weight gain (between 0.2 to 0.53x control) was again reduced throughout the lactation period. This could be partly contributed to Female No. 67, which showed severe body weight loss on Day 4 of lactation and subsequent limited body weight gain. However, overall body weight gain in 33 mg/kg/day treated females remained low compared to concurrent controls during lactation. In males at 90 mg/kg/day, decreased body weight gain was observed (from Day 8 onwards, ranging from 0.56x to 0.73x control), resulting in statistically significantly decreased body weights on Day 22 (0.95x control) and at termination (0.94x control). In females at 90 mg/kg/day, overall slight body weight loss was observed after one week of treatment and absent body weight gain in the subsequent week, resulting in decreased body weight on Day 8 (0.96x control, not statistically significant), on Day 1 of the mating period (0.91x control), during the post coitum period (up to 0.88x control), and during lactation (up to 0.87x control) and at termination (0.84x control).

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

In males at all dose levels and in females at 11 mg/kg/day, absolute and relative food consumption was similar between treated and control animals. In females at 33 mg/kg/day, decreased absolute food consumption was noted during the lactation period (up to 0.83x control, not always statistically significant). For relative food consumption a similar trend (up to 0.88x control, not statistically significant) was noted. In females at 90 mg/kg/day, decreased absolute food consumption was noted during the premating period (up to 0.73x control, not statistically significant), and during the postcoitum and lactation periods (up to 0.74x control, not always statistically significant). A similar trend was noted ror relative food consumption (up to 0.84x control, not always statistically significant).

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

The following (statistically significant) changes were noted in treated males at 90 mg/kg/day.
Relative differences in mean values as compared to the control group are indicated between parentheses.
- Increased absolute monocyte levels (3.0x; exceeding historical control range).
- Increased haemoglobin levels (1.06x; within historical control range).
- Increased platelet levels (1.25x; within historical control range).
See section 'Any other information on results' for historical control data

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

The following (statistically significant) changes were noted in treated males at 33 and 90 mg/kg/day and females at 90 mg/kg/day.
Relative differences in mean values as compared to the control group are indicated between parentheses.
- Increased albumin levels in males (1.05x and 1.08x at 33 and 90 mg/kg/day, respectively; exceeding historical control range).
- Increased bilirubin levels in males at 90 mg/kg/day (1.62x; exceeding historical control range).
- Increased urea levels in males at 90 mg/kg/day (1.44x; exceeding historical control range).
- Increased glucose levels in males at 90 mg/kg/day (1.32; within historical control range).
- Increased cholesterol levels in males at 90 mg/kg/day (1.41x; within historical control range).
- Increased bile acid levels in males at 90 mg/kg/day (2.05x; exceeding historical control range).
- Increased calcium levels in males at 90 mg/kg/day (1.05x; within historical control range).
- Increased inorganic phosphate levels in males at 90 mg/kg/day (1.15x; exceeding historical control range).
- Increased sodium levels in females at 90 mg/kg/day (1.02x; within historical control range).
-Thyroid hormone analyses: Serum levels of T4 in F0 males were considered to be unaffected by treatment.
See section 'Any other information on results' for historical control data

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Functional observation parameters were considered unaffected by treatment. Hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all examined animals. The motor activity in treated animals was comparable to controls. All groups showed a similar habituation profile with a decreasing trend of activity over the duration of the test period

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):

Test item-related microscopic findings after treatment with the test substance were noted in the stomach of both sexes, olfactory bulbs (brain level 1) and bone marrow (sternum and femur) of males and cecum of females of the 90 mg/kg/day groups.
A combination of findings were recorded for the stomach (glandular mucosa) and were seen in males at 33 and 90 mg/kg/day and in females at 90 mg/kg/day. Test item-related alterations consisted of an increased incidence and severity (minimal-slight) of mixed inflammatory cell infiltrate in the glandular mucosa in males at 33 and 90 mg/kg/day and in females at 90 mg/kg/day, eosinophilic globules (minimal to moderate) in the glandular mucosa of a few males at 33 and 90 mg/kg/day and a single female at 90 mg/kg/day and increased mitosis in a few males (minimal) and a single female (slight). Minimal inflammatory infiltrates of the glandular mucosa can be seen as a spontaneous finding and the minimal severity recorded in two males at 11 mg/kg/day was regarded within background pathology. Minimal or slight mucosal hypertrophy was recorded in the cecum of 3 females at 90 mg/kg/day. Mononuclear inflammatory infiltrate surrounding the olfactory bulbs was recorded at a minimal degree in all males at 90 mg/kg/day. An increased number of adipocytes in the bone marrow was recorded in two surviving males at 90 mg/kg/day in femur and/or sternum at minimal degree. Except for the unusual microscopic findings recorded in the three premature decedents described under mortality, the remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

effects observed, non-treatment-related

Description (incidence and severity):

Length and regularity of the estrous cycle were considered to have not been affected by treatment with the test item. Most females had regular cycles of 4 to 5 days. An irregular cycle occurred in Female No. 51 at 11 mg/kg/day (not pregnant) and in Female No. 77 at 90 mg/kg/day (with normal litter). Given their incidental nature, absence of a dose-related incidence, these findings did not indicate a relation with treatment.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

Stage dependent qualitative evaluation of spermatogenesis in the testis was performed. The testis revealed normal progression of the spermatogenic cycle and the expected cell associations and proportions in the various stages of spermatogenesis were present.

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

Reproduction data:
- Mating index was considered unaffected by treatment with the test item. The mating indices were 100% for the control group, and 11 and 33 mg/kg/day groups. One female (No. 78) at 90 mg/kg/day did not show evidence of mating, resulting in a mating index of 90% in this group which was considered to be within normal ranges.
- Precoital time was considered unaffected by treatment with the test item up to 33 mg/kg/day. At 90 mg/kg/day, precoital time was extended (median 5.1 days compared to 3.0 days in concurrent controls). Only 4 females showed evidence of mating within 4 days, the remaining 5 mated females showed evidence of mating after 5, 7 or 13 days. Notably, 4 out of these 5 females did not show evidence of mating during the first estrus of the mating period, but were mated during a subsequent estrus. Most females at 0, 11 and 33 mg/kg/day showed evidence of mating within 4 days, except for one control female (No. 45) and one female at 11 mg/kg/day (No. 59). As this variation in precoital time remained within the normal range of biological variation, occurred at low incidence and in absence of a dose-related trend, this was considered to be unrelated to treatment.
- Number of implantation sites were 11.2, 12.1, 8.6 and 9.9 for the control group, and 11, 33 and 90 mg/kg/day groups, respectively. All mean values remained within the normal range.
- Fertility index was considered not to be affected by treatment with the test item up to 33 mg/kg/day. The fertility indices were 90, 90, 90 and 78% for the control, and 11, 33 and 90 mg/kg/day groups, respectively. The number of non-pregnant females versus mated females was 9/10 in the control, 11 and 33 mg/kg/day groups, and 7/9 in the 90 mg/kg/day group. As the incidence of non-pregnancy in the high dose group was below the historical control range11 a treatment related effect could not be excluded.

Developmental Data :
As Female Nos. 73 and 80 (90 mg/kg/day) were not pregnant and Female No. 78 (90 mg/kg/day) was not mated, developmental data is available of only 7 females in the high dose group.
- Gestation index and duration of gestation were unaffected by treatment. All pregnant females had live offspring, except for one female at 33 mg/kg/day (No. 68), that had at total litter loss on Day 1 (3 dead pups). The gestation index was 100% for the control, and 11 and 100 mg/kg/day groups, and 89% for the 33 mg/kg/day group. There were no histopathological changes in the reproductive organs to explain the failed pregnancy of Female No. 68.
- No signs of difficult or prolonged parturition were noted among the pregnant females. Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No deficiencies in maternal care were observed.
- The total number of offspring born compared to the total number of uterine implantations was considered to be unaffected by treatment. Post-implantation survival index (total number of offspring born as percentage of total number of uterine implantation sites) was 90% for the control, and 11, 33 and 90 mg/kg/day groups, and 100% for the 90 mg/kg/day group.
- Live litter sizes were 9.9, 10.6, 7.0 and 9.1 living pups/litter for the control, 11, 33 and 90 mg/kg/day groups, respectively. At 33 mg/kg, the number of living pups per litter appeared reduced, which was the result a decrease in the live birth index.
- Live birth index (number of live offspring on PND 1 as percentage of total number of offspring born) was 98, 97, 91 and 93% for the control, and 11, 33 and 90 mg/kg/day groups, respectively. Two pups of the control group, three pups at 11 mg/kg/day, six pups at 33 mg/kg/day, and five pups at 90 mg/kg/day were found dead at first litter check. Female No. 68 had a total litter loss, with 3 dead pups at first litter check. Although no dose-related trend was observed in mortality incidence, the live birth index at 33 and 90 mg/kg/day was below the historical control range and therefore a treatment related effect could not be excluded.
- Viability indices were 100, 100, 87 and 92% for the control, and 11, 33 and 90 mg/kg/day groups, respectively. Postnatal loss was significantly increased at 33 and 90 mg/kg/day. Two pups at 33 mg/kg/day were sacrificed in extremis: one on PND 3, based on absence of milk in the stomach, and a pale, lean and moribund appearance, and one on PND 4, based on lean appearance. In addition, six pups of the 33 mg/kg/day group, and five pups at 90 mg/kg/day were found dead or missing between PND 2 and 4. Pups missing were most likely cannibalised. Female No. 69 had total litter loss on Day 3, with two dead pups at first litter check, three pups were missing on Day 2, and one pup was sacrificed in extremis on Day 3 (included in the numbers described above). Although no dose-related trend was observed, the viability indices at 33 and 90 mg/kg/day were below the historical control range13, and therefore a treatment related effect could not be excluded.
- Lactation index: The number of live offspring on Day 13 after littering compared to the number of live offspring on Day 4 (after culling) was considered to be unaffected by treatment. One pup at 11 mg/kg/day (litter No. 57) was found missing on PND 7 (cannibalised), resulting in lactation indices of 88, 99, 100 and 100% for the control, 11, 33 and 90 mg/kg/day groups, respectively. The eight pups of Female No. 42 were sacrificed after she was found dead on Day 10 of lactation, resulting in slightly lower lactation index for the controls. No toxicological relevance was attributed to the dead/sacrificed pups as the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.
See section 'Any other information on results' for historical control data

Details on results (P0)

Analysis of Dose Preparations:
-Accuracy of preparation: The corrected mean accuracies analysed in the formulations of Groups 2, 3 and 4 were in agreement with target concentrations (i.e. mean accuracies between 75.00% and 125.00%). The accuracies of the formulations were corrected with the mean accuracy of the QC samples to account for any bias introduced by inaccurate calibration solutions. No test item was detected in the Group 1 formulations.
-Homogeneity: The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation ≤ 10.00%).

Parental results:
There were two unscheduled deaths (one control female and one female at 33 mg/kg/day) that were regarded to be unrelated to the treatment with the test item. For one high dose male that was euthanized in extremis, it is unlikely that the observed granulocytic inflammation of the meninges is related to the test item, but this could not be fully excluded.
General toxicity was observed parental females treated at 90 mg/kg/day. A significant decrease in body weight (gain) and food consumption were noted from start treatment onwards and piloerection observed in the majority of females from Day 22 onwards. A similar trend in body weight observed in males was considered non-adverse, as the effect was only slight and no effect on food consumption was observed. No parental toxicity was observed in rats treated at 11 or 33 mg/kg/day.

Reproductive results:
An extended precoital time was observed at 90 mg/kg/day. Notably, 4 out of 5 females that were not mated within 4 days, did not show evidence of mating during the first estrus of the mating period, but were mated during a subsequent estrus. Additionally, at 90 mg/kg/day, a decreased fertility index compared to concurrent controls and historical control data was observed, two mated females were not pregnant. Taken together, these findings indicate a test item-related effect on reproduction that should be considered adverse. No reproduction toxicity was observed at 11 and 33 mg/kg/day.

Effect levels (P0)

1

Key result

Dose descriptor:

NOAEL

Remarks:

Parental toxicity

Effect level:

33 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

2

Key result

Dose descriptor:

NOAEL

Remarks:

Reproductive toxicity

Effect level:

33 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive performance

Target system / organ toxicity (P0)

Key result

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No clinical signs occurred among pups that were considered to be related to treatment. The nature and incidence of clinical signs remained within the range considered normal for pups of this age, and were therefore considered not to be toxicologically relevant.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

The number of live offspring on Day 13 after littering compared to the number of live offspring on Day 4 (after culling) was considered to be unaffected by treatment. One pup at 11 mg/kg/day (litter No. 57) was found missing on PND 7 (cannibalised), resulting in lactation indices of 88, 99, 100 and 100% for the control, 11, 33 and 90 mg/kg/day groups, respectively. The eight pups of Female No. 42 were sacrificed after she was found dead on Day 10 of lactation, resulting in slightly lower lactation index for the controls. No toxicological relevance was attributed to the dead/sacrificed pups as the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weights of pups were considered unaffected by treatment up to 33 mg/kg/day. Body weights of pups at 90 mg/kg/day were decreased in both sexes from PND 1 onwards (statistically significant for males on PND 7 and 13). On PND 13, body weights were 0.85x and 0.89x of controls, in males and females, respectively.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Serum T4 levels in male and female PND 14-16 pups were considered unaffected by treatment with the test item up to 90 mg/kg/day. At 90 mg/kg/day, T4 serum levels were 1.22x of controls. As difference was not statistically significant and all mean values remained within the historical control range, it was considered not toxicologically relevant.

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

Anogenital distance (absolute and normalized for body weight) in male and female pups was considered to be unaffected by treatment

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

Treatment up to 90 mg/kg/day had no effect on areola/nipple retention. For none of the examined male pups nipples were observed at PND 13.

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No macroscopic findings were noted among pups that were considered to be related to treatment. The nature and incidence of macroscopic findings remained within the range considered normal for pups of this age, and were therefore considered to be not related to treatment.

Histopathological findings:

not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Details on results (F1)

Sex ratio was considered to be unaffected by treatment. An opposite effect in sex ratio in animals treated at 33 mg/kg/day, when compared to control, resulted in a statistically significant difference. As the sex ratio was within the normal range and occurred in the absence of a dose-relationship, this was considered to be of no toxicological relevance.

Developmental results:
The live birth and viability indices at 33 and 90 mg/kg/day were decreased compared to concurrent control and were below historical control data. The observed decrease in early pup viability at 33 mg/kg/day could be partly contributed two females with total litter loss, however four additional litters were affected. At 90 mg/kg/day five pups from three litters were dead at first litter check and five pups from four litters went missing. Under the conditions of this screening study, the observed effects should be considered indicative of an adverse effect on early development of the pups at both dose levels, even though no dose response was obsered for this effect. Additionally, a marked reduction of body weight of the pups was observed at 90 mg/kg/day, which was considered adverse. No developmental toxicity was observed at 11 mg/kg/day.

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Remarks:

Developmental toxicity

Generation:

F1

Effect level:

11 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

yes

Lowest effective dose / conc.:

33 mg/kg bw/day (nominal)

Treatment related:

yes

Relation to other toxic effects:

not specified

Dose response relationship:

not specified

Relevant for humans:

not specified

Any other information on results incl. tables

Historical control data for Wistar Han rats (2017 -2019):

Monocytes (10E9/L) - males: mean = 0.1; P5 -P95 = 0.09 -0.22 (n=259)

Haemoglobin (mmol/L) - males: mean = 10.1; P5 -P95 = 9.4 -10.75 (n=260)

Platelets (10E9/L) - males: mean = 735; P5 -P95 = 577 -890 (n=260)

Albumin (g/L) - males: mean = 32.2; P5 -P95 = 30.2 -34.0 (n=270)

Total bilirubin (umol/L) - males: mean = 2.3; P5 -P95 = 1.6 -3.0 (n=270)

Urea (mmol/L) - males: mean = 6.7; P5 -P95 = 4.8 -9.2 (n=270)

Glucose (mmol/L) - males = 9.05; P5 -P95 = 6.40 -12.22 (n=270)

Cholesterol (mmol/L) - males: mean = 1.95; P5 -P95 = 1.41 -2.59 (n=270)

Bile acids (umol/L) - males: mean = 25.6; P5 -P95 = 9.7 -53.7 (n=264)

Calcium (mmol/L) - males: mean = 2.62; P5 -P95 = 2.48 -2.75 (n=270)

Inorganic phosphate (mmol/L) - males: mean = 2.04; P5 -P95 = 1.70 -2.37 (n=270)

Sodium (mmol/L) - males: mean = 139.0; P5 -P95 = 135.6 -142.3 (n=210)

Historical control data for Wistar Han rats (2017 -2018):

prostate weight (gram): mean = 0.802; P5 -P95 = 0.5590 -1.0530 (n=318)

prostate weight (organ/bodyweight ratio): mean = 0.238; P5 -P95 = 0.1653 -0.3128 (n=318)

Historical control data for Wistar Han rats (period 2017 -2019)

Mating index: mean = 99; P5 -P95 = 90 -100 (n=120 studies)

Fertility index: mean = 95; P5 -P95 = 80 -100 (n=120 studies)

Applicant's summary and conclusion

Conclusions:

The test substance was administered by daily oral gavage to male and female Wistar Han rats at dose levels of 11, 33 and 90 mg/kg. Based on the results, the following No Observed Adverse Effect Levels (NOAEL) were derived: Parental NOAEL 33 mg/kg/day, Reproduction NOAEL 33 mg/kg/day, Developmental NOAEL 11 mg/kg/day.