Registration Dossier

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-09-22 to 2016-12-27
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
other: EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
other: OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
other: EPA OPPTS 870.3050 (Repeated dose 28-day oral toxicity study in rodents)
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
other: EPA OPPTS 870.3550 (Reproduction/Developmental Toxicity Screening Test)
Deviations:
no
Principles of method if other than guideline:
No testing guidelines were applicalbe for the pilot phase, as this part of the study was intended for dose level selection purposes only.
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
- Physical state: solid
- Appearance: white powder
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: I16CB1581
- Expiration date of the lot/batch: 2019-04-04 (retest date).

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature (+15 to +25 °C).
- Stability under test conditions: Stability of the test item under test conditions was demonstrated in the method validation study (study AB21450).
- Solubility and stability of the test substance in the solvent/vehicle: Stability of formulations over 6 hours at room temperature and 15 days refrigerated at concentration of 1 to 200 mg/mL based on results of stability study no. AB21450.

FORM AS APPLIED IN THE TEST: suspension

OTHER SPECIFICS: No correction factor was applied.

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Crl:WI(Han)
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 and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories France, Domaine des Oncins, 69210 Saint-Germain-Nuelles, France.
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: males approx. 9 weeks (at start F0-treatment); females approx. 9 weeks (at start pretest) and approx. 11 weeks (at start F0-treatment).
- Weight at study initiation: 237.9 - 258.7 g (males, day -5), 191.1 - 236.9 g (females, day 1)
- Fasting period before study: No
- Housing:
Pretest: Animals were housed in groups of 5 sex/cage.
Pre-mating: Animals were housed in groups of 5 sex/cage.
Mating: Males and females were cohabitated on a 1:1 basis.
Post-mating: Males were housed in their home cage with a maximum of 5 animals/cage. Females were individually housed.
Lactation: Pups were housed with the dam.
- Diet (e.g. ad libitum): Free access to pelleted complete rodent diet
- Water (e.g. ad libitum): Free access to softened and filtered (0.2 μm) mains drinking water,
- Acclimation period: 7 days prior to start of pretest (females) or treatment (males).

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25 °C
- Humidity (%): >35 %
- Air changes (per hr): at least 10 room air changes/hour
- Photoperiod (hrs dark / hrs light): 12-hour light/12-hour dark cycle

IN-LIFE DATES:
From: 2016-10-11 (start pretest period females), 2016-10-25 (start treatment males) TO: 2016-11-25 (necropsy males), 2016-12-27 (necropsy females)

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
propylene glycol
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Formulations (w/v) were prepared weekly (based on stability study no. AB21450) and were homogenized to a visually acceptable level. No adjustment was made for specific gravity/density of the test item, vehicle and/or formulation. A correction was made for the purity/composition of the test item. A correction factor of 1 was used.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on trial formulations performed at Charles River Lyon.
- Concentration in vehicle: 0, 22, 66, 200 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg body weight. Actual dose volumes were calculated according to the latest body weight.
- Lot/batch no. (if required): no data
- Purity: no data
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, one female was cohabitated with one male of 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 on formulations used on the first day of treatment during the main phase (25 October 2016), according to a validated method (Test Facility Study no. AB21450). Three sets of duplicate samples (2 x 1 g) were collected. Two sets of duplicate samples were stored in a refrigerator (+2 to +8 °C) as reserve samples.
Once analytical results were approved in the raw data by the Principal Scientist for analytical chemistry, the reserve samples were destroyed.
Samples of formulations were analyzed for homogeneity (highest and lowest concentrations) and accuracy of preparation (all concentrations).
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 % compared to those obtained during the method validation.
The accuracy of preparation was considered acceptable if the mean measured concentrations were 85-115 % of the target concentration for suspensions. Homogeneity was demonstrated if the coefficient of variation was ≤10 %.
Stability of formulations over 6 hours at room temperature and 15 days refrigerated under normal laboratory light conditions (concentration range 1-200 mg/mL) was confirmed as part of the analytical method development and validation study (Test Facility Study no. AB21450).
Duration of treatment / exposure:
Males 31 days
Females that delivered: 51-63 days
Females which failed to deliver: 39-41 days.
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 less than 5 hours difference between the earliest and latest dose; via oral gavage using a plastic cannula
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Group 1 (Control)
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
Group 2
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
Group 3
Dose / conc.:
1 000 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 results of a dose range finding study (Test Facility Study No. 514054) in which animals were dosed for 10 days at 500 and 1000 mg/kg. In summary, no clinical signs (indicative of toxicity) were observed. No mortality occurred, clinical appearance was considered normal, there were no effects on body weight and food consumption, no macroscopic abnormalities were noted, and kidney and liver weights were considered normal. Based on these results, dose levels of 100, 300 and 1000 mg/kg were selected for the main study. Therefore, clinical observations in the main study were conducted after dosing, and functional observation tests were conducted after dosing at no specific time point, but within a similar time period after dosing for the respective animals.
- 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 (at the beginning and at the end of the working day, including weekends and public holidays)

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: before and at least once after dosing (between 1 and 3 hours), from start of treatment onwards up to the day prior to necropsy. Animals were observed once daily on non-treatment days. A full clinical examination was performed on each weighing day. The time of onset, grade (where applicable) and duration of any observed sign was recorded.

BODY WEIGHT: Yes
- Time schedule: Males and females were weighed on the first day of treatment 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 and calculated body weight gain were reported.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): yes
- Time schedule: 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 on PND 1, 4, 7 and 13. Both absolute food consumption and food consumption relative to body weight were reported.
- compound intake calculated as time-weighed averages from the consumption and body weight gain data: not applicable

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

HAEMATOLOGY: Yes
- 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 under anaesthesia using isoflurane. The animals were deprived of food overnight (with a maximum of 24 hours) before blood sampling, but water was available. Blood samples were drawn from the retro-orbital sinus and collected into tubes with K2-EDTA for hematology parameters, and with citrate for clotting tests
- parameters assessed: white blood cells, differential white blood cell count (neutrophils, lymphocytes, monocytes, eosinophils, basophils, large unstained cells), red blood cell count, reticulocytes count, red blood cell distribution width, haemoglobin, haematocrit, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, platelet count, prothrombin time, activated partial thromboplastin time

CLINICAL CHEMISTRY: Yes
- 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 under anaesthesia using isoflurane. The animals were deprived of food overnight (with a maximum of 24 hours) before blood sampling, but water was available. Blood samples were drawn from the retro-orbital sinus and collected into tubes with Li-heparin for clinical biochemistry parameters. An additional blood sample was collected into serum tubes for determination of bile acids.
- parameters checked: alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, total protein, albumin, total bilirubin, bile acids, urea, creatinine, glucose, cholesterol, sodium, potassium, chloride, calcium, inorganic phosphate,
- thyroid hormone analysis: Blood sampling at the end of study from all animals at planned necropsy; this included females on day 14 of lactation, all non-mated females, non-pregnant females and all males after 4 weeks of treatment (including all males that failed to sire). No samples were collected from any females that were sacrificed in extremis or found dead and females with total litter loss. Blood samples were collected, under anesthesia using isoflurane. The animals were deprived of food overnight (less than 24 hours) before blood sampling, but water was available. Blood samples (0.9 mL, target volume) were drawn from the retro-orbital sinus and collected into tubes without anticoagulant. After clotting and centrifugation, serum was used as:
- males 1 aliquot of 150 µL serum was used for measurement of T4 and the remaining volume of serum was kept for possible future measurement of thyroid-stimulating hormone (TSH)
- females: the serum was kept for possible future measurement of T4 and/or TSH

FUNCTIONAL OBSERVATIONS
- Time schedule: The selected males were tested once during week 5 of treatment and the selected females were teted once during the last week of lactation, on PND 13. These tests were performed approx 1.5 hour and 3 hours for females and males, respectively, after observation for clinical signs.
- Dose groups that were examined: selected 5 animals/sex/group
- battery of functions tested: hearing ability, pupillary reflex, static righting reflex, fore- and hind-limb grip strength recorded as the mean of 3 measurements per animal, locomotor activity in an open field test
- parameters: hearing ability, pupillary reflex, static righting reflex, fore and hind-limb grip strength recorded as mean of three measurements per animal, locomotor activity
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 any 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 F0 male parental generation: additional slides of the testes to examine staging of spermatogenesis; testis weight
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; excess pups were killed and discarded.

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.
- 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 14. Sex ratio (% male pups on PND 1) was calculated per group.
- 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.
on PND4 and PND13, pups were sacrificed by intraperitoneal injection of sodium pentobarbitone. Pups that died were necropsied and their stomach examined for the presence of milk, if possible. Defect or cause of death were evaluated, if possible.

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: no
ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: no
Postmortem examinations (parental animals):
SACRIFICE
Adult animals were deprived of food overnight (less than 24 hours) before scheduled necropsy, but water was available.
The 5 selected males and females were deeply anaesthetized using isoflurane (AErrane, Baxter) and subsequently exsanguinated.
Other adult animals surviving to the end of the observation period, any moribund adult animals and females with total litter death were killed by carbon dioxide inhalation and exsanguination then necropsied. Any found dead adult animals were also submitted to necropsy procedures.
Necropsy was conducted according to the following schedule:
- Males: following completion of the mating period (after 31 days of dose administration).
- Females which delivered: on PND 14.
- Females which failed to deliver: on post-coitum Day 26 (females with evidence of mating) or 25 days after the last day of the mating period (female without evidence of mating).
- Females with total litter loss: within 24 hours of litter loss.
- 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: Yes
- After sacrifice, all animals were subjected to a full post mortem necropsy, with special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities were recorded.
- Samples of the following tissues and organs were collected and fixed in 10% buffered formalin, for selected 5 animals/sex/group and all animals that died spontaneously or were killed in extremis: 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), (Cowper’s gland) (M), Duodenum (M/F), Epididymides (M), Eyes (with optic nerve (if detectable) and Harderian gland) (M/F), Mammary gland area (M/F), Femur including joint (M/F), (Glans penis) (M), (Levator ani plus bulbocavernosus muscle complex (LABC)) (M), 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), Liver (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), Preputial gland (M), Prostate gland (M), Rectum (M/F), (Salivary glands - mandibular, sublingual) (M/F), Sciatic nerve (M/F), Seminal vesicles (F), 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), Cowper’s glands (M), Epididymides (M), Glans penis (M), Levat or ani plus bulbocavernosus muscle complex (LABC) (M), Mammary gland area (M/F), Ovaries (F), Preputial gland (M), Prostate gland (M), Seminal vesicles (M), Testes (M), Thyroid including pa rathyroid if detectable (M/F), Uterus (F), Vagina (F), All gross lesions (M/F)

ORGAN WEIGHT
- 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, Cowper’s glands, Epididymides, Glans penis, Heart, Kidneys, Levator ani plus, bulbocavernosus muscle complex (LABC), Liver, Ovaries, Prostate, Seminal vesicles including coagulating glands, Spleen, Testes, Thymus, Thyroid, Uterus (including cervix)
- The following organ weights and terminal body weight were recorded from all remaining animals on the scheduled day of necropsy: Cowper’s glands, Epididymides, Glans penis, Levator ani plus bulbocavern osus muscle complex (LABC), Testes, Thyroid

HISTOPATHOLOGY: Yes
- All organ and tissue samples were processed, embedded and cut at a thickness of 2-4 micrometers. These slides were stained with haematoxylin and eosin. The additional slides of the testes (to exam
ine staging of spermatogenesis) were stained with PAS/haematoxylin.
- The following slides were examined by a pathologist: The preserved organs and tissues of the selected 5 animals/sex of Groups 1 and 4; Additional slides of the testes of the selected 5 males
of Groups 1 and 4 and of all males that failed to sire to examine staging of spermatogenesis; The preserved organs and tissues of one female at 1000 mg/kg (no. 72) that was euthanized in extremis;
The mammary gland of one female at 100 mg/kg (no. 54) with total litter loss; All gross lesions of all animals (all dose groups); Thyroid gland of all selected 5 animals of Groups 2 and 3 (males and
females), based on (possible) treatment-related changes in this organ in Group 4; The reproductive organs of all males that failed to sire and all females that failed to deliver healthy pups.
- All abnormalities were described and included in the study report.
Postmortem examinations (offspring):
SACRIFICE
On PND 4 and PND 13, pups were sacrificed by intraperitoneal injection of sodium pentobarbitone (CEVA Santé Animale).
Pups that died were necropsied and their stomach examined for the presence of milk, if possible. Defects or cause of death were evaluated, if possible.

GROSS NECROPSY
On PND 4 or PND 13 all pups were sexed by both external as well as internal examination. Descriptions of all external abnormalities were recorded. Particular attention was paid to the external reproductive genitals to examine signs of altered development.
At terminal sacrifice (PND 13), the thyroid from 1 male and 1 female pup per litter, i.e. the same pups as selected for blood sampling, was preserved in 10% buffered formalin.
The stomach of all pups was examined for the presence of milk, if possible.

HISTOPATHOLOGY / ORGAN WEIGTHS
not examined
Statistics:
Statistical analyses were performed by the Provantis data acquisition system, where appropriate, as follows:
The best transformation for the data (none, log or rank) was determined depending upon
- the kurtosis of the data
- the probability of the Bartlett's test for homogeneity of the variances and
- an assessment of whether the size of the groups were approximately equal or not.
Non- or log-transformed data were analysed by parametric methods.
Rank transformed data were analysed using non-parametric methods.
Data were then analysed to test for a dose-related trend to detect the lowest dose at which there was a significant effect, based on the Williams test for parametric data or the Shirley's test for non-parametric data.
Homogeneity of means was assessed by analysis of variances (ANOVA) for parametric data or Kruskal-Wallis test for non parametric data.
If no trend was found and means were not homogeneous, the data were analysed by parametric or non-parametric Dunnett's test to look for significant differences from the control group.
Selected incidence data were analysed using the Provantis data acquisition system and/or a SAS software package. A chi2 test was used for all groups followed by Fisher’s two-tailed test with Bonferroni correction for each treated group versus the control if the chi2 was significant.
The locomotor activity in an open field and the oestrous cycle, pre-coital interval and anogenital distance data were analysed using a SAS software package. Levene’s test was used to test the equality of variance across groups and Shapiro-Wilk's test was used to assess the normality of the data distribution in each group. Data with homogenous variances and normal distribution in all groups were analysed using ANOVA followed by Dunnett’s test. Data showing non-homogenous variances or a non-normal distribution in at least one group were analysed using Kruskal-Wallis test followed by the Wilcoxon's rank sum test.
Reproductive indices:
For each group, the following calculations were performed:
Mating index (%) = (Number of females mated/Number of females paired) x 100
Fertility index (%) = (Number of pregnant females/Number of females mated) x 100
Gestation index (%) = (Number of females bearing live pups/Number of pregnant females) x 100
Duration of gestation = Number of days between confirmation of mating and the beginning of parturition
Offspring viability indices:
Survival indices:
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% when the number of offspring exceeded the number of implantation sites recorded.
Live birth index (%) = (Number of live offspring on Day 1 after littering/Total number of offspring born) x 100
Viability index (%) = (Number of live offspring on Day 4 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.
Sex ratio (percentage males) = (Number of males in litter/Total number of offspring in litter) x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related clinical changes in any group.
One male (no. 166) from Day 25 until the end treatment and one female (no.153) from Day 20 to Day 22 had abnormal breathing. This isolated finding was considered incidental. Incidental findings that were noted included localised hairloss, purple area(s) on the tail, bent tail, sore(s) or scab(s), occasionally noted for both sexes in all groups. 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 the study.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
No mortality occurred during the study period that was considered to be related to treatment with the test item. There were 2, 0, 0 and 3 unscheduled deaths in the control, 110, 330 and 1000 mg/kg/day groups, respectively. None of them were considered test item-related.
Female no. 180 given 1000 mg/kg/day was found dead before dosing on Day 6 of treatment. No clinical signs were noted for this female. Although pale foci were seen in the kidneys and lungs, which also had dark lobes, only moderate diffuse congestion was seen histologically and no changes could account for the death of this animal.
Female no. 178 in the same group was found dead on gestation Day 20 (G20), 14 implantations sites were observed at the autopsy. This female lost 14g between G14 and G17. The most significant macroscopic findings were pale raised areas on the left kidney, which correlated with slight bilateral purulent ascending pyelonephritis. The ascending pyelonephritis was associated with marked purulent cystitis, which was considered to be the cause of death. Other macroscopic findings included, distension of the stomach with fluid and several dark foci on the glandular mucosa, which correlated histologically with slight focal erosions and dark foci on the lungs, which correlated histologically with moderate diffuse congestion. Other microscopic changes included, minimal periportal vacuolation in the liver and slight increased cellularity of the bone marrow, which was considered a likely consequence of the higher recruitment of inflammatory cells in the urinary tract.
Control female no. 118 was euthanized for ethical reasons after showing severe clinical signs on treatment Day 6. These included decreased activity, laboured breathing, pilorection and cold to the touch. There were no macroscopic or histological changes that could account for the moribund status of the animal.
Female nos. 114 and 174 in the control and 1000 mg/kg/day groups, respectively, were euthanized on L0 and L1, respectively, after the death of their entire litter.
Female no. 114 had abnormal vocalization, decreased activity and marked piloerection. These observations occurred before delivery. No macroscopic or histological findings were noted for this female.
Female no.174 did not show any clinical signs or difficulty to litter prior the death of her entire litter. In addition, no macroscopic or histological findings were noted. This premature sacrifice was therefore considered incidental.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Males: Mean body weights and body weight gain of treated males remained in the same range as controls over the pre-mating treatment period. Non-dose-related minor differences in mean body weight gain were observed but the magnitude of the changes was similar to the pre-treatment differences. These were therefore considered incidental.
Females: Body weights and body weight gains were not obviously affected by treatment for females during pre-mating or lactation periods. During the gestation period, there was a lower, though not statistically significant mean body weight gain in treated than in control females (i.e. 120.6, 126.8 and 114.6g in the 110, 330 and 1000 mg/kg/day groups, respectively, versus 132.5g for controls). Mean body weights were slightly lower in all treated females than in controls at the end of the gestation period. However, differences in mean body weights were already present on Gestation Day 0. Since this change was not dose related and was not associated with any effect on food consumption, it was not considered as toxicologically relevant.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Males: The mean food consumption for males in the treated groups between Day 1 and Day 15 of the premating period was comparable with, or superior to that in the control.
Females: There was no treatment related effect on mean food consumption during the premating, gestation or lactation periods at any dose level.
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):
There were no differences noted in haematological parameters between control and treated rats that were considered to be related to treatment with the test item. Minor statistically significant changes arising between control and treated animals in white blood cell or platelet counts (i.e. slightly increased total white blood cell count including all subsets in males or increased platelet count in females) were considered not to be toxicologically relevant as they were of low magnitude and remained within the range considered normal for rats of this age and strain.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
No toxicologically relevant changes occurred in clinical biochemistry parameters of treated rats.
Any statistically significant changes arising between control and treated animals were not considered to be toxicologically significant as they occurred either in the absence of a treatment-related distribution, were of low magnitude or remained closed or within the range considered normal for rats of this age and strain.
No statistical differences in total T4 levels were noted between control and treated F0 males.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
No toxicologically relevant effects on hearing ability, pupillary reflex, static righting reflex grip strength and open field test were observed.
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):
Hypertrophy of the zona glomerulosa of the adrenal glands was seen in two females (minimal or slight) and in one male (minimal) given 1000 mg/kg/day. In the absence of clinical disease, adrenal weight changes, clinical chemistry, in particular electrolyte changes, this finding was not considered to be of any toxicological significance and might reflect normal background physiological variations in the appearance of this zone of the cortex. In addition, as the outermost zone, it is prone to handling artefacts. There was considered to be no test item-related alteration in the prevalence, severity, or histologic character of the remaining histologic changes, which were considered to be incidental findings.
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
Length and regularity of the estrous cycle were not considered to have been affected by treatment. Most females had regular cycles of 4 days.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
Spermatogenic staging profiles were normal (at least unilateral) for all males examined
Reproductive performance:
effects observed, non-treatment-related
Description (incidence and severity):
REPRODUCTION DATA
No toxicologically relevant effects on reproductive parameters were noted.
-Mating index : There were 9, 10, 9 and 9 mated pairs of animals in the control, 110, 330 and 1000 mg/kg/day groups. Except for control female no. 113 with a pre-coital interval of 13 days, all mated female showed evidence of insemination within the first 4 days of pairing (approximate duration of a normal oestrus cycle). The mating index was therefore not affected by treatment.
-Fertility index : One female in each of the 330 (no. 160) and 1000 (no. 173) mg/kg/day groups was not pregnant. The fertility index was therefore lower (89 %) than in the control and 110 mg/kg/day groups (100 %). In isolation, these findings were not considered to be related to treatment.
Pre-coital interval : The mean pre-coital interval was not affected by treatment.
Number of implantation sites : Number of implantation sites was not considered to be affected by treatment. The number of implantation sites was slightly lower in the 1000 mg/kg/day group (10.0) than in the control, 110 and 330 mg/kg/day groups (13.2, 11.3 and 13.6, respectively). This was due to an atypical female (no. 174) that had only one implantation site and one single pup at birth. When this value excluded, the re-calculated mean value was increased to 11.5 for this group, which was comparable to the low dose group. All mean values (including 10.0) were within the historical control range at the facility. This change was therefore considered incidental.

DEVELOPMENTAL DATA
No toxicologically relevant effects on developmental parameters were noted. There were 9, 10, 8 and 7 females that completed delivery in the control, 110, 330 and 1000 mg/kg/day groups, respectively.

-Gestation index and duration: The mean gestation index and duration were not affected by treatment in any group.
-Parturition/maternal care: 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.
-Post-implantation survival index: The percentage pre-birth loss (total number of offspring born compared to the total number of uterine implantations) was not considered to be affected by treatment. The number of dead, missing or cannibalised pups at birth was higher in the control than in treated groups. The live litter size was slightly lower at birth in the 1000 mg/kg/day. This was due to the atypical female no. 174 with a single pup that influenced the mean value for this group. This was therefore considered not toxicologically relevant.
-Live birth index: The number of live offspring at birth compared to the total number of offspring born was not considered to be affected by treatment. Control female no. 114 and female no. 174 given 1000 mg/kg/day had total litter loss. A total of 19 pups of the control group and 1 pup in the 1000 mg/kg/day groups compared with none in the 110 and 330 mg/kg/day groups were found dead or missing. No toxicological relevance was attributed to these dead/missing pups since the mortality incidence occurred mainly in the control group.
-Viability index: The number of live offspring on Day 4 before culling compared to the number of offspring alive at birth was not considered affected by treatment. There were 2, 1 and 2 pups in the control, 110 and 1000 mg/kg/groups, respectively that was found dead or missing between lactation Days 0 and 4, compared with none in the 330 mg/kg/day group. No toxicological relevance was attributed to these dead/missing pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age. 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 not considered to be affected by treatment. One pup from dam no. 176 in the 1000 mg/kg/day group was found dead/missing between lactation Days 5 and 13. In isolation, this was considered incidental.

Details on results (P0)

Parental results:
No treatment-related changes were noted in any of the parental parameters investigated in this study (i.e. clinical appearance, functional observations, body weight, food consumption, clinical laboratory investigations, macroscopic examination or organ weights, and microscopic examination). Minimal hypertrophy of the zona glomerulosa of the adrenal glands was seen in a few animals at 1000 mg/kg/day. In the absence of clinical disease, adrenal weight changes, clinical chemistry, in particular electrolyte changes, this finding was not considered to be of any toxicological significance and might reflect normal background physiological variations in the appearance of this zone of the cortex. In addition, as the outermost zone, it is prone to handling artefacts.

Reproductive results:
No treatment-related changes were noted in any of the reproductive parameters investigated in this study (i.e. mating, fertility, pre-coital interval, number of implantations and histopathological examination of reproductive organs).

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
Parental toxicity
Effect level:
>= 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Dose descriptor:
NOAEL
Remarks:
Reproductive toxicity
Effect level:
>= 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects

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.
Incidental clinical symptoms of pups consisted of incomplete hair growth, haematoma, one pup with limping attitude and one with an external abnormality. The nature and incidence of these clinical signs remained within the range considered normal for pups of this age, and were therefore not considered 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 4 before culling compared to the number of offspring alive at birth was not considered affected by treatment. There were 2, 1 and 2 pups in the control, 110 and 1000 mg/kg/groups, respectively that was found dead or missing between lactation Days 0 and 4, compared with none in the 330 mg/kg/day group.
No toxicological relevance was attributed to these dead/missing pups since 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:
no effects observed
Description (incidence and severity):
Body weights of pups were not affected by treatment.
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:
effects observed, non-treatment-related
Description (incidence and severity):
Slightly higher Total T4 levels were noted among individual 1000 mg/kg/day treated PND 13 pups compared to control PND 13 pups but this difference did not reach statistical significance.
Urinalysis findings:
not examined
Sexual maturation:
not examined
Anogenital distance (AGD):
no effects observed
Description (incidence and severity):
Anogenital distance (normalized for body weight) in male and female pups was not affected by treatment.
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
Treatment up to 1000 mg/kg bw/day had no effect on areola/nipple retention. For none of the examined male pups nipples that were observed at PND13.
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined
Other effects:
not specified

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Details on results (F1)

Developmental results:
No treatment-related changes were noted in any of the developmental parameters investigated in this study (i.e. gestation, viability and lactation indices, gestation index and duration, parturition, sex ratio, maternal care and early postnatal pup development consisting of mortality, clinical signs, body weight, anogenital distance (PND 1), areola/nipple retention (PND 13 males), T4 thyroid hormone levels (PND 13) and macroscopy.

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Remarks:
Developmental toxicity
Generation:
F1
Effect level:
>= 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Any other information on results incl. tables

Analysis of dose preparations

All formulations at 22, 66 and 200 mg/mL of test substance in vehicle (propylene glycol), including the vehicle, used on the first day of treatment of the main study, were in agreement with acceptance criteria. The formulations at 22 and 200 mg/mL were homogenous. The deviations from the nominal concentrations ranged from -2.0% to -0.8% , and the RSD was <=0.6%.

No significant amount of test item was detected in the vehicle sample.

Applicant's summary and conclusion

Conclusions:
In conclusion, treatment with JNJ-39453349-AAA (T003066) by oral gavage in male and female Wistar Han rats at dose levels of 110, 330 and 1000 mg/kg revealed no adverse parental, reproduction and developmental toxicity at any dose level.
Based on these results, the following No Observed Adverse Effect Levels (NOAEL) were derived:
Parental NOAEL: at least 1000 mg/kg
Reproduction NOAEL: at least 1000 mg/kg
Developmental NOAEL: at least 1000 mg/kg
Therefore, the substance is not classified as a reproductive toxicant according to the CLP Regulation.