Registration Dossier

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2015-10-05 to 2016-02-02
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

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:
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: OECD 421, Reproduction/Developmental Toxicity Screening Test
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
other: EPA, OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test
Deviations:
no
Qualifier:
equivalent or similar to
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. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
- Name of test material (as cited in study report): JNJ-16479489-AAA (T001159)
- Physical state: solid
- Appearance: off-white powder
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: M14IB5308
- Expiration date of the lot/batch: 2016-02-03 (retest date)
- Purity test date: 2015-04-23

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Solubility and stability of the test substance in the solvent/vehicle: Stability of formulations over 6 hours at room temperature 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. 509777)

FORM AS APPLIED IN THE TEST (if different from that of starting material): Light tan hazy formulation (Groups 2 and 3), tan turbid formulation (Group 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 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. 11 weeks (at start F0-treatment); females approx. 11 weeks (at start pretest) and approx. 13 weeks (at start F0-treatment).
- Weight at study initiation: 303-339 g (males) and 191-246 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)
Mating: Females were caged together with males on a one-to-onebasis in Macrolon plastic cages (MIII 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)
- Use of restrainers for preventing ingestion (if dermal): no, not applicable
- Diet (e.g. ad libitum): Free access to pelleted rodent diet
- 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): 18 to 24°C
- Humidity (%): 40 to 70%
- 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: 2015-10-05 To: 2016-02-02

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
propylene glycol
Remarks:
specific gravility 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. Adjustment was made for specific gravity of the vehicle. A correction was made for the purity of the test item. A correction factor of 1.00 was used. Formulations were placed on a magnetic stirrer during dosing

VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on trial formulations pe rformed at Charles River Den Bosch.
- Concentration in vehicle: 10 - 100 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 postcoitum. Once mating was confirmed, the males and females were separated. A maximum of 14 days was allowed for mating, after which females who had not shown evidence of mating were separated from their males. Since less than 9 females of Group 4 showed evidence of mating, each non-mated female was re-mated once with a proven male of the same group for 5 days.
- Further mating 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 (2015-12-09; day 6 of treatment), according to a validated method (Test Facility Study no. 509777). Three sets of duplicate samples were collected. Two set of duplicate samples were stored as reserve samples. Samples of formulations were analyzed for homogeneity (highest and lowest concetnration) and accuracy of pr eparation (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. Homogeneity was demonstrated if the coefficient of variation was <=10%. Once analytical results were approved in the raw data by the Principal Scientist, the reserve samples were destroyed.
Duration of treatment / exposure:
31 days (males) except for male nos. 6, 7 and 26: 33 days
52-60 days (females that delivered), 44 and 39 days (female nos. 63 and 77 with total litter loss, resp), 42 days (females which failed to deliver)
Pups were not dosed directly but could have potentially been 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 / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
group 1 (control group)
Dose / conc.:
50 mg/kg bw/day (nominal)
Remarks:
group 2
Dose / conc.:
150 mg/kg bw/day (nominal)
Remarks:
group 3
Dose / conc.:
500 mg/kg bw/day (nominal)
Remarks:
group 4
No. of animals per sex per dose:
10 rats/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. 509045) in which animals were dosed for 10 days at 500 and 1000 mg/kg. In summary, no mortality occurred and there were no clear effects on body weight and food consumption. Clinical signs consisted of occasional hunched posture on 1-3 days during treatment at 500 and 1000 mg/kg and rales in one animal at 500 mg/kg on the last days of treatment. Kidney weights were within normal range, however a clear increase in liver weight and liver to body weight ratio was noted at both 500 and 1000 mg/kg. Based on the increased liver weights, dose levels of 50, 150 and 500 mg/kg were selected for the main study in consultation with the Sponsor
- Rationale for animal assignment: 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: Daily from treatment onwards up to the day prior to necropsy, detailed clinical observations were made in all animals, at least immediately after dosing. 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 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 gain was calculated and reported.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: 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
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Not applicable

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.

FUNCTIONAL OBSERVATIONS:
- Time schedule: between 1 and 3 hours after dosing on the selected 5 animals/sex/group. Selected males were tested during week 4 of treatment and the selected females were tested once during the last week of lactation. These tests were performed after observation for clinical signs (incl. arena observation, if applicable)
- parameters: hearing ability, pupillary reflex, static righting reflex, fore and hind-limb grip strength recorded as the mean of three measurements per animal, locomotor activity.

HAEMATOLOGY
- 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 between 7.00 and 10.30 a.m. 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 K3-EDTA for hematology parameters, and with citrate for clotting tests
- parameters assessed: white blood cells, differential leukocyte counts, red blood cells, reticulocytes, red blood cell distribution width, haemoglobin, haematocrit, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, platelets, prothrombin, activated partial thromboplastin time

CLINICAL BIOCHEMISTRY
- 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 between 7.00 and 10.30 a.m. 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
- parameters assessed: 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

Estrous 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. At the end of the pretest phase, 40 females with at least two regular estrous cycles were selected at random1 and continued in the study. The remaining females were removed from the study, and estrous cycle results of these nonselected females were not reported but kept in the raw data. On the day of 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)
- IMaximum 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
- body weights: live pups were weighed on PND 1, 4, 7 and 13
- Sex: sex was determined for all pups on PND1 and 14
- 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
- Male animals: All surviving animals, following completion of the mating period (a minimum of 28 days of dose administration)
- Maternal animals: All surviving animals, on PND 14-16 (females that delivered) , on days 25-27 (females which failed to deliver, with evidence of mating), within 24 hours of litter loss (females with total litter loss)

GROSS NECROPSY
- All animals surviving to the end of the observation period were deeply anaesthetized using isoflurane and subsequently exsanguinated. After sacrifice, all animals were subjected to a full post mortem examination, with special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities were recorded.
- Necropsy was conducted as soon as possible after spontaneous death and always within 24 hours.
- 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), (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), 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), Preputial 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), Cowper’s glands (M), Epididymides (M), Glans penis (M), Levator 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 parathyroid if detectable (M/F), Uterus (F), Vagina (F), All gross lesions (M/F)

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, 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 bulbocavernosus muscle complex (LABC), Testes, Thyroid

HISTOPATHOLOGY
- 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 examine 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; The additional slides of the testes of the selected 5 males of Groups 1 and 4 and all males suspected to be infertile to examine staging of spermatogenesis; All gross lesions of all animals (all dose groups); Thyroid glands and liver of all selected 5 males and females of Groups 2 and 3, and the spleen, ovaries and sternal bone marrow of all selected 5 females of Groups 2 and 3, based on (possible) treatment-related changes in these organs in Group 4; The reproductive organs of all males that failed to sire and all females that failed to deliver healthy pups.
- histopathological examination of the mammary gland was also conducted for the female nos. 63 and 77 that had a total litter loss.
- A peer review on the histopathology data was performed by a second pathologist.
Postmortem examinations (offspring):
SACRIFICE
- Pups, younger than 7 days were killed by decapitation
- The F1 offspring were sacrificed at PND 4 (by decapitation between 7.00 and 10.30 a.m.), and at PND 7-15 (using Euthasol 20% by intraperitoneal injection).

GROSS NECROPSY
- All pups were sexed both externally and internally. description of all abnormalities were recorded.
- At terminal sacrifice (PND 13-15), the thyroid from 2 pups per litter, i.e. the same pups as selected for blood samplinfg, was 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 t-test) 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 was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
- The Kruskal-Wallis nonparametric ANOVA test was applied to motor activity data to determine intergroup differences.
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 and 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
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 and 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
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 impl antation 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 animals)

General toxicity (P0)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No clinical signs or abnormalities during weekly arena observations were noted that were considered to be related to treatment.
One female at 500 mg/kg (no. 72) showed piloerection during three consecutive days in post-coitum. This sign was absent among other animals of the same dose group, and resolved while treatment continued. This clinical sign was therefore not considered to be related to treatment with the test item.
Salivation seen after dosing among all test item treated groups was not considered toxicologically relevant, taking into account the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). This sign was considered to be a physiological response related to taste of the test item rather than a sign of systemic toxicity.
Other clinical signs noted during the treatment period occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and did not show any apparent dose-related trend. At the incidence observed, these were considered to be unrelated to treatment.

Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
No mortality occurred during the study period that was considered to be related to treatment with the test item.
One female at 150 mg/kg (no. 63) and one female at 500 mg/kg (no. 77) were sacrificed due to total litter loss and/or difficult parturition.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 500 mg/kg, a treatment related but non-adverse lower body weight and body weight gain was noted for females during the second half of the post-coitum phase (from day 11 to 20), along with a lower food intake during the post-coitum and lactation phase. These effects were considered secondarily to the lower number of pups born at this dose level.
Body weights and body weight gain of other dose groups were considered unaffected by treatment; any other statistically significant changes in body weight gain occurred in the absence of a treatment related trent, and were therefore not considered to be related to treatment.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
At 500 mg/kg, food consumption was lower throughout the lactation phase, being statistically significant over Days 4-13. This was attributed to two litters (nos. 78 and 80) that each had only one pup. Food consumption was also statistically significantly lower at 500 mg/kg over Days 17-20 of the post coitum phase. Food consumption before and after correction for body weight of other dose groups remained in the same range as controld over the treatment period.
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):
One female at 500 mg/kg (no. 75) had lower red blood cell counts, haemoglobin and haematocrit, and higher reticulocyte counts and red cell distribution width. Only for haemoglobin, the lower mean was statistically significant (mean approximately 18% lower than control mean)
Any other statistically significant changes in haematology parameters were not considered to be related to treatment as they occurred in the absence of a dose-related trend
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
The following (statistically significant) changes in clinical biochemistry parameters distinguished treated animals from control animals
- Lower aspartate aminotransferase activity (ASAT) in males at 50, 150 and 500 mg/kg (approximately 28%, 44% and 60% lower than control mean, respectively) and in females at 500 mg/kg (not statistically significant for females due to high standard deviation for the control mean).
- Higher creatinine in males at 500 mg/kg (approximately 9% higher than control mean).
- Higher total protein and albumin in females at 500 mg/kg (approximately 5% and 8% higher than control mean, respectively; not statistically significant).
- Lower urea in females at 500 mg/kg (approximately 24% lower than control mean).
- Higher sodium in females at 500 mg/kg (approximately 2% higher than control mean).
- Lower potassium in females at 500 mg/kg (approximately 18% lower than control mean).
One control female (no. 47) showed a very high aspartate aminotransferase activity and bile acid level. No cause could be found for this high value based on parameters assessed in this study. It was considered that this did not adversely compromise interpretation of the study results. Any other statistically significant changes in clinical biochemistry parameters were not considered to be related to treatment as they occurred in the absence of a dose-related trend.
Thyroid hormone analyses: at 50, 150 and 500 mg/kg, serum levels of total T4 were statistically significantly lower in males. Mean T4 levels were approx. 22, 29 and 50% lower than controls.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Hearing ability, pupillary reflex and static righting reflex were normal in all selected animals. Motor activity was considered unaffected by treatment. All groups showed a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period. Grip strength was similar across the groups for both sexes.The apparent higher mean motor activity of females at 500 mg/kg was due to a high value for one female (no. 74; both total movements and ambulations). Since individual values of other animals of the same group remained in the range of values for control animals, this was not considered to be related to treatment.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
The following test item differences in organ weights were observed between treated and control animals:
- Higher liver weight of males at 500 mg/kg (both absolute and relative to body weight) and females at 150 mg/kg (both absolute and relative to body weight) and 500 mg/kg (relative to body weight). Relative weight differences to control means were 33 and 27% for males and females at 500 mg/kg respectively, and 23% for females at 150 mg/kg. The increase in female liver weight was also evident at 50 mg/kg (relative weight increased with 19%; not statistically significant).
- Higher ovary weight at 50 and 500 mg/kg (absolute). Absolute weight difference to control mean was 23 and 25%, respectively (relative weight difference was 19 and 33%, respectively).
- Higher kidney weight of males at 500 mg/kg (relative to body weight; 18% higher than control mean).
Any other differences in organ weights, including those that reached statistical significance were considered not to be test item-related due to the direction of the change, lack of dose-related pattern, and/ or general overlap and variability in individual values.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
At 500 mg/kg, one female (no. 77) had pale discolouration of the liver. The remainder of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Test item-related microscopic findings were noted in:
- Thyroid gland: increased incidence and/or severity of hypertrophy of follicular cells in males at 500 mg/kg up to moderate degree and in females at 50, 150 and 500 mg/kg up to slight degree.
- Liver: Hepatocellular centrilobular scattered vacuolation in males and females at 150 and 500 mg/ kg up to moderate degree; Hepatocellular centrilobular hypertrophy in males at 150 and 500 mg/kg and in females at 50, 150 and 500 mg/kg up to slight degree.
- Ovaries: increased incidence and severity of interstitial cell hypertrophy in females at 500 mg/kg up to slight degree.
An increased severity of extramedullary hematopoiesis in the spleen was noted in two females at 500 mg/kg (nos. 75 and 77) at marked degree. This finding was considered to be secondary to the failed pregnancy in these two females.
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 of histologic character of those incidental tissue alterations.
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: estrous cycle:
effects observed, non-treatment-related
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. Extended di-estrus occurred in two control females, one female at 150 mg/kg and one female at 500 mg/kg (all with a regular cycle). An irregular cycle was noted for one female at 50 mg/kg (with normal litter), and for two females at 500 mg/kg (both either had no offspring or low litter size). Given their incidental nature and absence of a dose-related incidence and absence of an apparent correlation to pregnancy status, these findings did not indicate a relation with treatment.
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 up to 500 mg/kg.
- At 500 mg/kg, the mean number of implantations appeared lower than in the control group (8.4 vs 11.2 in the control group; not statistically significant). However, this was essentially ascribed to female nos. 72 and 73 which had only 2 or 3 implantation sites. Upon excluding the values of these females, the mean would be 10.3.
- Mating and fertility indices and precoital time were unaffected by treatment. All females showed evidence of mating. Two females at 50 mg/kg (nos. 55 and 57) were not pregnant. The incidence of non-pregnancy did not show any apparent dose-related trent, and as such this was considered to be unrelated to treatment.
- For one female at 150 mg/kg (no. 70), the number of pups was slightly higher than the number of implantations. This was considered caused by normal resorption of these areas as these enumerations were performed on Day 16 of lactation.
There were no morphological findigns in the reproductive organs of either sex which could be attributed to the test item and spermatogenic staging profiles were normal (at least unilateral) for all males examined.

DEVELOPMENTAL DATA
- At 500 mg/kg, an increased incidence of reproductive failure was noted; 5/10 females had no offspring (nos. 71, 72, 73, 75 and 76). Three of these pregnant females (nos. 72, 73 and 75) had implantation sites only. This resulted in a lower gestation index (63% vs. 100% in the control group, i.e. 5 out of 8 pregnant females delivered offspring). One female at 500 mg/kg (no. 77) had total litter loss. Although the incidence of total litter loss did not show an apparent dose-related trend, it could not be excluded that at 500 mg/kg this was related to treatment. No abnormalities were seen in the reproductive organs, which could account for their lack of healthy offspring.
- Post implantation survival index: was also lower at 500 mg/kg (28% vs 94% in the control group); for 4 out of 5 females that delivered offspring, the number of pups vs the number of implantation sites per litter was less than 50%. The mean number of pups per litter was also lower than controls at 500 mg/kg (3.4 vs 10.5 in the control group)
- One female at 500 mg/kg (no. 77) which had total litter loss on day 1 of lactation was found to have several fetuses in uterus at palpation that were not likely to be delivered. This indicated that this female had parturition difficulties. At necropsy, this female was shown to have a placenta in the left uterine horn and 4 dead fetuses in the right uterine horn
- One female at 500 mg/kg (no. 72) had 1 early resorption in the right uterine horn, and two late resorptions in the left uterine horn (without abnormalities)
- At 500 mg/kg, mean pup body weights of male and female pups combined were slightly lower than controls on days 4, 7 and 13 of the lactation period (statistically significant on most occasions). This was primarily ascribed to the lower weight gain of litter nos. 78 and 80 (each consisting of a single pup). This pup also had a lean appearance throughout lactation, and had no or insufficient milk in the stomach and/or appeared dehydrated on several occasions. At 50 and 150 mg/kg, mean pup body weights were similar to control levels.
- No toxicologically relevant effects on gestation duration, maternal care and early postnatal pup development (mortality, clinical signs, anogenital distance, areola/nipple retention, PND13-15 pup serum T4 levels and macroscopy) were observed. The statistically significant higher postnatal loss at 150 mg/kg was due to 8 dead pups in one litter that had total litter loss (no. 63). This was considered incidental in nature, given the absence of any treatment-related reproductive or developmental changes in this group. The apparent higher mean postnatal loss at 500 mg/kg (not statistically significant) was due to the low total number of pups at this dose.
- Parturition/maternal care: Apart from the single female at 500 mg/kg (no. 77), 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.
- early postnatal pup development: number of dead pups at first litter check, postnatal loss, viability index and sex ratio were unaffected by treatment, and clinical signs, body weight and external macroscopy did not reveal treatment-related findings
- viability index: A total of 2 pups of the control group, 2 pups at 50 mg/kg, 13 pups at 150 mg/kg (8 of which occurred in litter no. 63) and 3 pups at 500 mg/kg were found dead or missing between Days 0-4 of lactation. Pups missing were most likely cannibalised. No toxicological relevance was attributed to these dead/missing pups since the mortality incidence did not show a dose-related trent and remained within the range considered normal for pups of this age.
- live birth index: unaffected
- lactation index: unaffected

Details on results (P0)

Analysis of dose preparations:
- Accuracy of preparation: The concentrations analyzed in the formulations of Group 2, Group 3 and Group 4 were in agreement with target concentrations (i.e. mean accuracies between 85% and 115%). No test item was detected in the Group 1 formulation.
- Homogeneity: The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation ≤ 10%).

Parental results:
- Treatment up to 500 mg/kg was tolerated without mortality, toxicologically relevant clinical signs or changes in function observation tests. At 500 mg/kg, a treatment-related but non-adverse lower body weight and body weight gain was noted for females during the second half of the post-coitum phase, along with a lower food intake during the post-coitum and lactation phase. This was considered to have occurred secondarily to the lower number of pups born at this dose.
- histopathological examination of the liver showed an adverse centrilobular scattered vacuolation up to moderate degree in both sexes at 150 and 500 mg/kg. This finding correlated with pale discolouration of the liver noted for one female at 500 mg/kg. this was accompanied by a non-adverse hepatocellular centrilobular hypertrophy in males at 150 and 500 mg/kg/day and in females at 50, 150 and 500 mg/kg up to slight degree. In addition, increased liver weights were recorded for males and females at 500 mg/kg (33% and 27% higher, resp), and for females also to 50 and 150 mg/kg (19% and 23% higher, resp). Since these higher liver weights occurred in the absence of correlating histopathological findings in the liver, these were considered not adverse in nature.
- At 500 mg/kg, follicular cell hypertrophy in the thyroid gland was noted in males up to moderate degree, and in females this finding was recorded at 50, 150 and 500 mg/kg up to slight degree. For males, this finding was accompanied by lower total T4 levels of approx 50%. T4 levels were also lower for males at 50 and 150 mg/kg with approx 22 and 29% resp. Follicular cell hypertrophy in the thyroid gland was considered secondary to the hepatocellular hypertrophy in the liver. However, the magnitude of change in T4 levels at 500 mg/kg was considered adverse in nature.
- Other treatment-related changes that were not considered adverse were noted in the spleen, ovaries and kidneys, and in clinical biochemistry parameters. An increased severity of extramedullary hematopoiesis in the spleen was recorded in 2 females at 500 mg/kg that did not deliver healthy pups, but did show signs of pregnancy (implantation sites placenta or fetuses in the uterus). For one of these females this correlated to lower red blood cell counts, haemoglobin and haematocrit, and higher reticulocyte counts and red cell distribution width. These findings were considered to be secondary to the failed pregnancy.
- An increased incidence and severity of interstitial cell hypertrophy was noted in the ovaries of females at 500 mg/kg. This lesion was only minimal to slight in nature and occurred in the absence of any degenerative changes. This finding was accompanied by a higher absolute ovary weight at 50 and 500 mg/kg (23% and 25% higher, resp), which occurred in the absence of a dose-related trend. Overall, these changes in the ovaries were not considered to be adverse in nature.
- an 18% higher kidney weight was noted for males at 500 mg/kg. As this change occurred in the absence of any supportive microscopic lesions, this was not considered adverse in nature.
- changes in clinical biochemistry parameters that were considered to be related to treatment consisted of lower aspartate aminotransferase activity in males at 50, 150 and 500 mg/kg and in females at 500 mg/kg, higher creatinine in males at 500 mg/kg, and higher total protein, albumin and sodium and lower urea and potassium in females at 500 mg/kg. As these changes occurred in the absence of any (clear) morphological correlated and were considered not severe in nature, these were not considered adverse.

Reproductive results
- No toxicologically relevant changes in reproductive parameters were noted up to 500 mg/kg
- At 500 mg/kg, the mean number of implantations appeared lower than in the control group (8.4 vs 11.2 in the control group, not statistically significant). However, this was essentially ascribed to two females which had only 2 or 3 implantation sites. Upon excluding the values of these females, the mean would be 10.3. Given the slight nature of this change, it was not consdiered adverse in nature.
- no treatment-related changes were noted in any of the other reproductive parameters investigated in this study (i.e. mating and fertility indices, precoital time, estrous cycle, spermatogenic profiling, and histopathological examination of reproductive organs)

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
parental
Effect level:
50 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Dose descriptor:
NOAEL
Remarks:
reproduction
Effect level:
>= 500 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: No adverse effects observed up to highest dose tested

Target system / organ toxicity (P0)

Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
150 mg/kg bw/day (nominal)
System:
gastrointestinal tract
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified

Results: F1 generation

General toxicity (F1)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Clinical signs did not reveal treatment-related findings.
Apart from clinical signs noted for the single pup for litter no. 80 at 500 mg/kg (lean appearance, no or insufficient milk in the stomach and dehydrated appearance) , other clinical symptoms of pups were considered to be unrelated to treatment. THese clinical signs remained within the range considered normal for pups of this age, and consisted of blue discolouration of the left eye, alopecia, wounds, blue spot on the neck, absence of milk in the stomach, pallor, scabs and lean appearance. The nature and incidence of these clinical signs.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
A total of 2 pups of the control group, 2 pups at 50 mg/kg, 13 pups at 150 mg/kg (8 of which occurred in litter no. 63) and 3 pups at 500 mg/kg were found dead or missing between Days 0-4 of lactation. Pups missing were most likely cannibalised. 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. Live birth index and lactation index was unaffected; no breeding loss occurred (i.e. between lactation Days 5 and 13).
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Body weight did not reveal treatment-related findings
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 13-15 pups were considered unaffected by treatment.
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Incidental macroscopic findings of pups that were found dead included autolysis and cannibalism. The nature and incidence of these findings remained within the range considered normal for pups of this age, and were therefore considered to be of no toxicological relevance
Histopathological findings:
not examined
Other effects:
no effects observed
Description (incidence and severity):
Anogenital distance: Anogenital distance (absolute and normalized for body weight) in male and female pups was not affected by treatment. The statistically significantly higher meadian anogenital distance of male pups at 150 mg/kg occurred without a dose related-trend, and as such was not considered to be related to treatment.
Areola/nipple retention: Treatment up to 500 mg/kg/day had no effect on areola/nipple retention. For none of the examined male pups nipples were observed at PND13.

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Details on results (F1)

- sex ratio: sex ratio was not affected by treatment. the apparent shift towards a higher sex ratio at 500 mg/kg on lactation day 13 was attributed to a relatively low litter size at this dose.

Developmental results:
- At 500 mg/kg, out of 8 pregnant females, only 5 had offspring (one of which had total litter loss), and three had implantation sites only. Consequently, the gestation index was lower (63% vs 100% in the control group). Also, a lower post implantation survival index was recorded (28% vs 94% in the control group); for 4 out of 5 females that delivered offspring, the number of pups vs the number of implantation sites per litter was less than 50%. The mean number of pups per litter was also lower than controls at 500 mg/kg (3.4 vs 10.5 in the control group).
- One female at 500 mg/kg (which had total litter loss on day 1 of lactation) was considered to have parturition difficulties, indicated by the presence of a placenta and four dead fetuses in the uterus. The uterus of another female at this dose had early resorptions without abnormities.
- At 500 mg/kg, lower male and female pup body wieghts were recorded durng most of the lactation period. This was primarily ascribed to the lower weight gain of two out of four litters surviving until scheduled necropsy, that each consisted of a single pup. These pups also had a lean appearance throughout lactation, and had no or insufficient milk in the stomach and/or appeared dehydrated on several occassions.
- no toxicologically relevant effects in any of the other developmetnal parameters investigated in this study were noted, i. e. viability index, lactation index, duration of gestation, parturition, sex ratio, maternal care and other early postnatal pup development parameters consisting of mortality, clinical signs, anogenital distance (PND1), areola/nipple retention (PND13 males), T4 thyroid hormone levels (PND13-15) and macroscopy.

Effect levels (F1)

Dose descriptor:
NOAEL
Remarks:
developmental
Generation:
F1
Effect level:
150 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: based on lower gestation index, post implantation survival index, mean number of pups and mean pup body weights

Target system / organ toxicity (F1)

Critical effects observed:
not specified

Overall reproductive toxicity

Reproductive effects observed:
no

Applicant's summary and conclusion

Conclusions:
Based on the results, the following was derived
parental NOAEL: 50 mg/kg/day (based on hepatocellular centrilobular vacuolation at 150 and 500 mg/kg).
reproduction NOAEL: at least 500 mg/kg
developmental NOAEL: 150 mg/kg (based on lower gestation index, post implantation survival index, mean number of pups and mean pup body weights