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Diss Factsheets

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14 June 2017- 07 November 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
July 2016
Deviations:
yes
Remarks:
Minor deviations occurred which were determined not to have adversely affected the integrity of the study.
Qualifier:
according to guideline
Guideline:
other: EPA OPPTS 870.3650 (Combined Repeated Dose Toxicity Study with the Reproduction/Deve lopmental Toxicity Screening Test)
Version / remarks:
July 2000
Deviations:
yes
Remarks:
Minor deviations occurred which were determined not to have adversely affected the integrity of the study.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
July 2016
Deviations:
yes
Remarks:
Minor deviations occurred which were determined not to have adversely affected the integrity of the study.
Qualifier:
equivalent or similar to guideline
Guideline:
other: EPA OPPTS 870.3550 (Reproduction/Developmental Toxicity Screening Test)
Version / remarks:
July 2000
Deviations:
yes
Remarks:
Minor deviations occurred which were determined not to have adversely affected the integrity of the study.
Qualifier:
equivalent or similar to guideline
Guideline:
other: OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Version / remarks:
October 2008
Deviations:
yes
Remarks:
Minor deviations occurred which were determined not to have adversely affected the integrity of the study.
Qualifier:
equivalent or similar to guideline
Guideline:
other: EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Version / remarks:
May 2008
Deviations:
yes
Remarks:
Minor deviations occurred which were determined not to have adversely affected the integrity of the study.
Qualifier:
equivalent or similar to guideline
Guideline:
other: EPA OPPTS 870.3050 (Repeated dose 28-day oral toxicity study in rodents)
Version / remarks:
July 2000
Deviations:
yes
Remarks:
Minor deviations occurred which were determined not to have adversely affected the integrity of the study.
Principles of method if other than guideline:
No testing guidelines were applicable for the pilot phase, as this part of the study was intended for dose level selection purposes only.
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
α-(2,4-dichlorophenyl)-1H-imidazole-1-ethanol
EC Number:
246-042-5
EC Name:
α-(2,4-dichlorophenyl)-1H-imidazole-1-ethanol
Cas Number:
24155-42-8
Molecular formula:
C11H10Cl2N2O
IUPAC Name:
1-(2,4-dichlorophenyl)-2-(1H-imidazol-1-yl)ethan-1-ol
Test material form:
solid: particulate/powder
Details on test material:
- State of aggregation: Beige powder
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Sponsor; M15KB5305
- Expiration date of the lot/batch: 2019-11-24
- Purity test date: 2016-10-21

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability in vehicle: Stable for at least 6 hours at room temperature under normal laboratory light conditions and for at least 14 days in refrigerator (2-8°C) is confirmed over the concentration range 1 to 200 mg/mL (Project 517993).
- Stability under test conditions: Homogeneity and stability of the test item under test conditions was demonstrated in the analytical method development and validation study (Project 517993).

FORM AS APPLIED IN THE TEST (if different from that of starting material): Solution (Group 2-3) and suspension (Group 4).

OTHER SPECIFICS: Correction factor was 1.

Test animals

Species:
rat
Strain:
Wistar
Remarks:
rat
Details on species / strain selection:
This species and strain of rat has been recognized as appropriate for general and reproduction toxicity studies. Charles River Den Bosch has general and reproduction/developmental historical data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: Males approx. 10 weeks (at starat F0-treatment); Females approx. 11 weeks (at start pretest) and approx. 13 weeks (at start F0-treatment).
- Weight at study initiation: 269-313 g (males); 203-236 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 in Macrolon plastic cages (MIV type, height 18 cm).
Mating: Males and females were cohabitated on a 1:1 basis 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).
Lactation: Females were housed in Macrolon plastic cages (MIII type,height 18 cm). Pups were housed with the dam, except during locomotor activity monitoring of the dams, when the pups were kept warm in their home cage using bottles filled with warm water. In order to avoid hypothermia of pups, pups were not left without their dam or a bottle filled with warm water for longer than 30-40 minutes.
- Diet (e.g. ad libitum): Free access to pelleted rodent diet. During motor activity measurements, animals did not have access to food for a maximum of 2 hours
- Water (e.g. ad libitum): Free access to tap-water. During motor activity measurements, animals did not have access to water for a maximum of 2 hours.
- 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. The light/dark cycle was interrupted for study related activities. Any variations to these conditions were maintained in the raw data and had no effect on the outcome of the study.

IN-LIFE DATES
From: 2017-08-23 (start pretest, females); 2017-09-06 (start treatment, males); 2017-10-13 through 2017-10-17 and 2017-10-24 through 2017-10-25 (delivery of litters)
To: 2017-10-05 (necropsy males); 2017-10-18/23/26/27/30/31 and 2017-11-1/6/7 (necropsy females); through (necropsy pups)

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
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/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 Den Bosch.
- Concentration in vehicle: 0 mg/mL (group 1); 6 mg/mL (group 2); 20 mg/mL (group 3); 66 mg/ mL (group 4).
- 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): not reported
- Purity: not reported
Details on mating procedure:
- M/F ratio per cage: 1 animal/sex/cage
- Length of cohabitation: Following a minimum of 14 days of treatment for the males and females, the animals were cohabitated on a 1:1 basis within the same treatment group, avoiding sibling mating until mating was confirmed.
- Proof of pregnancy: Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating was confirmed, the males and females were separated.
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): Females were individually housed in Macrolon plastic cages (MIII type, height 18 cm).
- Any other deviations from standard protocol: no
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were conducted on a single occasion during the treatment phase (2017-09-06, Day 1 of treatment) according to a validated method (Test Facility Study No. 517993). Three sets of duplicate samples were collected. Two sets of duplicate samples were stored in the refrigerator as reserve samples. Once analytical results were approved in the raw data by the Principal Scientist, the reserve samples were destroyed.
Samples of formulations were analyzed for homogeneity (highest and lowest concentration) 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 ±10.00% compared to those obtained during the method validation.
The accuracy of preparation was considered acceptable if the mean measured concentrations were 85.00-115.00% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10.00%.
Stability of formulations over 6 hours at room temperature under normal laboratory light conditions (concentration range 1-200 mg/mL) was determined as part of the analytical method development and validation study (Test Facility Study No. 517993).
Duration of treatment / exposure:
Males were treated for 29 days, i.e. 2 weeks prior to mating, during mating, and up to the day prior to scheduled necropsy.
Females that delivered were treated for 50-62 days, i.e. during 2 weeks prior to mating (with the objective of covering at least two complete estrous cycles), the variable time to conception, the duration of the pregnancy and at least 13 days after delivery up to and including the day before scheduled necropsy.
Females which failed to deliver healthy offspring were treated for 41-47 days.

Pups were not treated directly but were potentially exposed to the test item in utero, via maternal milk or from exposure to maternal urine/feces.
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:
Control, group 1
Dose / conc.:
30 mg/kg bw/day (nominal)
Remarks:
group 2
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
group 3
Dose / conc.:
330 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 based on results of a 10-day dose range finding study (Test Facility Study No. 517995). During the DRF phase, oral (gavage) administration of T00824 to female Wistar rats for 10 days (500 mg/kg) or 4 days (600 mg/kg). Dose limiting effects occurred at 600 mg/kg (clinical signs of toxicity, body weight loss and reduced food consumption, leading to early sacrifice at Day 4). Based on these results, dose levels of 0, 30, 100, and 330 mg/kg/day were selected for the main study.
- 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

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once daily from start of treatment onwards up to the day prior to necropsy, detailed clinical observations were made in all animals at 0-15 minutes and 1 hour (±15 minutes) afterdosing. 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. Arena observations were conducted at least 1 hour (± 15 min) after dosing. The time of onset, grade and duration of any observed sign was recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: Males and females were weighed on the first day of treatment (prior to first dosing) and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on PND 1, 4, 7 and 13. Females of Group 4 were also weighed on Day 41 in order to aid in confirmation of pregnancy. Body weight gain was calculated and reported.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- 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.

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

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood samples were collected at the end of the treatment period on the day of scheduled necropsy from the selected 5 animals/sex/group under anaesthesia using isoflurane between 7.00 and 10.30 a.m. Blood samples were drawn from the retroorbital sinus and collected into tubes prepared with K3-EDTA for haematological parameters, with citrate for clotting tests, and tubes treated with Li-heparin for clinical biochemistry parameters.
- Animals fasted: F0-Female animals were not fasted prior to blood sampling. F0-Male animals were fasted prior to blood sampling, but water was available.
- Parameters examined: White blood cells (WBC); Differential leucocyte count: Neutrophils, Lymphocytes, Monocytes, Eosinophils, Basophils, Red blood cells, Reticulocytes, Red blood cell distribution width (RDW), Haemoglobin, Haematocrit, Mean corpuscular volume (MCV), Mean corpuscular haemoglobin (MCH), Mean corpuscular haemoglobin concentration, Platelets, Prothrombin Time (PT), Activated Partial Thromboplastin Time (APTT).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood samples were collected at the end of the treatment period on the day of scheduled necropsy from the selected 5 animals/sex/group under anaesthesia using isoflurane between 7.00 and 10.30 a.m. Blood samples were drawn from the retroorbital sinus and collected into tubes prepared with K3-EDTA for haematological parameters, with citrate for clotting tests, and tubes treated with Li-heparin for clinical biochemistry parameters.
- Animals fasted: F0-Female animals were not fasted prior to blood sampling. F0-Male animals were fasted prior to blood sampling, but water was available.
- How many animals: 5 animals/sex/group
- Parameters examined: Alanine aminotransferase (ALAT), Aspartate aminotransferase (ASAT), Alkaline Phosphatase (ALP), Gamma glutamyl transferase (GGT), Total protein, Albumin, Total Bilirubin, Bile acids, Urea, Creatinine, Glucose, Cholesterol, Sodium, Potassium, Chloride, Calcium, Inorganic Phosphate (Inorg. Phos).

FUNCTIONAL OBSERVATIONS: Yes
- Time schedule for examinations: The selected males were tested once during Week 4 of treatment. The selected females of Groups 1 and 2 were tested once during the last week of lactation (PND 6-13) and selected females of Group 3 were tested once at PND 5-8. Selected females of Group 4 (no offspring) were tested once at Day 23-26 post-coitum. These tests were performed after observation for clinical signs (incl. arena observation, if applicable).
- Dose groups that were examined: 5 animals/sex/group
- Battery of functions tested: hearing ability (HEARING), pupillary reflex (PUPIL L/R), and static righting reflex (STATIC R); fore- and hind-limb grip strength, recorded as the mean of three measurements per animal; locomotor activity (recording period: 1-hour under normal laboratory light conditions, using a computerized monitoring system. Total movements and ambulations are reported. Ambulations represent movements characterized by a relocation of the entire body position like walking, whereas total movements represent all movements made by the animals, including ambulations but also smaller or finer movements like grooming, weaving or movements of the head.
Oestrous cyclicity (parental animals):
Daily vaginal lavage was performed to determine the stage of estrous beginning 14 days prior to treatment (pretest), the first 14 days of treatment and during mating until evidence of copulation was observed. Vaginal lavage continued for those females with no evidence of copulation until termination of the mating period. During pretest, this was done for 48 females. On the day of scheduled necropsy, a vaginal lavage was taken to determine the stage of estrous.
Sperm parameters (parental animals):
Parameters examined in F0 male parental generation: For the testes of the selected 5 males of Groups 1 and 4 and all males that failed to sire or which died before mating, detailed qualitative examination was made, taking into account the tubular stages of spermatogenic cycle. The weight of the testes was recorded.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- On PND 1, all pups were randomized per litter and individually identified by means of subcutaneous injection of Indian ink. When general hair growth blurred the identification, the pups were identified by tattoo on the feet.
- To reduce variability among the litters, on PND 4 eight pups from each litter of equal sex distribution (if possible) were selected. Blood samples were collected from two of the surplus pups. Selective elimination of pups, e.g. based upon body weight, or anogenital distance was not done. Whenever the number of male or female pups prevented having four of each sex per litter, partial adjustment (for example, five males and three females) was acceptable.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
- Mortality/viability: The numbers of live and dead pups were determined on PND1 and daily there after. If possible, defects or cause of death were evaluated. Animals showing pain, distress or discomfort, which was considered not transient in nature or was likely to become more severe, were sacrificed for humane reasons.
- 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 tables 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 PND 1 and 4.
- Anogenital distance: Anogenital distance (AGD) was measured for all live pups on PND 1. The AGD was normalized to the cube root of body weight.
- Areola/nipple retention: On PND 13, all males in each litter were examined for the number of areola/nipples.

GROSS EXAMINATION OF DEAD PUPS:
- Descriptions of all abnormalities were recorded. If possible, defects or cause of death were evaluated.

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: no

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: no
Postmortem examinations (parental animals):
SACRIFICE
F0-Female animals were not fasted prior to necropsy. F0-Male animals (except for male no.32 which was sacrificed in extremis) were deprived of food overnight (with a maximum of 24 hours) prior to necropsy, but water was available. Necropsy was conducted on the following days:
- Males: Following completion of the mating period (a minimum of 28 days of doseadministration).
- Females which delivered: PND 14 or 16.
- Females which failed to deliver: Post-coitum Days 25-27 (females with evidence of mating).
- Females with total litter loss: Within 24 hours of litter loss.
- Euthanized in extremis: When pain, distress or discomfort was considered not transient in nature or was likely to become more severe.

GROSS NECROPSY:
- All animals surviving to the end of the observation period and all moribund animals were deeply anaesthetized using isoflurane and subsequently exsanguinated.
- 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. The number of former implantation sites were recorded for all paired females. In case no macroscopically visible implantation sites were present, nongravid uteri were stained using the Salewski technique in order to detect any former implantation sites (Salewski staining prepared at Charles River Den Bosch using Ammoniumsulfide-solution 20% and Milli-Ro water), and the number of corpora lutea were recorded in addition. Samples of the following tissues and organs 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), Colon (M/F), Coagulation gland (M), Duodenum (M/F), Epididymides (M), Eyes (with optic nerve (if detectable) and Harderian gland) (M/F), Mammary gland area, inguinal region with skin (M/F), Femur with bone marrow including joint (M/F), Heart (M/F), Ileum (M/F), Jejunum (M/F), Kidneys (M/F), (Lacrimal gland, exorbital) (M/F), (Larynx) (M/F), Liver (M/F), Lung, infused with formalin (M/F), Lymph nodes (mandibular, mesenteric) (M/F), (Nasopharynx) (M/F), Esophagus (M/F), Ovaries (F), (Pancreas) (M/F), Peyer's patches [jejunum, ileum] if detectable (M/F), Pituitary gland (M/F), 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.

HISTOPATHOLOGY:
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 following were examined:
- The preserved organs and tissues of the selected 5 animals/sex of Groups 1 and 4
- For the testes of the selected 5 males of Groups 1 and 4 and all males that failed to sire or which died before mating detailed qualitative examination was made, taking into account the tubular stages of spermatogenic cycle.
- The preserved organs and tissues of the animals of all dose groups which were euthanized in extremis.
- The mammary gland of all females with total litter loss.
- All gross lesions of all animals (all dose groups).
- Liver, spleen and kidneys of all selected 5 animals of Groups 2 and 3 (males and females), adrenal glands, lung, ovaries, uterus, cervix, vagina and mammary gland of all selected 5 females of Groups 2 and 3, based on (possible) treatment-related changes in these organs in Group 4.
- Nasal cavity level 3, 4 and 4 caudal and larynx of all selected 5 females of Groups 1 and 4 and Male no. 32 and Female no. 74 since the observation of rales may have been related to the moribundity of these animals.
- 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 report. An attempt was made to correlate gross observations with microscopic findings.
- A peer review on the histopathology data was performed by a second pathologist.

ORGAN WEIGHTS:
- Absolute organ weights were reported and organ to terminal body weights and organ to brain weights were calculated.
- The following organ weights and terminal body weight were recorded from the selected 5 animals/sex/ group on the scheduled day of necropsy: Adrenal glands, Brain, Epididymides, Heart, Kidneys, Liver, Ovaries, Prostate, Seminal vesicles including coagulating glands, Spleen, Testes, Thymus, Thyroid (including parathyroid if detectable), Uterus (including cervix).
- The following organ weights and terminal body weight were recorded from all remaining animals on the scheduled day of necropsy: Epididymides, Testes, Thyroid (including parathyroid if detectable), Prostate, and Seminal vesicles including coagulating glands.
- Paired organs were weighed together.
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 7-16 (using Euthasol 20% by intraperitoneal injection).

GROSS NECROPSY
- All pups were sexed by both external as well as internal examination. Descriptions of all abnormalities were recorded. Particular attention was paid to the external reproductive genitals to examine signs of altered development.
- At terminal sacrifice (PND 14 or 16), the thyroid from 2 pups per litter, i.e. the same pups as selected for blood sampling, 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 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. In case intergroup differences were seen, the Wilcoxon test was applied to compare the treated groups to the control group. 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. 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 with living pups on Day 1/Number of pregnant females) x 100
Duration of gestation = Number of days between confirmation of mating and the beginning of parturition
Offspring viability indices:
Survival indices:
Post-implantation survival index (%) = (Total number of offspring born/Total number of uterine implantation sites) x 100
Live birth index (%) = (Number of live offspring on Day 1 after littering/Total number of offspring born) x 100
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, treatment-related
Description (incidence and severity):
Clinical signs in the animals that were sacrificed prematurely are described in the section on mortality. Findings in animals that survived until scheduled termination are described below.
At 330 mg/kg, the main clinical sign of toxicity was piloerection of the fur which started after about one week of treatment and was seen most frequently in females. Less frequent findings included rales (in several males and females on a few occasions), hunched posture (in 8 out 10 females, starting on treatment Day 6 for approximately 1-2 weeks), and slight lethargy, ptosis, pale appearance and red discharge/discoloration at the vulva (in a single female (no. 789) towards the end of the treatment period). Slight lethargy was further noted once (Day 3) in two males (without other clinical signs. Additionally, yellow discoloration of the faeces was seen in all 330 mg/kg animals in Weeks 2-3. Piloerection of the fur and rales also occurred at 100 and 30 mg/kg, particularly in females. The incidence and time of onset of piloerection of the fur showed a dose-related trend. Such trends were not evident for rales (which occurred less frequently than piloerection of the fur). Salivation was noted after dosing at all dose levels, starting after seven (low dose) or three (higher dose levels) days of treatment. This salivation was considered to be a physiological response rather than a sign of systemic toxicity considering its minor severity (generally slight) and the time of occurrence (i.e. immediately after dosing), and may be related to the irritancy of the test item. Any other clinical signs noted incidentally 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 showed no dose-related trend. At the incidence observed, these were considered to be unrelated to treatment.
No additional findings were noted during the weekly arena observations.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, treatment-related
Description (incidence):
At 330 mg/kg there were three premature decedents, all sacrificed in moribund condition, which were considered to be related to treatment with the test item. Male no. 32 and female no. 74 were euthanized at study Day 8. These animals showed clinical signs of toxicity (hunched posture, piloerection of the fur, rales, ptosis) and body weight loss (10-12% between Days 1-8). Main necropsy findings consisted of gas distended parts of the intestines (no. 32) and emaciated appearance (no. 74). Moderate hepatocellular vacuolation of the liver was noted in both rats, similar as in the scheduled sacrificed rats. Examination of the nasal cavity revealed in both rats mucosal lesions indicative of reflux-related effects in the form of (sub)acute erosion/necrosis of the respiratory and/or olfactory epithelium, luminal exudate and granulocytic inflammatory cell infiltrate. These findings were considered to have attributed to the moribundity of these animals.
The third decedent (female no. 79) at 330 mg/kg was euthanized at Day 38 (Day 19 postcoitum) when she showed red discoloration in the inguinal region (blood was seen in her cage), increased respiratory rate, pale appearance (moderate), hunched posture, lethargy, piloerection of the fur7 and body weight loss (nearly 10% between Days 17-19 post-coitum). Main morphologic findings in female no. 79 consisted of two early resorptions and one dead fetus in the left uterus horn and slight inflammation of the endometrium of the uterus. This reproductive failure was considered to be related with the moribundity.
At 100 mg/kg, one female (no. 63) was sacrificed for humane reasons at Day 21 (Day 1 postcoitum). On the day of sacrifice, after dosing, she showed laboured respiration, rales, ptosis, felt cold8 and excessive salivation. Necropsy findings consisted of an emaciated appearance (body weight was normal) and gastric changes (distended with gas, isolated red foci in the glandular stomach mucosa). Microscopic examination revealed lesions indicative of a dosing error in the form of moderate to marked necrosis of bronchus and tracheal epithelium and the moribundity of this animal was therefore not considered test-item related.
Three females at 330 mg/kg were sacrificed due to total litter loss (nos. 62, 66 and 70 at PND 1, 3 and 4, respectively).
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Males and females at 330 mg/kg showed statistically significantly reduced body weight gain during the first week of treatment. Significant weight loss (between 6-12% of the initial weight) occurred in the two animals that were euthanized on Day 8 (male no. 32, female no.74) and in two other animals (male no. 36, female no. 73). Mean body weights at Day 8 were statistically significantly reduced in males (9% difference) while the difference in females (5%) did not attain statistical significance. Growth rate and mean body weights had recovered to normal values at Day 1 of the mating period and remained normal until the end of the treatment period in males and until Day 7 post-coitum in females. Thereafter, 330 mg/kg females gained less weight than controls (mean body weights were statistically significantly lower on post-coitum Days 11-20, 25% difference in body weight gain at Day 20 post coitum). This reduced gestational body weight gain was due to their abnormal pregnancies (i.e. 3 non-pregnant females and six females with implantations only).
No toxicologically relevant changes in body weight (gain) were observed in males and females treated up to 100 mg/kg. The statistically significantly higher body weight gain values noted in 30 mg/kg females at Days 4 and 7 of the lactation period (associated with higher food consumption values between lactation Days 1-4) were judged to be non-adverse as weight gain of these females was not significantly different at other time points. Moreover, mean body weights of 30 mg/kg females remained close to control values.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food consumption before and after allowance for body weight was reduced at 330 mg/kg in the first week of treatment in both sexes (by about 25%) and, to a lesser extent, in the second treatment week in females. Food consumption of 330 mg/kg females was also reduced during the post-coitum period, which was related to their abnormal pregnancy (i.e. 3 non-pregnant females and six females with implantations only). At 100 mg/kg, mean food consumption (before and after allowance for body weight) was statistically significantly reduced between Days 7-13 of the lactation period. The lower group means resulted from the lower food consumption of two females with very small litters (nos.64 and 68). The remaining three lactating females at 100 mg/kg showed normal food consumption (and had normal litter sizes). This finding was therefore not regarded as toxicologically relevant, but a secondary effect due to the small litter sizes. No treatment-related changes in food consumption before or after allowance for body weight were observed in males treated up to 100 mg/kg and females treated at 30 mg/kg. The higher food consumption noted in 30 mg/kg females between Days 1-4 of the lactation period was considered unrelated to treatment as food consumption of these females was similar to that of controls at all other time points.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Note: Females at 330 mg/kg had no offspring. Several haematology parameters are known to be influenced by physiological status (lactating versus implantations only). This was taken into account when assessing the possible relation with treatment of differences in haematology values between females of the 330 mg/kg group (all selected females had implantation sites, suggestive of former pregnancy, but no offspring) and controls (all lactating). The following changes in haematology parameters distinguished treated animals from control animals. The differences were statistically significant unless indicated otherwise. Relative changes in mean values relative to the concurrent control group are indicated between parentheses.
Male rats treated up to 330 mg/kg:
- Lower mean corpuscular haemoglobin (MCH) at 330 mg/kg (7%). The statistically significantly lower number of platelets noted at 100 mg/kg was considered unrelated to treatment due to the absence of a dose-related trend and a somewhat high control value (resulting from an outlying value in one control male).
Female rats treated at 30 or 100 mg/kg:
- Lower mean corpuscular haemoglobin (MCH) at 100 mg/kg (8%).
- Higher red blood cell distribution width (RDW) at 100 mg/kg (11%). This change was not statistically significant.
Female rats treated at 330 mg/kg:
Note: Due to the difference in physiological status (lactating versus implantations only), no percentage differences from control values were calculated.
- Higher total white blood cell count (WBC; consisting of neutrophils, lymphocytes, monocytes, eosinophils and basophils)
- Higher number of reticulocytes.
- Lower haemoglobin concentration.
- Lower mean corpuscular haemoglobin volume (MCV), mean corpuscular haemoglobin (MCH) and mean corpuscular haemoglobin concentration (MCHC).
- A tendency towards higher red blood cell distribution width (RDW) and lower haematocrit.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Note on females at 330 mg/kg: see haematology section.
The following changes in clinical biochemistry parameters distinguished treated animals from control animals. The differences were statistically significant unless indicated otherwise. Relative changes in mean values relative to the concurrent control group are indicated between parentheses.
Male rats treated up to 330 mg/kg:
- Lower aspartate aminotransferase activity (ASAT) at 330 mg/kg (30%).
- Higher gamma glutamyl transferase activity (GGT) at 330 mg/kg. Statistical significance and magnitude of difference from control could not be determined as all control values were below the limit of detection (0.5 U/L). Values at 330 mg/kg were 2-3 fold higher than this detection limit and were outside the historical control range. Values at 30 and 100 mg/kg were within the historical control range.
- Lower total bilirubin at 330 mg/kg (20%).
Female rats treated at 30 or 100 mg/kg:
Clinical biochemistry parameters in lactating females were considered not to be affected by treatment up to 100 mg/kg.
Female rats treated at 330 mg/kg:
Note: Due to the difference in physiological status (lactating versus implantations only), no percentage differences from control values were calculated.
- Lower alanine aminotransferase activity (ALAT), alkaline phosphatase (ALP) activity (not
statistically significant), bile acids (not statistically significant), urea, potassium and chloride.
- Higher gamma glutamyl transferase activity (GGT) (not statistically significant), glucose, total protein, albumin, creatinine, cholesterol (not statistically significant), sodium, calcium and inorganic phosphate (not statistically significant).
Thyroid hormone analyses:
Mean serum T4 levels of F0 males were statistically significantly decreased from 30 mg/kg onwards (relative differences from controls: 24, 40 and 48% at 30, 100 and 330 mg/kg, respectively). The mean value at 330 mg/kg, 2.75 ug/dL, was below the historical control range.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related findings were noted for functional observations up to 330 mg/kg. Mean hind limb grip strength values of treated males were statistically significantly lower compared with the control value. The differences showed no dose-related response. Moreover, the concurrent control value was higher than normal. Therefore, the differences in hind limb grip strength in males were judged to be the result of a high control value rather than an effect of the test item. Fore limb grip strength in males and grip strength values in females were unremarkable. The non-statistical reduction in fore limb grip strength of females treated with 330 mg/kg was within normal range for nulliparous females.
Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals. The variation in motor activity did not indicate a relation with treatment. All groups showed a similar habituation profile with a decreasing trend in activity over the duration of the test period. The higher mean values for total movements and ambulations noted in females at 100 mg/kg were considered unrelated to treatment since these values were within normal range and due to the lack of consistency in time (values were particularly high during the fifth interval). Moreover, the significant outcome at 100 mg/kg was likely the result of the lower control values in this study which were in the lower limit of the historical control data.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Results of males (treated for 29 days), females of the control, 30 and 100 mg/kg groups (selected females treated for 50 to 55 days and with normal offspring), and females of the 330 mg/kg group (selected females treated for 42 days and without healthy offspring) were evaluated separately.

Male rats of all groups:
Test item-related microscopic findings were noted in the liver of 100 and 330 mg/kg males and kidneys of 330 mg/kg males.
Liver:
- Hepatocellular vacuolation was observed in males at 100 mg/kg (minimal) and 330 mg/kg (up to moderate). This vacuolation was variable in size (micro- and macro-vesicular) and in general present in zone 1 and 2.
- Hepatocellular hypertrophy was observed in males at 100 mg/kg (minimal, particularly centrilobular) and 330 mg/kg (up to slight, diffuse).
Kidneys:
- Increased severity of hyaline droplets accumulation was noted in males at 330 mg/kg (all slight).

Selected female rats of the control, 30 and 100 mg/kg groups:
Test item-related microscopic findings were noted in the spleen of 30 and 100 mg/kg females. A dose-related increase of yellow-brown pigmentation of the spleen was observed in 30 and 100 mg/kg group females (up to moderate). This pigmentation was considered to represent hemosiderin, a degradation product of haemoglobin.

Female rats of the 330 mg/kg group (without living offspring):
Test item-related microscopic findings were noted in the liver, kidneys and lung of 330 mg/kg females as listed in the text below (shaded gray). Furthermore, there were possible test item related findings in the ovaries and adrenal glands of 330 mg/kg females.
Liver:
- Hepatocellular vacuolation was observed in 330 mg/kg females (up to slight). This vacuolation was variable in size (micro- and macro-vesicular) and in general present in zone 1 and 2.
- Hepatocellular hypertrophy was observed at increased incidence and severity in 330 mg/kg females (up to slight, diffuse). The presence of this finding in a single 100 mg/kg female at a minimal degree was considered to be a background finding.
Kidneys and lung:
- Intravascular macrophage-like cellular emboli attached to the endothelium were observed at minimal degree in veins of the kidneys, and up to moderate degree in veins of the lung of 330 mg/kg females. The cells were characterized by oval to polygonal nuclei and a foamy or finely vacuolated cytoplasm, with frequent pigment and occasional eosinophilic granules. The cellular emboli were attached to the intima layer of the vessel wall, sometimes lining a larger area and sometimes more focally protruding into the lumen. Since they were only present in females, and in particular those that had experience embryo loss, a uterine or
implantation site origin of the cellular emboli could not be ruled out. The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.
Histopathological findings: neoplastic:
not examined
Other effects:
not specified

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
effects observed, treatment-related
Description (incidence and severity):
Length and regularity of the estrous cycle were adversely affected by treatment at 330 mg/kg. Only 3/10 females (nos. 72, 75, 78) at this dose level had regular cycles of five days during the premating phase (during the pre-treatment phase, they had cycles of four days). Four 330 mg/kg females (nos. 71, 73, 77 and 80) had an irregular cycle (of six days) and two females (nos. 76 and 79) were acyclic (persistent diestrus). None of these females had live offspring (they were not pregnant or had implantation sites only). Estrous stage/length of the animal euthanized in extremis on treatment Day 8 (no. 74) could not be determined.

Length and regularity of the estrous cycle at the lower dose levels were not affected by treatment. All females of the control and 30 and 100 mg/kg groups had regular cycles of four or five days. Extended di-estrus during pairing was noted in two control females (no. 44 and 47) and one 30 mg/kg female (no. 52) which had normal litters. This is a normal background finding in females of this strain and age.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
There were no test-item related findings in the reproductive organs of males, and all spermatogenic profiles were normal.
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
REPRODUCTION DATA
- Mating index: Mating index was not affected by treatment. All paired females showed evidence of mating.
- Fertility index: Fertility index was reduced to 67% at 330 mg/kg. Three of the nine mated females in this group were not pregnant. No abnormalities were seen in the reproductive organs which could account for these cases of non-pregnancy. Fertility index was unaffected by treatment up to 100 mg/kg. The fertility index was 90, 100 and 100% for the control, 30 and 100 mg/kg groups, respectively. One control female was not pregnant despite evidence of mating.
- Precoital time: Precoital time was considered not to be affected by treatment. Most females showed evidence of mating within five days.
- Number of implantation sites: The mean number of implantation sites was statistically significantly decreased at 330 mg/kg (8.5 versus 13.2 in controls). A treatment related reduction is suspected at 100 mg/kg (11.0 versus 13.2 in controls). Treatment at 30 mg/kg was considered not to have affected the number of implantation sites.

DEVELOPMENTAL DATA
- Gestation index and duration: Gestation index was reduced to 78% and 0% at 100 and 330 mg/kg, respectively. At 330 mg/kg, none of the six pregnant females had live pups at first litter check. At 100 mg/kg, one female (no. 62) had only dead pups and another female (no. 61) had implantation sites only. These females had histopathological changes in the uterus (mostly inflammation of the endometrium) related to their reproductive failure. Taken in the context of the other effects on viability at 100 mg/kg (reduced post-implantation survival and early postnatal viability) and the many failed pregnancies at 330 mg/kg (no pregnant females with offspring), the two failed pregnancies at 100 mg/kg were judged to be related to treatment. At 30 mg/kg and in the control group all pregnant females had live offspring (gestation index 100%). Duration of gestation was considered not to be affected by treatment. Compared with concurrent controls, mean duration of gestation was statistically significantly higher at 30 and 100 mg/kg. There was no dose-related trend and duration of gestation was normal16 (mostly 22 days) in all treated females. Moreover, mean duration of gestation in concurrent controls was slightly lower than the historical control mean (21.0 versus 21.4). Therefore, these differences were judged to be chance findings and unrelated to treatment.

- Parturition/maternal care: Female no. 79 showed signs of delivery problems and was euthanized for humane reasons at Day 19 post coitum. No signs of difficult or prolonged parturition were noted among the other 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: Post-implantation survival index (total number of offspring born as percentage of total number of uterine implantation sites) was reduced to 52% and 0% at 100 and 330 mg/kg, respectively. At 30 mg/kg, post-implantation survival index was lower than that in the control group (83% versus 90%) and was just outside of the historical control range. The individual difference between number of implantation sites and number of pups born (the so-called unaccounted for sites) that has led to this reduction in post-implantation survival index was somewhat higher in this dose group. Whereas the unaccounted for sites varied from 0-1 in 6 females and 2-4 for 3 females in the control group, 3 females had 0-1 unaccounted for sites and 7 females 2-5 unaccounted for sites at dose 30 mg/kg. Although there were no related histopathology changes in reproductive organs or effects on other developmental parameters in this dose
group, the lower post-implantation survival index at 30 mg/kg was considered treatmentrelated as this parameter was affected in a dose related manner.

- Live birth index: Live birth index (number of live offspring on PND 1 as percentage of total number of offspring born) was reduced to 76% at 100 mg/kg. One female at 100 mg/kg (no. 62) had four dead pups at first litter check and total litter loss at PND 1. Female (no. 68) had a litter with 10 pups of which 7 pups were found dead and for female no. 70 had one out of 3 pups were dead at first litter check. The numbers of dead pups at first litter check in the 30 mg/kg group (one dead pup, litter no. 53) and in the control group (two dead pups, litter nos. 45 and 47) were within normal limits. The live birth indices for these groups were 99 and 98%, respectively. The incidental pup mortality at 30 mg/kg was judged to be unrelated to treatment.

- Viability index: Viability index (number of live offspring on PND 4 before culling as percentage of number of live offspring on PND 1) was reduced to 90% at 100 mg/kg and was considered treatment related. The postnatal loss in this dose group (in total 4/39 pups) occurred in three small litters: 1/3 pups of litter no. 68 went missing on PND 2; the single pup of no. 66 went missing on PND 3 (total litter loss); 2/2 pups of litter no. 70 were euthanized in extremis on PND 3-4 (total litter loss). The pups of the remaining four litters survived until scheduled sacrifice. At 30 mg/kg, no pups died on PND 2-4 (viability index 100%). In the control group three pups (all of litter no. 42) went missing (presumably cannibalized) on PND 2 (viability index 97%). This incidental postnatal loss remained within the range considered normal for pups of this age.

- Lactation index: No pups were lost between PND 5-13. The lactation index (number of live offspring on PND 13 as percentage of number of live offspring on PND 4 after culling) was 100% in all groups with litters (control, 30 and 100 mg/kg).

Details on results (P0)

Parental results:
- Three animals treated at 330 mg/kg were prematurely euthanized for humane reasons. One male and one female were sacrificed at Day 8. They had lesions in the mucosa of the nasal cavity, likely attributing to their moribundity, which were indicative of reflux-related effects and considered to represent unintended local adversity (Ref. 7; Ref. 8). Gavage-related reflux may also explain the respiratory symptoms (rales) noted in these animals. Another female was
sacrificed in moribund condition at Day 19 post-coitum and showed signs of delivery problems. No test item related mortality was noted up to 100 mg/kg.
- The main treatment-related clinical sign of toxicity was piloerection of the fur which occurred in a dose-related manner, starting at 30 mg/kg, most frequently in females. The piloerection of the fur at 30 mg/kg occurred at a low incidence without other signs of toxicity and was, therefore, considered to be not toxicologically relevant. At 100 mg/kg, piloerection of the fur was considered adverse as this sign was noted in several females for approximately 1-2 weeks. The majority of the females of the 330 mg/kg group also showed a hunched posture.
- Other treatment-related clinical findings included yellow discoloration of the faeces (in all 330 mg/kg animals in Weeks 2-3), slight salivation after dosing (all dose levels) and rales (all dose levels). Based on its transient nature, the discoloration of the faeces was regarded as non-adverse. The salivation was considered a physiological response rather than a sign of systemic toxicity. The rales, which occurred at a low incidence without dose-related trend, were likely due to gavage-related reflux.
- Body weight gain was reduced at 330 mg/kg in both sexes in the first treatment week (a few animals lost some weight). This was accompanied by a (transient) decrease in food consumption. As the resulting reductions in mean body weights were less than 10% and growth rate had returned to the control level by the end of the second treatment week, the transient growth retardation at 330 mg/kg was regarded as non-adverse.
- Serum levels of thyroid hormone T4 (measured only in males) were decreased from 30 mg/kg onward in a dose-related manner (by 24, 40 and 48% at 30, 100 and 330 mg/kg, respectively). There were no treatment-related changes in the morphology or weight of the thyroid (in either sex). A decrease in T4 may be due to increased T4 turnover resulting from metabolic enzyme induction in the liver (hepatocellular hypertrophy, present in males at 100 and 330 mg/kg).
- Microscopic examination revealed treatment-related changes in several organs as described below.
- Hepatocellular vacuolation (up to moderate) and hepatocellular hypertrophy (up to slight) were observed in the liver at 100 mg/kg (males) and 330 mg/kg (both sexes). This correlated with enlargement of the liver in 330 mg/kg males (liver weight was increased by about 40%, absolute and relative to body weight). Some changes in clinical biochemistry parameters noted at 330 mg/kg might be related to the effect on the liver (statistically lower aspartate aminotransferase activity in males, non-statistically higher gamma glutamyl transferase activity in both sexes and lower bile acids and cholesterol in females). In the absence of any degenerative or inflammatory hepatic changes, these liver (related) findings were considered to be non-adverse.
- An increased severity (up to slight degree) of hyaline droplet accumulation was noted in the kidneys of male rats treated at 330 mg/kg. The hyaline droplet accumulation likely represented alpha2u-globulin, a normal protein in male rats which undergoes reabsorption in the proximal cortical tubules. This male rat specific protein is not present in female rats nor in higher mammals, including man (Ref. 12). The severity of this finding was slight and it was not accompanied by other histologic hallmarks of renal toxicity in males. Therefore, the increased hyaline droplet accumulation in 330 mg/kg males was considered to be non-adverse.
- Increased severity of yellow pigmentation was observed in the spleen of 30 and 100 mg/kg females (up to moderate degree). This pigmentation was considered to represent hemosiderin, a degradation product of haemoglobin. The haemoglobin concentration in blood of 30 or 100 mg/kg treated females was not affected by treatment. Splenic hemosiderin pigmentation can be seen as a background finding (generally up to slight degree) in female rats subjected to 28-day toxicity studies combined with reproduction/developmental toxicity screening test. Based on the modest increase in severity and the absence of adverse haematological changes, the splenic hemosiderin pigmentation observed in this study was regarded as non-adverse.
- An unusual microscopic change was observed in the kidneys and lung of females treated at 330 mg/kg. This change consisted of macrophage-like cellular emboli attached to the intima layer of veins and was most obvious in the lung (up to moderate degree in the lung and slight in the kidneys). This change has not been described earlier and the underlying mechanism is unclear. Since they were only present in females, and in particular those that had experience embryo loss, a uterine or implantation site origin of the cellular emboli could not be ruled out. However, this change indicates obstruction of the veins which could be considered an adverse finding.
- For the microscopic changes noted at high incidence in the adrenals (single cell necrosis of the zona fasciculate, minimal degree) and ovaries (cysts, up to slight degree) of females treated at 330 mg/kg it remained unclear whether they were related with direct organ toxicity or were secondary and related with the (stress-full) physiological state of these animals. Based on their high incidence and/or degenerative nature, these findings were regarded as adverse.
- Females at 100 and 330 mg/kg which had total litter loss (3 at 100 mg/kg) or failed pregnancy (one at 100 mg/kg, six at 330 mg/kg) showed microscopic changes in reproductive organs, mostly the uterus, which were related to the reproductive failure and/or time of pregnancy/lactation. The main finding was inflammation of the endometrium of the uterus. The observed increase in white blood cell count in females at 330 mg/kg may have been related to the inflammation of the endometrium. Additional microscopic findings were endometrial hemorrhage and increased mucification of the vagina epithelium. Lobuloalveolar development of the mammary gland was noted in one 330 mg/kg female. At necropsy, a thickened horn, one or two early resorptions and/or a dead (large) fetus were observed in the uterus of four 330 mg/kg females.
- Treatment-related changes in red blood cell parameters were observed at 100 mg/kg (females only) and 330 mg/kg (both sexes). The changes at 100 mg/kg consisted of a decrease in mean corpuscular haemoglobin (MCH) and an increase in red blood cell distribution width (RDW). As these changes were slight and not accompanied by changes in haematological parameters that would impact tissue oxygenation (e.g. significantly reduced haemoglobin), they were regarded as non-adverse. For the same reasons the red blood cell changes observed at 330 mg/kg were considered non-adverse (lower MCH in both sexes; higher number of reticulocytes and RDW, and lower haemoglobin concentration, mean corpuscular haemoglobin (MCH) and mean corpuscular haemoglobin concentration (MCHC) in females).
- Clinical biochemistry results showed changes in some liver-related parameters in males and females at 330 mg/kg (see above). Additional findings noted in 330 mg/kg females consisted of higher plasma levels of albumin, total protein, creatinine, sodium, calcium and inorganic phosphate, and lower levels of alkaline phosphatase (ALP), potassium and chloride. These findings were inferred from a comparison with control data due to the different physiological state. Therefore, it could not be determined with certainty whether these findings were related to treatment with the test item. Also, their toxicological relevance remains unclear.

Reproductive results:
- Reproductive toxicity was observed at 100 mg/kg indicated by reduced number of implantation sites and at 330 mg/kg as indicated by abnormalities in estrous cyclicity in most females, reduced number of implantation sites and reduced fertility index. There were no morphological findings in the reproductive organs of either sex which could explain these effects.
- No treatment-related changes were noted in the other reproductive parameters investigated in this study (i.e. mating index, precoital time, spermatogenic profiling, and histopathological examination of reproductive organs).

Analysis of Dose Preparations:
- Accuracy of preparation: The concentrations analyzed in the formulations of Groups 2, 3 and 4 were in agreement with target concentrations (i.e. mean accuracies between 85.00% and 115.00%). No test item was detected in the Group 1 formulation.
-Homogeneity: The formulations of Groups 2 and 4 were homogeneous (i.e. coefficient of variation ≤ 10.00%).

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
parental toxicity
Effect level:
30 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
Remarks on result:
other: T4 was reduced in all treated groups (males) and considered to be compound-related. However, possible adversity of this effect could not be assessed within this type of screening study and was therefore not considered in determining the parental NOAEL.
Key result
Dose descriptor:
NOAEL
Remarks:
reproduction
Effect level:
30 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
reproductive performance

Target system / organ toxicity (P0)

Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
100 mg/kg bw/day (nominal)
System:
female reproductive system
Organ:
uterus
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):
- At 100 mg/kg, dehydration was noted in one of the two surviving pups (litter no. 68) and in the two pups sacrificed in extremis on PND 3 or 4 (litter no. 70). Additionally, both prematurely sacrificed pups had a lean appearance and one of them had less milk in the stomach. The clinical signs of these pups likely contributed to the reduced viability index. The scab in the neck noted for 1 pup (litter 65) at first litter check was considered a normal background finding.
- No treatment-related clinical signs were noted among pups of females treated at 30 mg/kg. The clinical signs observed incidentally in pups of the control and 30 mg/kg groups remained within the range considered normal for pups of this age.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
- The number of live offspring on PND 4 before culling as percentage of number of live offspring on PND 1) was reduced to 90% at 100 mg/kg and was considered treatment related.
- The postnatal loss in this dose group (in total 4/39 pups) occurred in three small litters: 1/3 pups of litter no. 68 went missing on PND 2; the single pup of no. 66 went missing on PND 3 (total litter loss); 2/2 pups of litter no. 70 were euthanized in extremis on PND 3-4 (total litter loss). The pups of the remaining four litters survived until scheduled sacrifice.
- At 30 mg/kg, no pups died on PND 2-4 (viability index 100%). In the control group three pups (all of litter no. 42) went missing (presumably cannibalized) on PND 2 (viability index 97%). This incidental postnatal loss remained within the range considered normal for pups of this age.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Body weights of pups were considered not to be affected by treatment up to 100 mg/kg. The isolated statistically significant difference noted at 30 mg/kg (higher weight of female pups on PND 1) was judged to be a chance finding.
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, treatment-related
Description (incidence and severity):
- Female PND 14-16 pups at 100 mg/kg showed a statistically significantly higher serum T4 level compared with the control group (relative difference 36%). The mean T4 value in these female pups almost exceeded the historical control range. Male pups at 100 mg/kg showed a similar tendency (25% higher mean T4) but the difference from controls was not statistically significant and remained within the historical control range.
- Serum T4 in pups at 30 mg/kg was judged to be unaffected by treatment.
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Histopathological findings:
not examined
Description (incidence and severity):
- Anogenital distance: Anogenital distance (absolute and normalized for body weight) in male and female pups was considered not to be affected by treatment up to 100 mg/kg.
- Areola/nipple retention: Treatment up to 100 mg/kg had no effect on areola/nipple retention. For none of the examined male pups nipples were observed at PND 13.
- Sex ratio: not affected by treatment.

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Details on results (F1)

Developmental results:
- Developmental toxicity was observed at 30, 100 and 330 mg/kg. There were no explanatory morphological findings in reproductive organs.
- At 330 mg/kg, none of the six pregnant females had live pups at first litter check and the post-implantation survival index was 0%. At necropsy, one or two early resorptions and/or a dead fetus were noted in the uterus of three females, and the remaining three had implantation sites only.
- At 100 mg/kg, the gestation index was reduced to 78% and post-implantation survival index was reduced to 52%. Further developmental effects consisted of reduced live litter size, reduced live birth index and reduced viability index. Most pups found dead at first litter check showed beginning autolysis with cannibalism in one of them. Noteworthy clinical findings consisted of dehydration in a surviving pup and two prematurely sacrificed pups, and a lean appearance and/or less milk in the stomach in the latter pups. Female PND 14-16 pups showed a statistically significant increase in serum T4 (36%, exceeding the normal range). T4 levels in male pups were also higher (on average 25%) but statistical significance was not achieved.
- At 30 mg/kg, the post-implantation survival index was reduced to 83% and the number of unaccounted for implantation sites was increased, which was considered an adverse finding. No treatment-related changes were noted in the other developmental parameters examined (duration of gestation, parturition, lactation index, maternal care, sex ratio, anogenital distance (PND1) and body weight).

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Remarks:
developmental toxicity
Generation:
F1
Effect level:
< 30 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Reduction in post-implantation survival (increase in number of unaccounted for implantation sites) at 30 mg/kg.

Target system / organ toxicity (F1)

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

Overall reproductive toxicity

Key result
Reproductive effects observed:
yes
Lowest effective dose / conc.:
100 mg/kg bw/day (nominal)
Treatment related:
yes
Relation to other toxic effects:
not specified
Dose response relationship:
yes
Relevant for humans:
not specified

Applicant's summary and conclusion

Conclusions:
In conclusion, treatment with JNJ-123955-AAA (T000824) by oral gavage in male and female Wistar Han revealed toxicological effects in several monitored
endpoints. Based on these results, the following No Observed Adverse Effect Levels (NOAEL) were reported:
- Parental NOAEL: 30 mg/kg, based on clinical signs of toxicity (piloerection of the fur) and inflammation of the endometrium of the uterus (related to reproductive failure) at 100 mg/kg in females.
- Reproduction NOAEL: 30 mg/kg, based on decreases in the number of implantation sites at 100 mg/kg.
- Developmental NOAEL: <30 mg/kg, based on the reduction in post-implantation survival (increase in number of unaccounted for implantation sites) at 30 mg/kg.

Therefore, the substance is classified as a reproductive toxicant (Repro, Category 2) according to the CLP Regulation.