1-benzhydrylpiperazine

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016-08-08 to 2016-12-01

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

Test guideline 4

Qualifier:

equivalent or similar to guideline

Guideline:

other: EPA OPPTS 870.3550 (Reproduction/Developmental Toxicity Screening Test)

Deviations:

no

Test guideline 5

Qualifier:

equivalent or similar to guideline

Guideline:

other: EU Method B.7 (Repeated Dose (28 days) Toxicity (oral))

Deviations:

no

Test guideline 6

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

no

Test guideline 7

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

1-benzhydrylpiperazine

EC Number:

212-667-7

EC Name:

1-benzhydrylpiperazine

Cas Number:

841-77-0

Molecular formula:

C17H20N2

IUPAC Name:

1-(DIPHENYLMETHYL)PIPERAZINE

Test material form:

solid: particulate/powder

Details on test material:

- Name of test material (as cited in study reports): JNJ-130806-AAA (T000750)
- Physical state: solid (powder)
- Appearance: white to slight beige powder

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: M15BB1451
- Expiration date of the lot/batch: 2021-01-30
- Purity test date: not specified

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability of the test substance in the solvent/vehicle: Stability of formulations for at least 6 hours at room temperature and 14 days in the refrigerator was confirmed over the concentration range 1 to 200 mg/mL during Test Facility Study No. 510921.

FORM AS APPLIED IN THE TEST (if different from that of starting material): Suspension

OTHER SPECIFICS: Correction factor: No correction was made for the purity/composition of the test item.

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

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

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: females approx. 11 weeks (at start pretest) and approx. 13 weeks (at start F0-treatment); males approx. 11 weeks (at start F0-treatment)
- Weight at study initiation: 304-347 g (males) and 205-242 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-one 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).
- Diet (e.g. ad libitum): Free access to pelleted rodent diet. During motor activity measurements, anim als 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

IN-LIFE DATES: From: 2016-09-15 (start pretest, females); 2016-09-29 (start treatment, males); 2016-11-05/06/07/08/09/18 (delivery of litters) To: 2016-10-28 (necropsy males); 2016-11-18/19/22 and 2016-12-01 (necropsy females); 2016-11-17/18/21/30 (necropsy pups)

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

1% aqueous

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily within 6 hours prior to dosing and were homogenized to a visually acceptable level. No adjustment was made for specific gravity/density of the test item, vehicle and/or formulation. Formulations were placed on a magnetic stirrer during dosing. No correction was made for the purity/composition of the test item. 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 performed at Charles River Den Bosch.
- Concentration in vehicle: 0 mg/mL (group 1); 0.6 mg/mL (group 2); 2.0 mg/mL (group 3); 6.0 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): 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, until detection of mating was confirmed.
- Proof of pregnancy: Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating was confirmed, the males and females were separated. A maximum of 14 days was allowed for mating.
- 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 during the treatment according to a validated method (Test Facility Study No. 510921). Sextuplicate samples (i.e. 3 sets of duplicate samples). Two sets of duplicate samples were stored in the refrigerator as reserve samples. On Day 5 of treatment (03 October 2016), samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability of Group 2 formulations over 5 hours at room temperature was also determined since the concentration of Group 2 was below the range over which stability was confirmed as part of the analytical method development and validation (Test Facility Study No. 510921). A new Group 4 formulation was analyzed on Day 43 of treatment (10 November 2016) since initial accuracy results were outside specifications. 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% for suspensions. Homogeneity was demonstrated if the coefficient of variation was ≤ 10.00%. Formulations were considered stable if the relative difference before and after storage was maximally 10.00%. Once analytical results were approved in the raw data by the Principal Scientist, the reserve samples were destroyed.

Duration of treatment / exposure:

29 days (males); 50-63 days (females that delivered); 42-53 days (females that failed to deliver). Routinely, females that are littering are left undisturbed. In this study, female nos. 41 and 43 (Group 1), and nos. 65, 66 and 70 (Group 3) were not dosed for one day as they were littering at the moment of dosing. The omission of one day of dosing over a period of several weeks was not considered to affect the toxicological evaluation.

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 / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Group 1 (Control)

Doses / concentrations 2

Dose / conc.:

3 mg/kg bw/day (nominal)

Remarks:

Group 2

Doses / concentrations 3

Dose / conc.:

10 mg/kg bw/day (nominal)

Remarks:

Group 3

Doses / concentrations 4

Dose / conc.:

30 mg/kg bw/day (nominal)

Remarks:

Group 4

No. of animals per sex per dose:

10 animals/sex/dose

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. 510918) in which animals were dosed for 10 days at 25 and 50 mg/kg. In summary, there were no mortalities for both dose groups. Tremors and piloerection were observed in all animals in both dose groups, while 1/3 females in the 50 mg/kg group exhibited restlessnes and all animals in the high dose group had hunched posture, were fearful and exhibited hypersensitivity to touch; additionally, one animal in the 25 mg/kg group had thickened area of the right ear and salivation. Body weight loss was observed for all animals in the 50 mg/kg group and one animal in the 25 mg/kg group. For the 50 mg/kg group, food consumption was lower than considered normal over Days 5-10 and was not determined over Days 1-5; most likely no or very low consumption of food occurred during this period. For the 25 mg/kg group, food consumption was lower than considered normal over Days 1-5, and normal over Days 5-10. Macroscopic examination of both dose groups showed no abnormalities. Liver and kidney weights were considered to be normal for both dose groups. Based on the results of this range finding study, dose levels for the main study were selected to be 3, 10 and 30 mg/kg body weight in consultation with the Sponsor. The peak effect of occurrence of relevant clinical signs at 25 mg/kg (tremors and piloerection) occurred between 1 and 3 hours after dosing. This time point was taken into account for conduct of clinical observations and functional observation tests in 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 for all animals, between 1 and 3 hours after treatment (i.e. on the anticipated peak period of effects after treatment, based on the dose range finding study. Once prior to start of treatment and at weekly intervals during the treatment period this was also performed outside the home cage in a standard arena.

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

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.

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 anesthesia using isoflurane between 7.00 and 10.30 a.m. The animals were deprived from 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 corpus cular haemoglobin, mean corpuscular haemoglobin concentration, platelets, prothombin time, activated 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

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 hindlimb grip strength recorded as the mean of three measurements per animal, locomotor activity

Oestrous cyclicity (parental animals):

Daily vaginal lavage was performed to determine the stage of estrous beginning 14 days prior to treatment (pretest), the first 14 days of treatment and during mating until evidence of copulation was observed. Vaginal lavage continued for 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 necropsy, a vaginal lavage was taken to determine the stage of estrous.

Sperm parameters (parental animals):

Parameters examined in F0 male parental generation: additional slides of the testes to examine staging of spermatogenesis; testis weight

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: No. All pups were randomized per litter and individually identified by means of subcutaneous injection of Indian ink on post-natal day 1 (= day the litter was found completed)
- Maximum of 8 pups/litter (4/sex/litter as nearly as possible) were selected for culling on PND 4; blood samples were collected from two of the surplus pups.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
- Mortality / Viability: The numbers of live and dead pups were determined on PND 1 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 of 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 14.
- 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:
Yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead if possible.

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).
- Female animals: All surviving animals, on PND 14-16 (females that delivered) or on post-coitum day 25 (females that failed to deliver, with evidence of mating).

GROSS PATHOLOGY: Yes
- 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 possibe 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 (F), Skeletal muscle (M/F), (Skin) (M/F), Spinal cord -cervical, midthoracic, lumbar (M/F), Spleen (M/ F), Sternum with bone marrow (M/F), Stomach (M/F), Testes (M), Thymus (M/F) , Thyroid including parathyroid if detectable (M/F), (Tongue) (M/F), Trachea (M/F), Urinary bladder (M/F), Uterus (F), Vagina (F), All gross lesions (M/F)
Tissues/organs mentioned in parentheses were not examined by the pathologist, since no signs of toxicity were noted at macroscopic examination.
- Samples of the following tissues and organs of all remaining animals, and females which failed 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, 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

HISTOPATHOLOGY: Yes
- All organ and tissue samples were processed, embedded and cut at a thickness of 2-4 micrometers. These slides were stained with haematoxylin and eosin. The additional slides of the testes (to 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 of Groups 1 and 4 males and of Group 1, 3 and 4 females; The additional slides of the testes of all males of Groups 1, 2, 3 and 4 and all males that failed to sire to examine staging of spermatogenesis; All gross lesions of all animals (all dose groups); Kidneys of all selected 5 animals of Group 2 (males and females) and Group 3 (males), and liver of all selected 5 males of Groups 2 and 3, based on (possible) treatment-related changes in these organs in Group 4 (males) or Group 3 (females); The reproductive organs of all animals of Group 4 (in this group, all males failed to sire and all females 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.

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 by both external and internal examination. Descriptions 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 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 WEIGHTS
not examined

Statistics:

The following statistical methods were used to analyze 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 (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
- 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.

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 paired) x 100
Gestation index (%) = (Number of females bearing live pups/Number of pregnant females) x 100
Duration of gestation = Number of days between confirmation of mating and the beginning of parturition

Offspring viability indices:

Survival indices:
Post-implantation survival index (%) = (Total number of offspring born/Total number of uterine implantation sites) x 100
Post-implantation survival index was expressed as 100% when the number of offspring exceeded the number of implantation sites recorded.
Live birth index (%) = (Number of live offspring on Day 1 after littering/Total number of offspring born) x 100
Viability index (%) = (Number of live offspring on Day 4 before culling/Number live offspring on Day 1 after littering) x 100
Lactation index (%) = Number of live offspring on Day 13 after littering/Number live offspring on Day 4 (after culling) x 100
Group mean values were calculated from individual litter values.
Sex ratio (percentage males) = (Number of males in litter/Total number of offspring in litter) x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

At 30 mg/kg, all females showed piloerection during one or more days in the post-coitum period. Two of these females (nos. 75 and 78) showed red discharge from the vagina on Day 14 or 13 of the postcoitum phase, respectively.

One female at 10 mg/kg (no. 68) showed hunched posture, abnormal gait and piloerection on four subsequent days during the first week of treatment of the premating phase and swelling of the right axillary region from the second week of the premating phase onwards. At necropsy gross examination of this animal revealed a nodule/mass (25 x 20 mm), which was confirmed histopathologically as a subcutaneous abscess. Since these findings were not recorded for the other animals of this dose group nor for animals at the highest dose group, these findings were not considered to be related to treatment.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

No mortality occurred during the study period.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 30 mg/kg, body weight gain of males was statistically significantly lower than controls throughout treatment. For most females of this dose group, weight loss (up to 9% of Day 1 premating body weights) was recorded during the first week of treatment (resulting in a statistically significantly lower mean body weight on Day 8), which recovered as treatment progressed. During the post-coitum phase, statistically significantly lower body weights (generally throughout this phase) and lower body weight gain (during the last week), was attributed to the fact that these animals did not deliver any pups.

Body weights and body weight gain of animals at 3 and 10 mg/kg remained in the same range as controls over the treatment period.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

At 30 mg/kg, food consumption before or after correction for body weight of females at 30 mg/kg was statistically significantly lower during the first week of the premating phase. This recovered during the subsequent treatment week.

The statistically significantly lower absolute food consumption and higher relative food consumption of females at 30 mg/kg over Days 14-17 and/or 17-20 of the post-coitum phase was attributed to the fact that these animals did not deliver any pups. Food consumption levels of these females were within the range expected for females that would not deliver pups.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

At 3 and 10 mg/kg, prothrombin time (PT) and activated partial thromboplastin time (APTT) of females were statistically significantly delayed compared to controls. Mean prothrombin time levels were approximately 12% and 7% higher than controls, and mean activated partial thromboplastin time levels were approximately 23% and 21% higher than controls at 3 and 10 mg/kg, respectively.

Note: No haematology data were available for females at 30 mg/kg.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

At 10 mg/kg, cholesterol of females was statistically significantly lower than controls (mean was approximately 26% lower than the control mean).

Note: No clinical biochemistry data were available for females at 30 mg/kg.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

Motor activity of females at 30 mg/kg was approximately a factor 3 higher than mean motor activity of the control group and was also distinctly higher than would be expected for non-mated females. For males at this dose level and for other female dose groups (trend), a much less pronounced and statistically non-significant higher mean motor activity was recorded. This higher motor activity was not accompanied by supportive changes in daily detailed clinical observations that would point towards an increased activity, or any other findings that would indicate that this higher motor activity had an adverse effect on functional integrity of these animals. However, given the degree of change in motor activity at 30 mg/kg and since this change could be indicative of neurotoxic potential of the test item, this was considered to be an adverse effect.

Results of hearing ability, pupillary reflex and static righting reflex that were conducted at the end of treatment were not recorded. It was considered unlikely that treatment-related changes would have occurred in these parameters since there were no signs of toxicity in related observations, such as daily detailed clinical signs (all groups), weekly arena observations (all groups), histopathological evaluation of eyes/neuronal tissues of Group 4 males and Group 3 females, and grip strength (all groups).

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):

Test item-related microscopic findings after treatment with T000750 were noted in males in the liver (starting at 10 mg/kg/day) and in the kidneys (at 30 mg/kg/day). Hepatocellular hypertrophy was present in males at 10 and 30 mg/kg up to minimal and slight degree, respectively. This was considered to be related to higher liver weights recorded at 30 mg/kg, which were approximately 20% higher than controls. Based on the absence of any additional test item-related degenerative, proliferative or inflammatory changes, the liver lesions are considered to be non-adverse. An increased incidence and severity of hyaline droplet accumulation up to marked degree was present in males at 30 mg/kg. This was considered to represent alpha2uglobulin, a normal protein in male rats which undergoes reabsorption in the proximal cortical tubules. A range of chemicals is known to increase hyaline droplet formation leading ultimately to proximal cortical tubule cell injury as manifested by formation of granular casts and increased tubular basophilia. This male rat specific protein is not present in female rats nor in higher mammals, including man. There were no additional degenerative or inflammatory changes in this study and therefore the hyaline droplet accumulation was considered non-adverse.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

effects observed, non-treatment-related

Description (incidence and severity):

Length and regularity of the estrous cycle were not considered to have been affected by treatment.

Most females had regular cycles of 4 days. Extended di-estrus occurred in one control female (no. 42) and one female at 30 mg/kg (no. 74), both with a regular cycle. One female at 10 mg/kg (no. 68) was acyclic, but did deliver a normal litter size. An irregular cycle was also noted for one female at 30 mg/kg (no. 76). Although none of the females at 30 mg/kg delivered healthy offspring, this was considered to be an incidental occurrence given that all of the other females at 30 mg/kg had a regular estrous cycle and pregnancy was confirmed for female no. 76.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

There were no histopathology findings in the male reproductive organs and spermatogenic staging profiles were normal.

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

REPRODUCTION DATA
There was a test item-related effect on the reproductive performance in all couples treated at 30 mg/kg/day which all failed to deliver healthy pups. Histopathology showed features of early pregnancy in most of these females (implantation sites in the uterus, foamy corpora lutea cells in the ovaries and slightly lobuloalveolar development in the female mammary gland) but there were no findings that could explain the lack of delivery of healthy pups. No treatment-related changes were noted in any of the reproductive parameters investigated in this study (i.e. mating and fertility indices, precoital time, estrous cycle, number of implantation sites, spermatogenic profiling, and histopathological examination of reproductive organs).

At 30 mg/kg, 2 out of 10 females were not pregnant (no implantation sites). This incidence of non-pregnant females may be encountered in this type of study, and the number of implantation sites of other females at this dose was normal. It was therefore considered that these findings were considered unrelated to treatment with the test item.

- Mating index: Mating index was not considered to be affected by treatment. All females showed evidence of mating.
- Fertility index: At 30 mg/kg, 2 out of 10 females were not pregnant resulting in a fertility index of 80% (vs. 100% in the control group). At 3 and 10 mg/kg, fertility index was not affected by treatment; all mated females were pregnant at these dose levels.
- Precoital time: Precoital time was not considered to be affected by treatment.
- Number of implantation sites: Number of implantation sites was not considered to be affected by treatment.

DEVELOPMENTAL DATA
At 30 mg/kg, none of the females delivered offspring. Since all females at this dose had a normal number of implantation sites and no fetuses were found in utero, it was considered that (early) post-implantation loss accounted for the lack in offspring at this dose. Histopathology showed features indicating that most of these females had been pregnant (implantation sites in the uterus, foamy corpora lutea cells in the ovaries and slightly lobuloalveolar development in the female mammary gland) but did not show any changes in the reproductive organs that could explain this high incidence in reproductive failure. At 10 mg/kg, body weights of male and female pups were lower than controls. Mean weights of both sexes combined were approximately 12-13% lower than controls throughout the lactation period. These lower pup body weights were not accompanied by lower parental body weights or food intake, which suggests that this is a primary developmental effect of the test item. Although this lower body weight occurred in the absence of other changes in developmental parameters, this change was considered to represent an adverse effect on pup development. No treatment-related changes were noted in any of the other developmental parameters investigated in this study (i.e. viability and lactation indices, duration of gestation, parturition, sex ratio, maternal care and early postnatal pup development consisting of mortality, clinical signs, anogenital distance (PND 1), areola/nipple retention (PND 13 males), T4 thyroid hormone levels (PND 13-15) and macroscopy.
- Gestation index and duration: At 30 mg/kg, none of the females delivered offspring, resulting in a gestation index of 0% (vs. 100% in the control group). Of the eight pregnant females, all had implantation sites only. Gestation duration up to 10 mg/kg was not considered affected by treatment.
- Parturition/maternal care: No signs of difficult or prolonged parturition were noted among the pregnant females. Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No deficiencies in maternal care were observed.
- Post-implantation survival index: At 30 mg/kg, no offspring was born, resulting in a post-implantation survival index of 0% (vs. 85% in the control group). At 3 and 10 mg/kg, the total number of offspring born compared to the total number of uterine implantations was not considered to be affected by treatment.
- Live birth index: At 3 and 10 mg/kg, the number of live offspring on Day 1 after littering compared to the total number of offspring born was not affected by treatment at 3 and 10 mg/kg. At 30 mg/kg, no offspring was born. A total of four pups at 10 mg/kg (one from litter no. 61 and three from litter no. 67) were found dead on Day 1. No toxicological relevance was attributed to these dead pups since the mortality incidence remained within the range considered normal for pups of this age.
- Viability index: The number of live offspring on Day 4 before culling compared to the number of offspring on Day 1 was not considered affected by treatment at 3 and 10 mg/kg. At 30 mg/kg, no offspring was born. Three pups of the control group (two from litter no. 43, and one from litter no. 44), two pups at 3 mg/kg (one each from litter nos. 56 and 59) and one pup at 10 mg/kg (litter no. 63) were found dead or missing. 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.
- Lactation index: The number of live offspring on Day 13 after littering compared to the number of live offspring on Day 4 (after culling) was not affected by treatment at 3 and 10 mg/kg. No pups were found dead/missing between lactation Days 5 and 13. At 30 mg/kg, no offspring was born.

Details on results (P0)

Parental results:
At 30 mg/kg, two females showed red discharge from the vagina on a single day of the post-coitum phase, and all females showed piloerection during one or more days in the post-coitum period. It is conceivable that these findings relate to the observation that none of the females at this dose delivered offspring (two were not pregnant and eight had implantation sites only). For most females of this dose group, weight loss (up to 9% of Day 1 premating body weights) along with lower food intake was recorded during the first week of treatment, which recovered as treatment progressed. For males at 30 mg/kg, body weight gain was slightly lower throughout treatment. Food intake levels however remained similar to control levels throughout treatment for both sexes. Motor activity of females at 30 mg/kg was approximately a factor 3 higher than mean motor activity of the control group and was also distinctly higher than would be expected for non-mated females. For males at this dose level and for other female dose groups (trend), a much less pronounced and statistically non-significant higher mean motor activity was recorded. This higher motor activity was not accompanied by supportive changes in daily detailed clinical observations that would point towards an increased activity, or any other findings that would indicate that this higher motor activity had an adverse effect on functional integrity of these animals. However, given the degree of change in motor activity at 30 mg/kg and since this change could be indicative of neurotoxic potential of the test item, this was considered to be an adverse effect. Results of hearing ability, pupillary reflex and static righting reflex that were conducted at the end of treatment were not recorded. It was considered unlikely that treatment-related changes would have occurred in these parameters since there were no signs of toxicity in related observations, such as daily detailed clinical signs (all groups), weekly arena observations (all groups), histopathological evaluation of eyes/neuronal tissues of Group 4 males and Group 3 females, and grip strength (all groups). Changes in blood parameters were confined to a delayed prothrombin time and activated partial thromboplastin time in females at 3 and 10 mg/kg, and lower cholesterol in females at 10 mg/kg. Means of these parameters remained in the range considered normal for rats of this age and strain, did not show a dose-related trend and occurred in the absence of supportive morphological lesions. As such, they were not considered to represent adverse changes. Hepatocellular hypertrophy was present in males at 30 mg/kg up to slight degree. This was considered to be related to higher liver weights recorded at 30 mg/kg, which were approximately 20% higher than controls. Based on the absence of any additional test item-related degenerative, proliferative or inflammatory changes, the liver lesions are considered to be non-adverse.

Reproductive results:
No treatment-related changes were noted in any of the reproductive parameters investigated in this study (i.e. mating and fertility indices, precoital time, estrous cycle, number of implantation sites, spermatogenic profiling, and histopathological examination of reproductive organs). At 30 mg/kg, 2 out of 10 females were not pregnant (no implantation sites). This incidence of non-pregnant females may be encountered in this type of study, and the number of implantation sites of other females at this dose was normal. It was therefore considered that these findings were considered unrelated to treatment with the test item.

Effect levels (P0)

1

Key result

Dose descriptor:

NOAEL

Remarks:

Parental toxicity

Effect level:

10 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: behaviour (functional findings)

Remarks on result:

other:

Remarks:

Based on higher motor activity in females at 30 mg/kg. Motor activity of females at 30 mg/kg was approximately a factor 3 higher than mean motor activity of the control group and was also distinctly higher than would be expected for nonmated females. For males at this dose level and for other female dose groups (trend), a much less pronounced and statistically non-significant higher mean motor activity was recorded.

2

Key result

Dose descriptor:

NOAEL

Remarks:

Reproduction

Effect level:

>= 30 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive performance

Target system / organ toxicity (P0)

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

30 mg/kg bw/day (nominal)

System:

central nervous system

Organ:

other: motor activity

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical signs occurred among pups that were considered to be related to treatment.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

The number of live offspring on Day 4 before culling compared to the number of offspring on Day 1 was not considered affected by treatment at 3 and 10 mg/kg. At 30 mg/kg, no offspring was born. Three pups of the control group (two from litter no. 43, and one from litter no. 44), two pups at 3 mg/kg (one each from litter nos. 56 and 59) and one pup at 10 mg/kg (litter no. 63) were found dead or missing. 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.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 10 mg/kg, body weights of male and female pups were lower than controls. Mean weights of both sexes combined were approximately 12-13% lower than controls throughout the lactation period. These lower pup body weights were not accompanied by lower parental body weights or food intake, which suggests that this is a primary developmental effect of the test item. Although this lower body weigh occurred in the absence of other changes in developmental parameters, this change was considered to represent an adverse effect on pup development.

At 3 mg/kg, pup body weights were not considered to be affected by treatment.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

effects observed, treatment-related

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 not considered to be affected by treatment.

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

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

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 considered to be affected by treatment at 3 and 10 mg/kg.

Areola/nipple retention: Treatment up to 10 mg/kg had no effect on areola/nipple retention. For none of the examined male pups nipples were observed at PND 13.

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Details on results (F1)

Developmental results:
At 30 mg/kg, none of the females delivered offspring. Since all females at this dose had a normal number of implantation sites and no fetuses were found in utero, it was considered that (early) post-implantation loss accounted for the lack in offspring at this dose. Histopathology showed features indicating that most of these females had been pregnant (implantation sites in the uterus, foamy corpora lutea cells in the ovaries and slightly lobuloalveolar development in the female mammary gland) but did not show any changes in the reproductive organs that could explain this high incidence in reproductive failure. At 10 mg/kg, body weights of male and female pups were lower than controls. Mean weights of both sexes combined were approximately 12-13% lower than controls throughout the lactation period. These lower pup body weights were not accompanied by lower parental body weights or food intake, which suggests that this is a primary developmental effect of the test item. Although this lower body weigh occurred in the absence of other changes in developmental parameters, this change was considered to represent an adverse effect on pup development. No treatment-related changes were noted in any of the other developmental parameters investigated in this study (i.e. viability and lactation indices, duration of gestation, parturition, sex ratio, maternal care and early postnatal pup development consisting of mortality, clinical signs, anogenital distance (PND 1), areola/nipple retention (PND 13 males), T4 thyroid hormone levels (PND 13-15) and macroscopy.

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Remarks:

developmental toxicity

Generation:

F1

Effect level:

3 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Remarks on result:

other:

Remarks:

Note that at 30 mg/kg, no offspring was born.

Target system / organ toxicity (F1)

Critical effects observed:

no

Overall reproductive toxicity

Reproductive effects observed:

yes

Lowest effective dose / conc.:

10 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-130806-AAA (T000750) by oral gavage in male and female Wistar Han rats at dose levels of 3, 10 and 30 mg/kg resulted in adverse changes in motor activity at 30 mg/kg. Motor activity of females at 30 mg/kg was approximately a factor 3 higher than mean motor activity of the control group and was also distinctly higher than would be expected for nonmated females. For males at this dose level and for other female dose groups (trend), a much less pronounced and statistically non-significant higher mean motor activity was recorded. No treatment-related changes were noted in any of the reproductive parameters investigated in this study (i.e. mating and fertility indices, precoital time, estrous cycle, number of implantation sites, spermatogenic profiling, and histopathological examination of reproductive organs). At 10 mg/kg, body weights of male and female pups were lower than controls. Mean weights of both sexes combined were approximately 12-13% lower than controls throughout the lactation period. These lower pup body weights were not accompanied by lower parental body weights or food intake, which suggests that this is a primary developmental effect of the test item. Although this lower body weight occurred in the absence of other changes in developmental parameters, this change was considered to represent an adverse effect on pup development.

Based on these results, the following No Observed Adverse Effect Levels (NOAELs) were derived:
Parental NOAEL: 10.0 mg/kg
Reproduction NOAEL: at least 30 mg/kg
Developmental NOAEL: 3.0 mg/kg

Therefore, the substance is classified as a Reproductive Toxicant-Category 2 according to the CLP Regulation.