(4aR*, 8aR*)-1-bromo-4a,5,9,10,11,12-hexahydro-3-methoxy-6H-benzofuro[3a,3,2-ef] [2]benzazepin-6-one hydrochloride (1:1)

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015-09-04 to 2015-11-16

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

1. On 2015-11-07, all remaining females of groups 2, 3 and 4 were treated by error with the formulations prepared with a different test item for another OECD422 study. This error was made during the second week of lactations, excluding any effects on reproduction, prenatal or early post-natal pup development. Moreover, the other test item did not indicate any developmental effect.
2. The weighing of food residues on observation day 29 was not performed. However, since no treatment-related changes were observed in the first 3 weeks of observation and no effects on body weights were observed in the complete observation period of 4 weeks, it is considered very unlikely that any treatment-related effects were missed.
3. No vaginal lavage was available for estrous cycle determination for female no. 58 (group 2) on 2015-10-08, however this was not needed since she delivered a healthy litter.

Deviations:

yes

Remarks:

See "Remarks"

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

rel-(4aR,8aR)-1-bromo-3-methoxy-4a,5,9,10,11,12-hexahydro-6H-[1]benzofuro[3a,3,2-ef][2]benzazepin-6-one hydrochloride (1:1)

EC Number:

700-867-1

Molecular formula:

C16H16BrNO3.HCl

IUPAC Name:

rel-(4aR,8aR)-1-bromo-3-methoxy-4a,5,9,10,11,12-hexahydro-6H-[1]benzofuro[3a,3,2-ef][2]benzazepin-6-one hydrochloride (1:1)

Test material form:

solid

Details on test material:

- Name of test material (as cited in study reports): T002102
- Physical state: solid
- Appearance: white (SDS)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: M15CB165
- Expiration date of the lot/batch: 2016-03-15 (retest date)
- Purity test date: 2015-04-12

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature
- Stability in vehicle: stability of formulations over 6 hours at room temperature under normal laboratory light conditions (concentration range 1-200 mg/mL) was confirmed as part of the analytical method development and validation study (Project 508955).
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: no data

FORM AS APPLIED IN THE TEST (if different from that of starting material): suspension (groups 2, 3 and 4)

OTHER SPECIFICS: correction factor of 1.10 was used.

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/develomental 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 Laboratories France, L'Arbresle Cedex, France
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation (start F0 treatment): females approximately 12-13 weeks; males approx. 10-11 weeks
- Weight at study initiation: 282-353 grams (males), 200-259 grams (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);
Lactation: pups were kept with the dam until termination in Macrolon plastic cages (MIII type, height 18 cm); general: sterilised sawdust as bedding material (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) and paper as cage-enrichment/nesting material (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) were supplied.
- Diet (e.g. ad libitum): ad libitum, free access to pelleted rodent diet
- Water (e.g. ad libitum): ad libitum, free access to tap-water
- Acclimation period: at least 5 days prior to start of pretest (females) or treatment (males), health inspection upon receipt of the animals

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-24°C
- Humidity (%): 40-70%
- Air changes (per hr): at least 10 room air changes/hour
- Photoperiod (hrs dark / hrs light): 12-hour light/12-hour dark cycle

IN-LIFE DATES:
From: 2015-09-04 (start pretest, females); 2015-09-18 (start treatment, males); 2015-10-25 through 2015-10-29 and 2015-11-1 (delivery of litters)
To: 2015-10-19 and 2015-10-21 (necropsy males), 2015-11-9 through 2015-11-12 and 2015-11-16 (necropsy females), 2015-11-6/9/11/13 (necropsy pups)

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Remarks:

specific gravity 1.036

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- Formulations (w/w) were prepared daily within 6 hours prior to dosing and were homogenized to a visually acceptable level. Adjustment was made for specific gravity of the vehicle (1.036). A correction was made for the purity/composition of the test item. A correction factor of 1.10 was used to correct for purity and salt/base conversion.
- 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: 2 mg/mL (10 mg eq/kg bw/day), 10 mg/mL (50 mg eq/kg bw/day) and 30 mg/mL (150 mg eq/kg bw/day)
- Amount of vehicle (if gavage): 5 mL/kg 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, 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 seperated.
- At the end of the first mating period of 14 days, female nos. 49 (group 1), 58 (group 2) and 62, 65 (group 3) had not shown evidence of mating. They were therefore remated with a male of proven fertility of the same group from 2015-10-16 onwards. Female no. 65 showed evidence of mating on 2015-10-18. The remaining females (49, 58, 62) were examined by palpation on 2015-10-19 and confirmed to be pregnant (i.e. mating was overlooked). Their mating dates were estimated at 21 days prior to the actual delivery dates. This day was designated day 0 post-coitum.
- 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:

Chemical analyses of dose preparations were conducted on a single occasion during the first treatment week (21 September 2015), according to a validated method (Project 508955). Sextuplicate samples (i.e. 3 sets of duplicate samples) were collected. Two sets of duplicate samples were stored as reserve samples. Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Once analytical results were approved in the raw data by the Principal Scientist, the reserve samples were destroyed.
In addition to the criteria mentioned in the validated analytical method, each calibration curve was accepted if the average of the retention times and response factors of the data points used to construct the calibration line were within a range of ±10% compared to those obtained during the method validation.
The accuracy of preparation was considered acceptable if the mean measured concentrations were 85-115% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%.
Stability of the test item under test conditions was determined as part of the method validation study (Project 508955).

Duration of treatment / exposure:

31-33 days (males), 52-59 days (females that delivered), 42 days (females that failed to deliver except for female no. 79 that was exposed for 52 days)
Female nos. 41 (Group 1), 50, 51, 53 and 58 (Group 2), 61 and 70 (Group 3) were not dosed on Day 1 of lactation as these females were littering at the time of dosing. The omission of one day of dosing over a period of several weeks was not considered to affect the toxicological evaluation.

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 (actual dose received)

Remarks:

group 1 (control group)

Doses / concentrations 2

Dose / conc.:

11 mg/kg bw/day (actual dose received)

Remarks:

10 mg eq/kg bw/day, group 2

Doses / concentrations 3

Dose / conc.:

55 mg/kg bw/day (actual dose received)

Remarks:

50 mg eq/kg bw/day, group 3

Doses / concentrations 4

Dose / conc.:

165 mg/kg bw/day (actual dose received)

Remarks:

150 mg eq/kg bw/day, 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 based on the results of an acute oral toxicity study in the rat (LD50 was 500 mg eq/kg; data on file at Sponsor) and a 28-day repeated toxicity study (NOAEL was 50 mg eq/kg/day; data on file at Sponsor). Dose levels of 10, 50 and 150 mg eq/kg/day (or 11, 55, 165 mg/kg bw/day) for the current reproductive screening study were selected in consultation with the Sponsor.
- Rationale for animal assignment (if not random): randomized

Positive control:

no

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS AND DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily from treatment onwards up to the day prior to necropsy, detailed clinical observations were made in all animals.
- The time of onset, grade and duration of any observed sign was recorded. Signs were graded for severity and the maximum grade was predefined at 3 or 4.

BODY WEIGHT: Yes
- Time schedule for examinations: Males and females were weighed on the first day of exposure 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.

FOOD CONSUMPTION: Yes
- Time schedule: weekly during treatment, 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.
- Both absolute food consumption and food consumtion relative to body weight were reported.

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

MORTALITY/VIABILITY:
- Time schedule: at least twice daily (early in the morning and close to the end of the working day).
- The circumstance of any death was recorded in detail.

GENERAL REPORDUCTION DATA:
- Male number paired with, mating date, confirmation of pregnancy, and delivery day were recorded. Palpation was used to aid in confirmation of pregnancy.
- Pregnant females were examined to detect signs of difficult or prolonged parturition.
- Any deficiencies in maternal care (such as inadequate construction or cleaning of the nest, pups left scattered and cold, physical abuse of pups or apparently inadequate lactation or feeding) were examined.

CLINICAL LABORATORY INVESTIGATIONS:
- F1-generation, PND 4 pups: from 2 pups per litter at culling. If there were insufficient pups in a litter to have two surplus pups, two of the pups that were typically retained were used for blood collection. When the litter size dropped below the culling target of eight pups/litter, preference was given to remove two female pups for PND 4 blood samples in order to retain more male pups for nipple retention on PND 13; however, retained female pups in each litter should not drop below 2. Blood samples were collected by decapitation, between 7.00 and 10.30 a.m. Blood samples from the 2 pups per litter (0.4 mL in total) were collected into one serum tube for possible future measurement of thyroxine (T4). After clotting and centrifugation, serum samples were stored at ≤-75°C. Under these storage conditions, the samples were stable for 6 months. Any samples remaining after 6 months will be discarded.
 - F1-generation, PND 13-15 pups: from 2 pups per litter (if possible from one male and one female) at planned necropsy. As part of the necropsy procedure, blood samples (0.9 mL) were drawn by aorta puncture under anaesthesia using isoflurane, between 7.00 and 10.30 a.m. Blood was collected into serum tubes. After clotting and centrifugation, serum from each sample was divided into 2 aliquots: 150 µL serum for measurement of thyroxine (T4) and the remaining volume of serum for possible future measurement of thyroid-stimulating hormone (TSH).
- F0-generation, males and females: End of study (Day 14-16 of lactation for females and after at least 4 weeks of treatment for males) from all animals at planned necropsy. Blood samples were collected 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(0.9 mL) were drawn from the retro-orbital sinus and collected into serum tubes. After clotting and centrifugation, serum was used as listed below: males: 1 aliquot of 150 µL serum was used for measurement of thyroxine (T4), and the remaining volume of serum was stored for possible future measurement of thyroid-stimulating hormone (TSH); females: the serum was stored for possible future measurement of thyroxine (T4) and/or thyroid-stimulating hormone (TSH).

Oestrous cyclicity (parental animals):

Daily vaginal lavage was performed to determine the stage of estrus beginning 14 days prior to treatment (pretest), the first 14 days of treatment and during mating until evidence of copulation was observed. Vaginal lavage was continued for those females with no evidence of copulation until termination of the mating period. During pretest, this was done for 48 females. At the end of the pretest phase, 40 females with at least two complete regular estrous cycles were selected at random and further used in the study. The remaining females were removed from the study, and estrous cycle results were not reported but kept in the raw data. On the day of necropsy, a vaginal lavage was taken to determine the stage of estrus.

Sperm parameters (parental animals):

Parameters examined in male parental generation (P0): testis weight, epididymis weight, and additional slides of the testes to examine staging of spermatogenesis.

Litter observations:

STANDARDISATION OF LITTERS
- 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. If there were insufficient pups in a litter to have two surplus pups, two of the pups, that were typically retained, were used for blood collection. When the litter size dropped below the culling target of eight pups/litter, preference was given to remove two female pups for PND 4 blood samples in order to retain more male pups for nipple retention on PND 13; however, retained female pups in each litter did not drop below 2.

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.
- 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:
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.

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY:
- not examined

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY:
- not examined

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals, following completion of the mating period (a minimum of 28 days of dose adminitration)
- Female animals, which delivered: PND 15-16
- Female animals, which failed to deliver (nos. 42, 45, 64, 66, 67 and all group 4 females): post-coitum days 25-27 (females with evidence of mating)
- Female animal no. 65 with suspected total litter loss: within 24 hours of suspected litter loss.
- Female no. 69 which was found dead on day 15 post-coitum: as soon as possible after death and within 24 hours
- The animals were deprived of food overnight (with a maximum of 24 hours) before blood sampling and necropsy, but water was available.
- All animals surviving to the end of the observation period were deeply anaesthetized using isoflurane and subsequently exsanguinated.

GROSS NECROPSY
- After sacrifice or death, all animals were subjected to a limited necropsy, with special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities were recorded. The number of implantation sites were recorded for all paired females.
- For animals found dead, necropsy was performed as soon as possible after death and within 24 hours.
- Samples of the following tissues and organs were collected and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution): cervix (F), clitoral gland (F), coagulation gland (M), (Cowper’s glands) (M), epididymides (M), mammary gland area (M/F), (Glans penis) (M), (Levator ani plus bulbocavernosus muscle complex (LABC)) (M), ovaries (F), preputial gland (M), pituitary gland (M/F), prostate gland (M), seminal vesicles (M), testes (M), thyroid including parathyroid if detectable (M/F), uterus (F), vagina (F), all gross lesions (M/F), identification marks: not processed (M/F). Tissues/organs mentioned in parentheses needed only be examined by the pathologist if changes in macroscopic appearance were indicative of (potential) toxicity.

ORGAN WEIGHTS
- The following fresh organ weights and terminal body weight were recorded from the surviving males on the scheduled day of necropsy: Cowper’s glands, epididymides, glans penis, levator ani plus bulbocavernosus muscle complex (LABC), testes, thyroid.
- Absolute organ weights and organ to body weight ratios were reported.
- Determination of thyroid organ weight for F0-females was not included in the original study plan, but added later for all F0-females that survived until scheduled necropsy. Since necropsies of these females were already completed, thyroid organ weight was determined after fixation in 10% buffered formalin. Only absolute organ weights were reported as no terminal body weight was determined for F0-females (not required by study plan).

HISTOPATHOLOGY
- All organ and tissue samples were processed, embedded and cut at a thickness of 2-4 micrometers. These slides were stained with haematoxylin and eosin. The additional slides of the testes (to examine staging of spermatogenesis) were stained with PAS/haematoxylin.
- The following slides were examined by a pathologist: ovaries, testes, epididymides and thyroid gland of all animals of Groups 1 and 4; testes, epididymides and thyroid gland of all males of Groups 2 and 3, based on (possible) treatment-related changes in these organs in Group 4; the additional slides of the testes of the males of all groups; the preserved organs and tissues of animal no. 69 which died spontaneously; all gross lesions of all animals (all dose groups); the reproductive organs of all males that failed to sire and all females that failed to deliver healthy pups.
- 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
- The F1 offspring were sacrificed at PND4 or PND 7-15.
- Pups, younger than 7 days were killed by decapitation. All remaining pups (PND 7-15) were sacrificed using Euthasol®20% by intraperitoneal (ip) injection.

GROSS NECROPSY
- Necropsy was conducted on the following days: culling: PND 4; terminal sacrifice: PND 13-15; spontaneous deths: as soon as possible after death and always within 24 hours.
- All pups were sexed by both external as well as internal examination. Descriptions of all external abnormalities were recorded.
- At terminal sacrifice (PND 13-15), the thyroid from 2 pups, 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 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.

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:

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

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No clinical signs of toxicity were noted up to 165 mg/kg/day.
Slight salivation occurred after dosing in males at all dose levels and in females at 55 and 165 mg/kg/day, generally starting after two or three weeks of treatment. The incidence increased dose-dependently. This salivation was considered to be a physiological response rather than a sign of systemic toxicity considering the nature and minor severity of the effect and its time of occurrence (i.e. after dosing).
Incidental findings that were noted included alopecia, scabbing or scales on different parts of the body, piloerection and rales. These findings occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study. At the incidence observed, these were considered signs of no toxicological relevance.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Two females in Group 3 (55 mg/kg bw/day) did not survive until scheduled necropsy. Neither of these two deaths was considered to be test item-related.
Female no. 69 was found dead on Day 15 post-coitum. This female showed no clinical signs of toxicity or abnormalities in body weight or food consumption. At necropsy, next to beginning autolysis watery-clear fluid was noted in the thoracic cavity. This latter observation could be indicative for an oral gavage accident. No cause of death could be established from the (limited number of) tissues examined microscopically. As no high dose animals died preterm the death of female no. 69 was considered not to be related to treatment with the test item.
Female no. 65 was sacrificed on Day 20 post-coitum because she was suspected to have a premature delivery. Blood was found in the vaginal smear prepared for estrous stage determination. Clinical observations, body weight and food consumption of this female did not indicate any signs of toxicity. At necropsy, she was noted with two normal fetuses in one uterus horn and no empty implantation sites. Based on this, it can be concluded that she had no premature delivery. Consequently, her premature sacrifice was not test item-related

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Body weights and body weight gain of treated males remained in the same range as controls over the treatment period.
In females, no toxicologically relevant changes in body weights and body weight gain were noted. It should be noted that all high-dose females (165 mg/kg/day) were non-pregnant. Consequently, body weight during gestation and lactation could not be evaluated at 165 mg/kg/day.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related changes in food consumption (absolute and relative to body weight) in treated males and females were noted.
It should be noted that all high-dose females (165 mg/kg/day) were non-pregnant. Consequently, food consumption during gestation and lactation could not be evaluated at 165 mg/kg/day.
The statistically significantly higher absolute food consumption noted in low-dose females between Days 0-4 post-coitum was considered incidental and most likely caused by a relatively low value for the control group.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

The serum concentration of the thyroid hormone thyroxine (total T4), measured in males at scheduled sacrifice, was not affected by treatment up to 150 mg eq/kg/day.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

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 testes lesions in males treated at 55 and 165 mg/kg/day consisted of:
- Abnormal stages present in 10 out of 10 males treated at 165 mg/kg/day.
- A dose- related increase in incidence and severity of atypical residual bodies up to an incidence of 10 out of 10 and severities of moderate degrees.
- A dose- related increase in incidence of retention of spermatids (step 19) / residual bodies up to an incidence of 10 out of 10.
- A dose- related increase in incidence of minimal degeneration of round spermatids up to an incidence of 10 out of 10.
- Additional tubular vacuolation at minimal degree in 3 out of /10 males at 165 mg/kg/day.

Test item-related epididymides lesions consisted of minimal to moderate degrees of luminal cell debris in 10 out of /10 males at 165 mg/kg/day.

The combination of these findings was considered to be adverse. This was based on the fact that all males were affected, severities (except the vacuolation) went up to moderate degrees, spermatogenic profiles were abnormal and all these males did not have produce offspring (while their females did not have test item-related findings in the reproductive organs).
The abnormal maturation of spermatids (noted as atypical residual bodies and retention of spermatids (step 19) / residual bodies) and degeneration of round spermatids starting at the meiotic phase, were regarded to have resulted in altered sperm quality and therefore related to lack of offspring and lower weight of the epididymides weights.

Test item-related thyroid gland lesions were seen in males only and consisted of a dose-related increased incidence of follicular cell hypertrophy (mostly at minimal degree, one at slight degree).
There were no test item-related adverse microscopic findings in the testes and epididymides of males at 11 mg/kg/day and ovaries and thyroid gland of females of the control group and at 165 mg/kg/day.

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.

Couples without offspring:
- Control Group 1: no abnormalities were seen in the reproductive organs which could account for the lack of offspring in two couples of the control group (female/male no. 42/02 and 45/05).
- Group 2 (11 mg/kg/day): all couples of this group had healthy offspring.
- Group 3 (55 mg/kg/day): in the 55 mg/kg/day group, pregnancy (late phase) was proven in female no. 64 by large implantation sites. Female no. 69 (found dead on Day 15 post-coïtum) showed early pregnancy, characterized by the detection of implantation sites noted at necropsy and histologic appearance of ovaries, uterus, vagina (mucification) and mammary gland (lobulo-alveolar development). No cause for the failure to sire or deliver healthy offspring for male no. 25, female no. 67 and the couple with male no 26 and female no. 66 treated at 55 mg/kg/day could be established from the sections examined. Spermatogenic staging profiles were normal for all males examined and the recorded test item-related findings in the testes of two males of the 55 mg/kg/day group (nos. 24 and 26) were present at only minimal severities.
- Group 4 (165 mg/kg/day): all couples treated at 165 mg/kg/day failed to deliver offspring despite proof of mating. A possible relation with test item-related findings observed in testes and epididymides was suspected.

Histopathological findings: neoplastic:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

effects observed, non-treatment-related

Description (incidence and severity):

Length and normality of the estrous cycle were not affected by treatment. Most females had regular cycles of 4 days. Extended di-estrus occurred in one or two females of each group, including the three females for which mating was overlooked, and irregular cycles occurred in three animals of the low-dose group (11 mg/kg/day) which all had normal litters. The incidence of these findings did not indicate a relation with treatment.

Reproductive function: sperm measures:

effects observed, treatment-related

Description (incidence and severity):

Test item-related microscopic findings were present in the testes and epididymides of all 10 males treated at 165 mg/kg/day.
Findings in the testes consisted of:
- Abnormal spermatogenic profiles, noted as abnormal stage.
- Atypical residual bodies up to moderate degrees.
- The presence of retention of spermatids (step 19) / residual bodies.
- Degeneration of round spermatids at minimal degrees.
Additional tubular vacuolation was observed in 3 out of 10 males at minimal degree. Findings in the epididymides consisted of:
- The presence of luminal cell debris up to moderate degree.

The combination of these findings was considered to be adverse. This was based on the fact that all males were affected, severities (except the vacuolation) went up to moderate degrees, spermatogenic profiles were abnormal and all males did not produce offspring (while their females did not have test item-related findings in the reproductive organs).

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

REPRODUCTION DATA:
- There were no pregnant females in the 165 mg/kg/day group. Consequently, the fertility and conception indices were reduced to zero at this high dose. This adverse reproductive outcome was attributed to the adverse effect of the test item on male fertility.
- The fertility and conception indices were unaffected by treatment up to 55 mg/kg/day.

- At 55 mg/kg/day, a higher incidence of females with low numbers of implantation sites was observed: 4 out of 8 pregnant females (nos. 62, 64, 65 and 69) had only 4, 3, 2 and 5 implantation sites, respectively. For comparison, in the concurrent control group there were 2 out of 8 females (nos. 46 and 49) with 4 and 5 implantation sites only. Mean numbers of implantation sites were 11.5, 12.2 and 8.5 for respectively the control, 11 and 55 mg/kg/day group.

- Precoital time and mating index were unaffected by treatment up to 165 mg/kg/day.

- For females no. 48, 49 (control) and 52 (11 mg/kg/day), the number of pups born was slightly higher than the number of implantations. This was considered to be due to the normal resorption of these areas as these enumerations were performed on Day 13 or 15 of lactation.

DEVELOPMENTAL DATA:
- Note: No developmental data were available for the 165 mg/kg/day group as none of the high dose females became pregnant.
- A lower index for gestation and post-implantation loss was noted at 55 mg/kg/day.
- No toxicologically relevant effects on the duration of gestation, parturition, maternal care and early postnatal pup development (postnatal endpoints evaluated are given below) were observed at 11 and 55 mg/kg/day.
- The gestation index was reduced to zero at 165 mg/kg/day (no pregnant females at this dose level).
- At 55 mg/kg/day, the gestation index was 83% versus 100% in the control and 11 mg/kg/day groups. This lower gestation index at 55 mg/kg/day was caused by one female (no. 64) which had been pregnant, but did not deliver any pups. On Day 24 post-coitum, she was noted with some blood in her cage, and at necropsy on Day 26 post-coitum, three empty implantation sites were found in her uterus. Based on the size of the implantation sites and the morphology of her uterus and vagina, it was concluded that she had lost her fetuses at a late stage in pregnancy. This is a very unusual finding and therefore a possible relation to treatment with the test item could not be excluded.
- The duration of gestation was not affected by treatment up to 55 mg/kg/day.
- No signs of difficult or prolonged parturition were noted among the pregnant females.
- No deficiencies in maternal care were observed.
- At 55 mg/kg/day, the post-implantation survival index was 82% versus 96% and 91% in the control and 11 mg/kg/day groups, respectively. This lower post-implantation survival index was mainly caused by the female that lost her three fetuses late during pregnancy. A possible relation to treatment with the test item could not be excluded.
- Postnatal pup develop could not be evaluated at 165 mg/kg/day because no litters were born at this dose level. At dose levels of 11 and 55 mg/kg/day, no treatment-related changes were noted in the live birth, viability and lactation indices, sex ratio, anogenital distance, areola/nipple retention, clinical signs, body weights, external macroscopy and serum concentration of the thyroid hormone T4 (PND 13-15).

Details on results (P0)

Analysis of dose preparations:
- accuracy of preparation: the concentrations analysed in the formulations of groups 2, 3 and 4 were in agreement with target concentrations (i.e. mean accuracies between 85% and 115%). No test item was detected in the group 1 formulaiton.
- homogeneity: the formulations of groups 2 and 4 were homogeneous (i.e. coefficient of variation <=10%)

Effect levels (P0)

1

Key result

Dose descriptor:

NOAEL

Remarks:

parental toxicity

Effect level:

>= 165 mg/kg bw/day (actual dose received)

Based on:

test mat.

Remarks:

150 mg eq/kg bw/day

Sex:

female

Remarks on result:

other: No adverse effects were noted in the examined parameters in the study

2

Key result

Dose descriptor:

NOAEL

Remarks:

parental toxicity

Effect level:

55 mg/kg bw/day (actual dose received)

Based on:

test mat.

Remarks:

50 mg eq/kg bw/day

Sex:

male

Basis for effect level:

organ weights and organ / body weight ratios

histopathology: non-neoplastic

reproductive function (sperm measures)

3

Key result

Dose descriptor:

NOAEL

Remarks:

reproduction toxicity

Effect level:

11 mg/kg bw/day (nominal)

Based on:

test mat.

Remarks:

10 mg eq/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

reproductive performance

Target system / organ toxicity (P0)

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

165 mg/kg bw/day (actual dose received)

System:

male reproductive system

Organ:

cauda epididymis

Sertoli cells

testes

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):

The control pup that went missing on PND 2 showed no clinical signs, and the Group 3 pup that was found dead on PND 3 had no milk in the stomach at the first litter check. Clinical signs in surviving pups were limited to general alopecia noted for several pups in two litters in the 11 mg/kg/day group. This was considered unrelated to treatment as no clinical signs were noted at the higher dose of 55 mg/kg/day and the nature and incidence of these clinical signs remained within the range considered normal for pups of this age.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

There were no dead pups at the first litter check. During lactation, one pup of the control group went missing on PND 2 and one pup at 55 mg/kg/day was found dead on PND 3. The missing pup was most likely cannibalised. This incidental mortality was not related to treatment.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

At 55 mg/kg/day combined body weights on PND 13 (male and female pups) were slightly (about 10%) lower compared to controls. The differences from controls were not statistically significant and values were considered to be within the normal range of biological variation.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

The serum level of the thyroid hormone T4 in male and female pups was not affected by treatment.
In the absence of a dose-related response, the statistically significantly lower T4 value noted in female pups of the low-dose group (11 mg/kg/day) was not attributed to treatment.

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Macroscopic findings were limited to absence of milk in the stomach of the Group 3 pup that died during lactation (PND 3). No toxicological relevance was attached to this isolated finding.

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Sex ratio: Sex ratio was not affected by treatment.
Anogenital distance: Anogenital distance in male and female pups was not affected by treatment.
Areola/nipple retention: Treatment up to 55 mg/kg/day had no effect on areola/nipple retention. The number of nipples in male pups at PND 13 was zero, except in one pup at 11 mg/kg/day (3 nipples were noted for male pup 1 in litter 54). This isolated observation in the low dose group was considered a chance finding and thus not related to treatment.

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Remarks:

developmental toxicity

Generation:

F1

Effect level:

11 mg/kg bw/day (actual dose received)

Based on:

test mat.

Remarks:

10 mg eq/kg bw/day

Sex:

male/female

Basis for effect level:

other: lower gestation and post implantation index

Target system / organ toxicity (F1)

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

55 mg/kg bw/day (nominal)

System:

other: not specified

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.:

11 mg/kg bw/day (actual dose received)

Treatment related:

yes

Relation to other toxic effects:

reproductive effects in the absence of other toxic effects

Dose response relationship:

yes

Relevant for humans:

not specified

Applicant's summary and conclusion

Conclusions:

In conclusion, treatment with JNJ-16520556-AAC (T002102) by oral gavage in male and female Wistar Han rats at dose levels of 11, 55 and 165 mg/kg/day (10, 50, 150 mg eq/kg bw/day) revealed parental toxicity at 165 mg/kg/day in male rats (lower epididymides organ weights, histopathological changes in testes and epididymides). No parental toxicity was observed in female rats up to 165 mg/kg/day. Reproduction toxicity (fertility and conception indices of 0% due to complete absence of implants in mated females) was observed at 165 mg/kg/day and attributed to the adverse effect of the test item on male fertility. At 55 mg/kg/day, the incidence of females with relatively low numbers of implantation sites was increased. In addition, developmental toxicity (a lower gestation and post-implantation index) was seen at this dose level.

Based on the results, the following No Observed Adverse Effect Levels (NOAEL) were derived:

Parental NOAEL: 165 mg/kg/day (females, 150 mg eq/kg/day); 55 mg/kg/day (males, 50 mg eq/kg/day).
Reproduction NOAEL: 11 mg/kg/day (10 mg eq/kg/day)
Developmental NOAEL: 11 mg/kg/day (10 mg eq/kg/day; note: only 5 litters could be evaluated at 55 mg/kg/day; no litters were available at 165 mg/kg/day).

The test substance is therefore considered as reprotoxic category 2 (H361) according to CLP Regulation.