Formamide, N-[(2-bromo-5-hydroxy-4-methoxyphenyl)methyl]-N-[2-(4-hydroxyphenyl)ethyl]-

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-01-31 to 2017-05-16

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test 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 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:

N-[(2-bromo-5-hydroxy-4-methoxyphenyl)methyl]-N-[2-(4-hydroxyphenyl)ethyl]formamide

Cas Number:

122584-18-3

Molecular formula:

C17H18BrNO4

IUPAC Name:

N-[(2-bromo-5-hydroxy-4-methoxyphenyl)methyl]-N-[2-(4-hydroxyphenyl)ethyl]formamide

Test material form:

solid: particulate/powder

Details on test material:

- Name of test material (as cited in study report): JNJ-28645409-AAA (T002078)
- Physical state: solid (powder)
- Appearance: White, brown grey powder

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: I15EB1917
- Expiration date of the lot/batch: 2017-09-07 (retest date)
- Purity test date: 2017-06-07

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability in vehicle: At least 6 hours at room temperature and 14 days in the refrigerator in the dark is confirmed over the concentration range 1 to 200 mg/mL (Test Facility Study No. 509975)

OTHER SPECIFICS: Correction factor was 1

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

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

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: Males approx. 10-12 weeks (at start F0-treatment); females approx. 10-12 weeks (at start pretest) and approx. 12-14 weeks (at start F0-treatment).
- Weight at study initiation: 253-317 g (males); 194-230 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: Males and females were cohabited 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, animals did not have access to food for a maximum of 2 hours.
- Water (e.g. ad libitum): Free access to tap-water. During motor activity measurements, animals did not have access to water for a maximum of 2 hours.
- Acclimation period: At least 5 days prior to start of pretest (females) or treatment (males).

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

IN-LIFE DATES
From: 2017-03-08 (start pretest, females); 2017-03-22 (start treatment, males); 2017-04-28 through 2017-05-02 (delivery of litters)
To: 2017-04-20 (necropsy males); 2017-05-11 through 2017-05-16 (necropsy females); 2017-05-10 through 2017-05-15 (necropsy pups)

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily within 6 hours prior to dosing and were homogenized to a visually acceptable level. Adjustment was made for specific gravity (1.036) of the vehicle. A correction was made for the purity/composition of the test item. A correction factor of 1 was used.

VEHICLE:
- Justification for use and choice of vehicle (if other than water): Based on trial formulations performed at Charles River Den Bosch.
- Concentration in vehicle: 0 mg/mL (group 1); 22 mg/mL (group 2); 66 mg/mL (group 3); 200 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): I15EB1917
- Purity: 98.9 %

Details on mating procedure:

- M/F ratio per cage: 1 animal/sex/cage
- Length of cohabitation: Following a minimum of 14 days of treatment for the males and females, one female was cohabitated with one male of the same treatment group until detection of mating was confirmed.
- Proof of pregnancy: Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating was confirmed, the males and females were separated.
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): Females were individually housed in Macrolon plastic cages (MIII type, height 18 cm).
- Any other deviations from standard protocol: no

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses were conducted on two occasions during the treatment phase (on 23 March 2017 and 21 April 2017, Day 2 and Day 31 of treatment, respectively) according to a validated method (Test Facility Study No.509975). The second occasion of analysis was included because the results of the first analysis did not meet acceptable criteria. Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). In addition to the criteria mentioned in the validated analytical method, each calibration curve was accepted if the average of the retention times and response factors of the data points used to construct the calibration line were within a range of ±10.00% compared to those obtained during the method validation.
The accuracy of preparation was considered acceptable if the mean measured concentrations were 85.00-115.00% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10.00%. Formulations were considered stable if the relative difference before and after storage was maximally 10.00%. Stability of formulations over 6 hours at room temperature under normal laboratory light conditions (concentration range 1-200 mg/mL) was determined as part of the analytical method development and validation study (Test Facility Study No. 509975).

Duration of treatment / exposure:

29 days (males); 50-55 days (females that delivered); 42 days (females that failed to deliver). In this study, two females from Group 2 and one female from Group 3 were left out from treatment for one day as they were littering at the moment of dosing.

Pups were not dosed directly but were potentially exposed to the test item in utero, via maternal milk or from exposure to maternal urine/faeces.

Frequency of treatment:

Once daily for 7 days per week, approximately the same time each day with a maximum of 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.:

110 mg/kg bw/day (nominal)

Remarks:

Group 2

Doses / concentrations 3

Dose / conc.:

330 mg/kg bw/day (nominal)

Remarks:

Group 3

Doses / concentrations 4

Dose / conc.:

1 000 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 the results of a 10-day dose range finding study. During the DRF phase, oral (gavage) administration of T002078 to female Wistar rats for 10 days at doses of 500 and 1000 mg/kg was not associated with any toxicologically relevant clinical signs. Based on these results, dose levels of 110, 330, and 1000 mg/kg were selected for the main study.
- Rationale for animal assignment (if not random): randomized

Positive control:

no

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once daily from start of treatment onwards up to the day prior to necropsy, detailed clinical observations were made for all animals after dosing at no specific time point, at least immediately after dosing. Once prior to start of treatment and at weekly intervals during the treatment period this was also performed outside the home cage in a standard arena. The time of onset, grade and duration of any observed sign was recorded.

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

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): No
- 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.

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

HAEMATOLOGY: Yes
- Blood samples were collected at the end of the treatment period on the day of the scheduled necropsy from the selected 5 animals/sex/group under 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 corpuscular haemoglobin, mean corpuscular haemoglobin concentration, platelets, prothombin time, activated thromboplastin time.

CLINICAL CHEMISTRY: Yes
- Blood samples were collected at the end of the treatment period on the day of the scheduled necropsy from the selected 5 animals/sex/group under anaesthesia using isoflurane. The animals were deprived of food overnight (with a maximum of 24 hours) before blood sampling, but water was available. Blood samples were drawn from the retro-orbital sinus and collected into tubes with Li-heparin.
- Parameters assessed: alanine aminotransferase, aspartate aminotrasnferase, alkaline phosphatase, total protein, albumin, total bilirubin, bile acids, urea, creatinine, glucose, cholesterol, sodium, potassium, chloride, calcium, inorganic phosphate, thyroid hormone analysis.

FUNCTIONAL OBSERVATIONS: Yes
- Time schedule: The selected males were tested during Week 4 of treatment and the selected females were tested once during the last week of lactation (e.g. PND 6-13). These tests were performed after observation for clinical signs (incl. arena observation, if applicable).
- Parameters: Hearing ability, pupillary reflex, static righting reflex, fore- and hind-limb grip strength recorded as the mean of three measurements per animal, locomotor activity (recording period: 1-hour under normal laboratory light conditions, using a computerized monitoring system).

Oestrous cyclicity (parental animals):

Daily vaginal lavage was performed to determine the stage of estrous beginning 14 days prior to treatment (pretest), the first 14 days of treatment and during mating until evidence of copulation was observed. Vaginal lavage continued for those females with no evidence of copulation until termination of the mating period. During pretest, this was done for 48 females. On the day of scheduled necropsy, a vaginal lavage was taken to determine the stage of estrous.

Sperm parameters (parental animals):

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

Litter observations:

STANDARDISATION OF LITTERS
- 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).
- To reduce variability among the litters, on PND 4 eight pups from each litter of equal sex distribution (if possible) were selected. Blood samples were collected from two of the surplus pups. Selective elimination of pups, e.g. based upon body weight, or anogenital distance was not done. Whenever the number of male or female pups prevented having four of each sex per litter, partial adjustment (for example, five males and three females) was acceptable.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
- Mortality / Viability: The numbers of live and dead pups were determined on PND 1 and daily thereafter. If possible, defects or cause of death were evaluated. One pup was sacrificed for humane reasons on PND 8.
- Clinical signs: At least once daily, detailed clinical observations were made for all animals. Only days on which clinical signs were present between first and last litter check are presented in the respective tables.
- 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:
Yes, for external and internal abnormalities.

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: no

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: no

Postmortem examinations (parental animals):

SACRIFICE:
Necropsy was conducted according to the following schedule:
- Males: All surviving animals, following completion of the mating period (a minimum of 28 days of dose administration).
- Females: All surviving animals, on PND 14-16 (females that delivered) or on post-coitum days 25-27 (females that failed to deliver, with evidence of mating)
- Pups: On PND 4 or 13-15. One pup was sacrificed for humane reasons on PND 8.
The 5 selected adult males and females for clinical pathology blood sampling and all surviving adult animals at the end of treatment were anaesthetized using isoflurane and subsequently exsanguinated. Pups intended for blood sampling on PND 4 were sacrificed via decapitation, while pups older than 7 days were sacrificed using Euthasol 20% by intraperitoneal (ip) injection. The stomach of pups not surviving to the scheduled necropsy date was examined for the presence of milk, if possible.

GROSS PATHOLOGY
- 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.
- Samples of the following tissues and organs of the selected 5 animals/sex/group and all animals that died spontaneously or were euthenized in extremis were collected and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution): Adrenal glands (M/F), (Aorta) (M/F), Brain - cerebellum, mid-brain, cortex (7 levels) (M/F), Caecum (M/F), Cervix (F), Colon (M/F), Coagulation gland (M), Duodenum (M/F), Epididymides (M), Eyes (with optic nerve (if detectable) and Harderian gland) (M/F), Mammary gland area, inguinal region with skin (M/F), Femur with bone marrow including joint (M/F), Heart (M/F), Ileum (M/F), Jejunum (M/F), Kidneys (M/F), (Lacrimal gland, exorbital) (M/F), (Larynx) (M/F), Liver (M/F), Lung, infused with formalin (M/F), Lymph nodes - mandibular, mesenteric (M/F), (Nasopharynx) (M/F), Esophagus (M/F), Ovaries (F), (Pancreas) (M/F), Peyer's patches [jejunum, ileum] if detectable (M/F), Pituitary gland (M/F), Prostate gland (M), Rectum (M/F), (Salivary glands - mandibular, sublingual) (M/F), Sciatic nerve (M/F), Seminal vesicles (M), Skeletal muscle (M/F), Skin (M/F), Spinal cord - cervical, midthoracic, lumbar (M/F), Spleen (M/F), Sternum with bone marrow (M/F), Stomach (M/F), Testes (M), Thymus (M/F) , Thyroid including parathyroid if detectable (M/F), (Tongue) (M/F), Trachea (M/F), Urinary bladder (M/F), Uterus (F), Vagina (F), All gross lesions (M/F).
Tissues/organs mentioned in parentheses were not examined by the pathologist, since no signs of
toxicity were noted at macroscopic examination.
- 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), Coagulation gland (M),
Epididymides (M), Glans penis (M), Mammary gland area inguinal region with skin (M/F), Ovaries (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)

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, Prostate, Seminal vesicles, including coagulation glands, Testes, Thyroid

HISTOPATHOLOGY
- All organ and tissue samples were processed, embedded and cut at a thickness of 2-4 micrometers.
These slides were stained with haematoxylin and eosin. The additional slides of the testes (to examine staging of spermatogenesis) were stained with PAS/haematoxylin.
The following slides were examined by a pathologist:
• The preserved organs and tissues of the selected 5 animals/sex of Groups 1 and 4
• Additional slides of the testes of the selected 5 males of Groups 1 and 4 and all males that failed to sire
• 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. Reproductive organs included the Cervix (F), Clitoral gland (F), Coagulation gland (M), Epididymides (M), Ovaries (F), Preputial gland (M), Prostate gland (M), Seminal vesicles (M), Testes (M), Uterus (F), and Vagina (F).

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.

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.
- 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 mated) x 100
Gestation index (%) = (Number of females with living pups on Day 1/Number of pregnant females) x 100
Duration of gestation = Number of days between confirmation of mating and the beginning of parturition

Offspring viability indices:

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

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No clinical signs of toxicity were noted during the observation period.
Salivation seen after dosing among animals of all test groups during the treatment period 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 red discoloration of the mouth, rales, hunched posture and alopecia. 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 not to be signs of toxicological relevance.
No test item-related findings were noted during the arena observations in this study.

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, non-treatment-related

Description (incidence and severity):

Body weights and body weight gain of treated animals remained in the same range as controls over the treatment period.
During the post-coitum phase, slightly higher body weight gain was observed in pregnant females at 1000 mg/kg when compared to controls, reaching levels of statistical significance on Days 14 and 17.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption, absolute and relative to body weight, was similar between treated and control animals.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Haematological parameters of treated rats were considered not to be affected by treatment.
In a single male at 1000 mg/kg and aberrant blood count was observed for the red blood cell parameters. This animal showed a slightly low value for the red blood cell count and a slightly high value for red blood cell distribution width. Moreover, the red blood cell indices MCV, MCH and
MCHC were also increased in this male. In the absence of any clinical signs and changes in other study parameters, these findings did not indicate a relation to treatment and were considered of no toxicological significance.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Clinical biochemistry parameters of treated rats were considered not to be affected by treatment.
Statistically significantly decreased total bilirubin values were observed in males at 1000 mg/kg/day, which were considered to have arisen as a result of a slightly high control value.
Mean values of treated animals remained within the normal range for rats of this age and strain and this difference was therefore considered not to be toxicologically relevant.
Other statistically significant changes, apparent for cholesterol, bile acids and sodium in females at 110 and/or 330 mg/kg/day, were considered not to be toxicologically significant as they occurred in the absence of a treatment-related distribution (bile acids and sodium) or were considered to have arisen as a result of a slightly low control value (cholesterol). Moreover, a relatively high mean value for bile acids was observed for control females which was caused by high values (but within normal range) in two individual females, which might have been attributed to the appearance of the statistical significance in the low dose females, when compared to controls.
Thyroid hormone analyses:
A dose dependent decrease was observed in serum levels of T4 in F0 males, achieving levels of stati
stical significance at 330 and 1000 mg/kg (approximately 20 and 32% decrease compared to the control mean, respectively). However, all mean T4 levels remained within the normal range for rats of this age and strain.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

No toxicologically relevant effects on hearing ability, pupillary reflex, static righting reflex and grip strength were observed.
The variation in motor activity did not indicate a relation with treatment. All groups showed
a similar habituation profile with very high activity in the first interval that decreased over the duration of the test period.
The relatively high mean value for total movements in Group 4 (1000 mg/kg) was caused by a highly active single male. In the absence of any corroborative finding in this male and as it concerned a single animal in this group, no toxicological relevance was attached to this finding.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related microscopic observations.
All of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Histopathological findings: neoplastic:

not examined

Other effects:

not 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 considered not to have been affected by treatment.
Most females had regular cycles of 4 days. An irregular cycle was noted for two females at 330 mg/kg (both with normal litter). Given their incidental nature, absence of a dose-related incidence and absence of an apparent correlation to pregnancy status, these findings did not indicate a relation with treatment.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

Stage aware evaluation of the testes did not show any indication for abnormal spermatogenesis.

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

REPRODUCTION DATA
- Mating index: Mating index was considered not to be affected by treatment. All females showed evidence of mating.
- Precoital time: Precoital time was considered not to be affected by treatment.
- Number of implantation sites: Number of implantation sites were considered not to be affected by treatment.
- Fertility index: Considered not to be affected by treatment. One control female and one female at 110 mg/kg were not pregnant. In the absence of a dose related incidence of non-pregnancy, this finding was considered not to be related to
treatment.

DEVELOPMENTAL DATA
- Gestation index and duration: Gestation index and duration were considered not to be 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: The total number of offspring born compared to the total number of uterine implantations was considered not to be affected by treatment.
For two control females, one dosed with 110 mg/kg, one dosed with 330 mg/kg, and one dosed with 1000 mg/kg, the number of pups were slightly higher than the number of implantations. This phenomenon is observed from time to time and is caused by normal resorption of these areas during lactation. No toxicological relevance was attached to this finding in the current study.
- Litter size: Litter size was considered not to be affected by treatment.
- Live birth index: The number of live offspring on Day 1 after littering compared to the total number of offspring born was considered not to be affected by treatment.
One pup at 110 mg/kg, 6 pups at 330 mg/kg (from 2 litters) and 3 pups at 1000 mg/kg (from 2 litters) were found dead at the first litter check. No toxicological relevance was attributed to these dead 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 considered not to be affected by treatment.
One pup at 110 mg/kg was sacrificed for humane reasons and another pup at 330 mg/kg went missing between lactation Days 5 and 13. 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.
- Viability Index: The number of live offspring on Day 4 before culling compared to the number of offspring on Day 1 was considered not to be affected by treatment.
Two pups of the control group (from 2 litters), 2 pups at 110 mg/kg (from 2 litters), 5 pups at 330 mg/kg (from 4 litters) and 1 pup at 1000 mg/kg were found dead or went 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.

Details on results (P0)

Parental results:
No parental toxicity was observed up to the highest dose level tested (1000 mg/kg).
No treatment-related changes were noted in all parental parameters investigated in this study (i.e. clinical appearance, functional observations, body weight, food consumption, clinical laboratory investigations, macroscopic examination, organ weights, and microscopic examination), except for thyroid hormones.
A slightly higher body weight gain was noted in pregnant females treated at 1000 mg/kg during the post-coitum period when compared to controls. This might be explained by the slightly larger litter size, i.e. 13.3 pups/litter compared to 12.4 pups/litter, in the high dose females compared to controls. In the absence of any corroborative changes in other study parameters, these changes were considered to have occurred by chance and not to be related to treatment.
A dose related decrease in mean T4 levels was observed in F0-males (T4 in females not investigated), but the levels remained within the normal range up to treatment at 1000 mg/kg.
In the absence of treatment related changes in thyroid weights and thyroid histopathology in both sexes, these changes were considered to be non-adverse and of no toxicological significance.

Reproductive results:
No treatment-related changes were noted in any of the reproductive parameters investigated
in this study (i.e. mating, fertility and conception indices, precoital time, and number of implantations, estrous cycle, spermatogenic profiling, and histopathological examination of reproductive organs).

Analysis of dose preparations: The concentrations analysed in the formulations of Group 2, Group 3 and Group 4 were in agreement with target concentrations (i.e. mean accuracies between 85.00% and 115.00%). No test item was detected in the Group 1 formulations. The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation ≤ 10.00%).

Effect levels (P0)

1

Key result

Dose descriptor:

NOAEL

Remarks:

parental toxicity

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

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

Remarks on result:

other: No treatment-related changes were noted in any of the parameters examined in the study

2

Key result

Dose descriptor:

NOAEL

Remarks:

reproduction toxicity

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

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

Remarks on result:

other: No treatment-related changes were noted in any of the parameters examined in the study

Target system / organ toxicity (P0)

Key result

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No clinical signs occurred among pups that were considered to be related to treatment.
One pup of a female dosed with 110 mg/kg was sacrificed early on Day 8 of lactation, due to lethargy and swelling and a red spot at the left flank.
For 7/12 pups from one female dosed with 330 mg/kg a missing tail apex was scored.
For five pups of one female (330 mg/kg) and one pup of another female (330 mg/kg) that were found dead on Day 1, and for one pup of one female (1000 mg/kg) that went missing on Day 3, absence of milk in the stomach was noted.
The nature and incidence of these and other clinical signs remained within the range considered normal for pups of this age, and were therefore considered not to be toxicologically relevant.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

The number of live offspring on Day 4 before culling compared to the number of offspring on Day 1 was considered not to be affected by treatment.
Two pups of the control group (from 2 litters), 2 pups at 110 mg/kg (from 2 litters), 5 pups at 330 mg/kg (from 4 litters) and 1 pup at 1000 mg/kg were found dead or went 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:

no effects observed

Description (incidence and severity):

Body weights of pups were considered not 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:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Serum T4 levels in male and female PND 13-15 pups were considered not 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:

effects observed, non-treatment-related

Description (incidence and severity):

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

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Sex ratio: Sex ratio was considered not to be affected by treatment.
Anogenital distance: Anogenital distance (absolute and normalized for body weight) in male and female pups was considered not to be affected by treatment.
Areola/nipple retention: Treatment up to 1000 mg/kg/day had no effect on areola/nipple retention. For none of the examined male pups were nipples 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:
No treatment-related changes were noted in any of the other developmental parameters investigated in this study (i.e. gestation, viability and lactation indices, duration of gestation, parturition, sex ratio, maternal care and early postnatal pup development consisting of mortality, clinical signs, body weight, anogenital distance (PND 1), areola/nipple retention (PND 13 males), T4 thyroid hormone levels (PND 4 and 13-15) and macroscopy.

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Remarks:

developmental toxicity

Generation:

F1

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

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

Remarks on result:

other: No treatment-related changes were noted in any of the parameters examined in the study

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Applicant's summary and conclusion

Conclusions:

In conclusion, treatment with JNJ-28645409-AAA (T002078) by oral gavage in male and female Wistar Han rats at dose levels of 110, 330 and 1000 mg/kg revealed no adverse parental, reproduction or developmental toxicity for treatment up to 1000 mg/kg.
Based on these results, the following No Observed Adverse Effect Levels (NOAEL) were derived:
Parental NOAEL: at least 1000 mg/kg
Reproduction NOAEL: at least 1000 mg/kg
Developmental NOAEL: at least 1000 mg/kg
Therefore, the substance is not classified as a reproductive toxicant according to the CLP Regulation.