Registration Dossier

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-06-23 to 2017-09-06
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA OPPTS 870.3550 (Reproduction/Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
- Name of test material (as cited in study reports): T002675, TIC876
- Physical state: liquid
- Appearance: colorless to yellowish liquid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: jsoontje-06-0456
- Expiration date of the lot/batch: 2018-01-31
- Purity test date: 2017-02-02


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Solubility and stability of the test substance in the solvent/vehicle: Stability for at least 6 hours at room temperature under normal laboratory light conditions was confirmed over the concentration range 1 to 200 mg/mL (suspensions), Project 514660



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


OTHER SPECIFICS: correction factor was 1.05

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
This species and strain of rat has been recognized as appropriate for general and reproduction toxicity studies. Charles River Den Bosch has general and reproduction/developmental historical data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: Males approx. 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: Males: 285-349 g; Females: 210-268 g;
- 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.
- Water (e.g. ad libitum): Free access to tap-water.
- Acclimation period: At least 5 days prior to start of pretest (females) or treatment (males).

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

IN-LIFE DATES:
From: 2017-06-27 (start pretest); 2017-07-11 (start treatment); 2017-08-17/18/19/20/21/23 (delivery of litters)
To: 2017-08-09 (necropsy males); 2017-08-29/30/31 and 2017-09-03/04/05 (necropsy pups); 2017-08-30/31 and 2017-09-01/04/05/06 (necropsy females)

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
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 test item. A correction was made for the purity/composition of the test item. A correction factor of 1.05 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); 1 mg/mL (group 2); 3 mg/mL (group 3); 10 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: 1:1 basis
- 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.
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): Females were individually housed in Macrolon plastic cages (MIII type, height 18 cm)
- Any other deviations from standard protocol: no
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were conducted on a single occasion during the treatment phase (11 July 2017, Day 01 of treatment) according to a validated method (Test Facility Study No. 514660). Three sets of duplicate samples were collected. Two sets of duplicate samples were stored in the refrigerator as reserve samples. Once analytical results were approved in the raw data by the Principal Scientist, the reserve samples were destroyed. Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). In addition to the criteria mentioned in the validated analytical method, each calibration curve was accepted if the average of the retention times and response factors of the data points used to construct the calibration line were within a range of ±10.00% compared to those obtained during the method validation. The accuracy of preparation was considered acceptable if the mean measured concentrations were 85.00-115.00% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10.00%. Formulations were considered stable if the relative difference before and after storage was maximally 10.00%.
Duration of treatment / exposure:
Groups 1-3: 29 days (males); 50-57 days (females that delivered). In the study, four females were left out from treatment for one day as they were littering at the moment of dosing.

Group 4 animals were treated for 15 days prior to euthanization.
Frequency of treatment:
Once daily for 7 days per week, approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose.
Doses / concentrationsopen allclose all
Dose / conc.:
0 other: mg eq/kg
Remarks:
Group 1 (Control)
Dose / conc.:
5 other: mg eq/kg
Remarks:
Group 2
Dose / conc.:
15 other: mg eq/kg
Remarks:
Group 3
Dose / conc.:
50 other: mg eq/kg
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 28-day study (Janssen reference number RMD953). This study showed severe toxicity at dose level 150 mg/kg resulting in the preterminal sacrifice of all rats in this group. A reduction in body weight and food consumption, elevated absolute and relative liver weights was observed at dose level 50 mg/kg. The increased liver weights correlated with elevated bilirubin values. Histopathological findings of the liver observed were increased fatty change at dose level 50 mg/kg and apoptosis, hypotrophy and pigment deposits were noted in animals treated with 150 mg/kg.

- 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 at least immediately after treatment (on the peak period of anticipated effects after treatment).

BODY WEIGHT: Yes
- Time schedule: 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
- Time schedule: Weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on PND 1, 4, 7 and 13. Relative food consumption was calculated and reported.

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

HAEMATOLOGY: No

CLINICAL CHEMISTRY: Yes, limited to thyroid hormone analysis.
- End of study from all animals at planned necropsy; this included females on Day 14-16 of lactation, all females that failed to deliver healthy pups and all males after at least 4 weeks of treatment.
- 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 approximately 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 described 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 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; testes weight.
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.
- 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:
- Mortality/viability: The numbers of live and dead pups were determined on PND1 and daily thereafter. If possible, defects or cause of death were evaluated.
- Clinical signs: At least once daily, detailed clinical observations were made for all animals. Only days on which clinical signs were present between first and last litter check were reported 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 PND1 and 4.
- Anogenital distance: Anogenital distance (AGD) was measured for all live pups on PND1. The AGD was normalized to the cube root of body weight.
- Areola/nipple retention: On PND13, all males in each litter were examined for the number of areola/nipples.

GROSS EXAMINATION OF DEAD PUPS:
Yes, all pups were sexed by both external as well as internal examination. Descriptions of all abnormalities were recorded.

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: no

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: no
Postmortem examinations (parental animals):
SACRIFICE
All parental animals surviving to the end of the observation period and all moribund animals were deeply anaesthetized using isoflurane and subsequently exsanguinated. Necropsy was conducted according to the following schedule:
- Males: following completion of the mating period (a minimum of 28 days of dose administration)
- Females which delivered: PND 14-16
- Euthanized in extremis: when pain, distress or discomfort was considered not transient in nature or was likely to become more severe. In consultation with the sponsor, Group 4 animals were necropsied on Day 15 based on body weight loss in the females.

GROSS NECROPSY
- 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.
- The number of former implantation sites were recorded for all paired females.
- Samples of the following tissues and organs were collected and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution): Cervix (F), Coagulation gland (M), Epididymides (M), Liver (M/F), Lungs (M/F), Mammary gland area (inguinal region with skin) (M/F), Ovaries (F), 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).

ORGAN WEIGHTS
- Absolute organ weights and organ to body weight ratios were reported.
- The following organ weights and terminal body weight were recorded from all animals of Groups 1-3 on the scheduled day of necropsy: Epididymides, Prostate gland, Seminal vesicles (including coagulation gland), Testes, Thyroid, Liver.

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 ovaries, testes, epididymides and thyroids of the animals of Groups 1 and 3; The preserved organs and tissues of the animals of all dose groups which were euthanized in extremis; All gross lesions of all animals (all dose groups).
- Of the testes of all males of Groups 1 and 3 and all males that failed to sire or which died before mating detailed qualitative examination was made, taking into account the tubular stages of spermatogenic cycle. The examination was conducted in order to identify treatment related effects such as missing germ cell layers or types, retained spermatids, multinucleate or apoptotic germ cells and sloughing of spermatogenic cells into the lumen. Any cell- or stage-specificity of testicular findings were noted.
- Preserved organs and tissues for Group 4 animals sent to necropsy at Day 15 of treatment, were not examined by a pathologist, with the exception of gross lesions observed in these animals.
- 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
- On PND 4 (at culling), 2 pups per litter were killed by decapitation for blood collection.
- Pups younger than 7 days were euthanized by decapitation.
- On PND 13-15, 2 pups per litter were anaesthetized using isoflurane for blood collection by aorta puncture followed by exsanguination.
- All remaining pups (PND 13-15) were sacrificed using Euthasol 20% by intraperitoneal (ip) injection.

GROSS NECROPSY
- All pups were sexed both externally and internally. Descriptions of all abnormalities were recorded
- At terminal sacrifice (PND 13-15), the thyroid from 1 male and 1 female pup per litter was preserved in 10% buffered formalin. If possible, these were the same pups as selected for blood sampling.
- The stomach of pups not surviving to the scheduled necropsy date were examined for the presence of milk, if possible. If possible, defects or cause of death were evaluated

HISTOPATHOLOGY / ORGAN WEIGTHS
- Not examined
Statistics:
The following statistical methods were used to analyse the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.

All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.
Reproductive indices:
For each group, the following calculations were performed:
Mating index (%) = (Number of females mated/Number of females paired) x 100
Fertility index (%) = (Number of pregnant females/ Number of females mated) x 100
Gestation index (%) = (Number of females bearing live pups on Day 1/Number of pregnant females) x 100
Duration of gestation = Number of days between confirmation of mating and the beginning of parturition
Precoital time = Number of days between initiation of cohabitation and confirmation of mating
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

Sex ratio (percentage males) = (Number of males in litter/Total number of offspring in litter) x 100

Percentage of live males at first litter check (%)

Percentage of live females at first litter check (%)

- Group mean values were calculated from individual litter values

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Two females dosed with 50 mg eq/kg showed hunched posture and/or piloerection for 1-2 days.
No clinical signs of toxicity were noted during the observation period at dose levels up to 15 mg eq/kg.
Incidental findings that were noted 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 not considered to be signs of toxicological relevance.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, treatment-related
Description (incidence):
At 50 mg eq/kg, one female was sacrificed in extremis on Day 14 of treatment, and all males and remaining females were sacrificed on Day 15 of treatment, based on significant body weight loss (up to 12% vs initial weight on premating Day 1), which was noted in almost all females, and severely reduced body weight gain in the males (mean gain was 0.21x of controls on Day 1 of the mating period). In addition, food consumption was lower for these animals (mean was 0.78 x and 0.79 x of controls for females and males, respectively) and a hunched posture and/or piloerection was noted for two females for 1-2 days.

Based on the severe body weight loss, it was considered that the animals were not sufficiently healthy for mating. Therefore, it was decided to terminate the high dose group of 50 mg eq/kg.

No mortality occurred in the other dose groups.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 15 mg eq/kg, body weight gain was slightly lower for the males during the mating period and for the females during the post coitum phase (achieving statistical significance on a single occasion only). Body weight gain was similar to controls for the males and females during premating and the females at 15 mg eq/kg during the lactation phase.

Body weights and body weight gain of animals at 5 mg eq/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):
Food consumption (absolute or relative to body weight) was slightly lower for females at 15 mg eq/kg during the post coitum and lactation phase, achieving statistical significance during post-coitum Days 7-11 (absolute mean 0.91x and and relative mean 0.95x of controls, respectively) and during lactation Days 7-13 (absolute mean 0.93x of controls).

Absolute and relative food consumption was similar between treated and control animals for males up to 15 mg eq/kg and females at 5 mg eq/kg.
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):
Serum levels of T4 in F0 males were not considered to be affected by treatment.
The statistically significantly increased serum T4 levels in males at 5 mg eq/kg were considered not to represent a sign of toxicological relevance, as mean values remained well within the range considered normal for rats of this age and strain, and as a dose-relationship was lacking.
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, non-treatment-related
Description (incidence and severity):
There were no test item-related microscopic observations up to 15 mg eq/kg. No test item related microscopic changes were observed for gross abnormalities at dose 50 mg eq/kg.
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 affected by treatment up to 50 mg eq/kg.

Most females had regular cycles of 4 to 5 days. Extended di-estrus occurred in one female (5 mg eq/kg) with a regular cycle. An irregular cycle was noted for one control female (with normal litter) and one female at 50 mg eq/kg. Given their incidental nature and absence of a dose-related incidence, 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
The couples at 50 mg eq/kg were sacrificed before mating and were not used to assess reproductive and developmental toxicity.
- Reproductive performance: There were no couples treated at 0, 5 and 15 mg eq/kg without offspring. There were no morphological findings in the reproductive organs of either sex which could be attributed to the test item and stage aware evaluation of the testes did not show any indication for abnormal spermatogenesis.
- Mating index was considered not to be affected by treatment up to 15 mg eq/kg. All females showed evidence of mating.
- Precoital time was considered not to be affected by treatment up to 15 mg eq/kg. All females showed evidence of mating within six days.
- Number of implantation sites was considered not to be affected by treatment. All values were within normal limits
- Fertility index was considered not to be affected by treatment up to 15 mg eq/kg. All mated females were pregnant.

DEVELOPMENTAL DATA
As all animals at 50 mg eq/kg were sacrificed in the premating period, developmental/reproduction data is available for females up to 15 mg eq/kg only.
- Gestation index and duration: Gestation index and duration of gestation were considered not to be affected by treatment up to 15 mg eq/kg.
- Parturition/maternal care: no signs of difficult or prolonged parturition were noted among the pregnant females up to 15 mg eq/kg. 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 up to 15 mg eq/kg. For one control female and three females at 15 mg eq/kg, the number of pups was 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 the 14-16 days of lactation. No toxicological relevance was attached to this finding in the current study. The slightly lower post-implantation survival index for females at 5 mg eq/kg (87% versus 95% in the control group) was considered not to be related to treatment, as a dose relationship was not established and as mean values remained within the range considered normal for rats of this age and strain
- Litter size was considered unaffected by treatment up to 15 mg eq/kg.
- Live birth index: The number of live offspring on Day 1 after littering compared to the total number of offspring born was considered unaffected by treatment up to 15 mg eq/kg. Three pups of the control group, two pups at 5 mg eq/kg and two pups at 15 mg eq/kg were found dead at 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 unaffected by treatment up to 15 mg eq/kg. No pups were found dead/missing between lactation Days 5 and 13.

Details on results (P0)

Parental results:
- At 50 mg eq/kg, significant body weight loss in the females (up to -12%) and severely reduced body weight gain in the males, accompanied by reduced food consumption was noted. In addition, a hunched posture and/or piloerection was noted for two females, which led to the early sacrifice of one female on Day 14 of treatment. Due to the observed body weight loss of the females, the females were not considered sufficiently healthy for mating. Since health issues with females could lead to secondary effects on reproduction, it was decided to terminate and sacrifice all males and remaining females of high dose group 50 mg eq/kg on Day 15 of treatment.
- At 15 mg eq/kg, body weight gain was slightly lower for the males during the mating period and for the females during the post coitum phase, which was accompanied by slightly lower (relative) food consumption for the females in the post coitum and lactation phase only. At the low magnitude observed, and in the absence of any other treatment-related findings, these changes were considered not adverse. No treatment-related toxicologically significant changes were noted in any of the remaining parental parameters investigated in this study (i.e. clinical appearance, serum T4 levels, macroscopic examination, organ weights, and microscopic examination).
- No parental toxicity was observed at a dose level of 5 mg eq/kg.

Reproductive results:
- As all animals at 50 mg eq/kg were sacrificed at the end of the premating period, no reproduction data was available at this dose level.
- No reproduction toxicity was observed up to a dose level of 15 mg eq/kg.
- No treatment-related toxicologically significant 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:
- Accuracy of preparation: The concentrations analyzed in the formulations of Group 2, Group 3 and Group 4 were in agreement with target concentrations (i.e. mean accuracies between 85.00% and 115.00%). For Group 2, two sets of reserve samples were analyzed because the mean accuracy during the first analysis was below the criterion range (i.e. 82.13%). The mean recovery of the two sets of reserve samples were in agreement with the target concentration (i.e. 94.34% and 94.19%) and the analysis was, therefore, accepted. No test item was detected in the Group 1 formulation.
- Homogeneity: The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation ≤ 10.00%).

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
Parental toxicity
Effect level:
15 other: mg eq/kg
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
body weight and weight gain
food consumption and compound intake
other:
Remarks on result:
other: At 50 mg eq/kg, significant body weight loss, reduced food consumption, hunched posture and/or piloerection,
Key result
Dose descriptor:
NOAEL
Remarks:
Reproduction toxicity
Effect level:
>= 15 other: mg eq/kg
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No reproduction toxicity was observed up to a dose level of 15 mg eq/kg.
Remarks on result:
other:
Remarks:
As all animals at 50 mg eq/kg were sacrificed at the end of the premating period, no reproduction data was available for these females. No reproduction toxicity was observed up to a dose level of 15 mg eq/kg. No treatment-related toxicologically significant 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).

Target system / organ toxicity (P0)

Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
50 other: mg eq/kg
System:
other: general systemic toxicity
Organ:
not specified
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):
No clinical signs occurred among pups that were considered to be related to treatment up to 15 mg eq/kg. For the pup of a control female who was missing on Day 2, a pale appearance and a blue spot on the snout was noted at first litter check.
The nature and incidence of these and other clinical signs remained within the range considered normal for pups of this age, and were therefore not considered 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 unaffected by treatment up to 15 mg eq/kg.
Two pups of the control group and one pup at 5 mg eq/kg were found missing on PND 2, and one pup at 15 mg eq/kg was found missing on PND 4. Pups missing were most likely cannibalized. No toxicological relevance was attributed to these 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, non-treatment-related
Description (incidence and severity):
Body weights of pups were considered not to be affected by treatment up to 15 mg eq/kg.
The slightly higher mean body weights, noted for pups at 5 mg eq/kg (not achieving statistical significance), was mainly caused by one litter, in which only male pups were born. No toxicological relevance was attached to this as it concerned only one litter in the low dose group.
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 up to 15 mg eq/kg.
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.
Histopathological findings:
not examined
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
- Sex ratio was not considered to be affected by treatment up to 15 mg eq/kg. The slightly higher sex ratio (males/females) noted at 5 mg eq/kg (not reaching statistical significance) was mainly caused by one female, who only had male pups. In the absence of a dose-related trend, this variation was considered not related to treatment but rather a chance finding.
- Anogenital distance in male and female pups was considered not to be affected by treatment up to 15 mg eq/kg.
- Areola/nipple retention: Treatment up to 15 mg eq/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 (As all animals at 50 mg eq/kg were sacrificed at the end of the premating period, no developmental data was available at this dose level.)
- No developmental toxicity was observed up to a dose level of 15 mg eq/kg.
- No treatment-related toxicologically significant changes were noted in any of the 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:
>= 15 other: mg eq/kg
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No developmental toxicity was observed up to a dose level of 15 mg eq/kg.
Remarks on result:
other: No reproduction and developmental toxicity was observed up to a dose level of 15 mg eq/kg.
Remarks:
As all animals at 50 mg eq/kg were sacrificed at the end of the premating period, no developmental data was available for these females. No treatment-related toxicologically significant changes were noted in any of the 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.

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Reproductive effects observed:
no

Applicant's summary and conclusion

Conclusions:
In conclusion, treatment with JNJ-26233415-AAA (T002675) by oral gavage in male and female Wistar Han rats at dose levels of 5, 15 and 50 mg eq/kg revealed adverse parental toxicity at 50 mg eq/kg. No reproduction and developmental toxicity was observed for treatment up to 15 mg eq/kg; due to the early termination of 50 mg eq/kg animals, this was not investigated at the high dose level.

Based on these results, the following NOAELs were derived:
Parental NOAEL: 15 mg eq/kg
Reproduction NOAEL: at least 15 mg eq/kg (not evaluated at 50 mg eq/kg)
Developmental NOAEL: at least 15 mg eq/kg (not evaluated at 50 mg eq/kg)

Therefore, the substance is not classified as a reproductive toxicant according to the CLP Regulation.