Registration Dossier

Administrative data

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Effects on fertility

Description of key information

In a combined repeated dose toxicity study with reproduction/developmental toxicity screening, the test substance was administered daily to rats at the dose level of 50, 150, and 500 mg/kg bw/day for 29 days for the males and 50-56 days for the females (OECD 422; Pels Rijcken, WR, 2018).

The parental NOAEL was considered to be at least 500 mg/kg/day. The reproductive NOAEL was considered to be at least 500 mg/kg/day as no reproduction toxicity was observed up to the highest dose level tested (500 mg/kg/day). 

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-09-20 to 2016-12-22
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EPA OPPTS 870.3650 (Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test)
Deviations:
no
Qualifier:
equivalent or similar to guideline
Guideline:
other: OECD 421 (Reproduction/Developmental Toxicity Screening Test)
Deviations:
no
Qualifier:
equivalent or similar to guideline
Guideline:
other: EPA OPPTS 870.3550 (Reproduction/Developmental Toxicity Screening Test)
Deviations:
no
Qualifier:
equivalent or similar to guideline
Guideline:
other: EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:
no
Qualifier:
equivalent or similar to guideline
Guideline:
other: OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
no
Qualifier:
equivalent or similar to guideline
Guideline:
other: EPA OPPTS 870.3050 (Repeated dose 28-day oral toxicity study in rodents)
Deviations:
no
Principles of method if other than guideline:
No testing guidelines were applicable for the pilot phase, as this part of the study was intended for dose level selection purposes only.
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: I16AB0081
- Expiration date of the lot/batch: 11 January 2018 (retest date)
- Purity test date: 2016-01-12

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 and 14 days in the refrigerator in the dark is confirmed over the concentration range 1 to 200 mg/mL, Project 513703.

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

OTHER SPECIFICS: Correction factor is 1
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: Females approx. 11 weeks (at start pretest) and approx. 13 weeks (at start F0-treatment); Males approx. 11 weeks (at start F0-treatment)
- 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: 5 days prior to start of pretest (females) or treatment (males)


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


IN-LIFE DATES:
From: 2016-10-13 (start pretest, females); 2016-10-27 (start treatment, males);
To: 2016-11-25 (necropsy males); 2016-12-16 and 2016-12-19/20/21/22 (necropsy females); 2016-12-15/16/17/18/19/20/21 (necropsy pups)
Route of administration:
oral: gavage
Vehicle:
polyethylene 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. 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); 10 mg/mL (group 2); 30 mg/mL (group 3); 100 mg/ mL (group 4)
- Amount of vehicle (if gavage): 5 mL/kg body weight (Actual dose volumes were calculated according to the latest body weight)
- Lot/batch no. (if required): no data
- Purity: no data
Details on mating procedure:
- M/F ratio per cage: 1 animal/sex/cage
- Length of cohabitation: Following a minimum of 14 days of treatment for the males and females, until detection of mating was confirmed.
- Proof of pregnancy: Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating was confirmed, the males and females were separated. A maximum of 14 days was allowed for mating. 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:
Analyses were conducted on a single occasion during the treatment phase (01 November 2016, Day 6 of treatment) according to a validated method (Test Facility Study No. 513703). Three sets of duplicate samples were collected. Two sets of duplicate samples were stored in the refrigerator as reserve samples. 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%. Once analytical results were approved in the raw data by the Principal Scientist, the reserve samples were destroyed. 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. 513703).
Duration of treatment / exposure:
29 days (males); 50-56 days (females that delivered); 41 days (females that failed to deliver). Routinely, females that are littering are left undisturbed. In this study, female nos. 48, 50 (Group 1), 51(Group 2), 66, 69 (Group 3), 73 and 79 (Group 4) were left out from treatment for one day as they were littering at the moment of dosing. The omission of one day of dosing over a period of several weeks was considered not to affect the toxicological evaluation.

Pups were not dosed directly but could have potentially been exposed to the test item in utero, via maternal milk or from exposure to maternal uring/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.
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Group 1 (control group)
Dose / conc.:
50 mg/kg bw/day (nominal)
Remarks:
Group 2
Dose / conc.:
150 mg/kg bw/day (nominal)
Remarks:
Group 3
Dose / conc.:
500 mg/kg bw/day (nominal)
Remarks:
Group 4
No. of animals per sex per dose:
10 animals/sex/dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were selected based on results of a dose range finding study (Test Facility Study No. 513699) in which animals were dosed for 10 days at 500 and 1000 mg/kg. In summary, a dose related increase in liver and kidney weights were observed at 500 and 1000 mg/kg/day. In comparison with laboratory background data, liver weights were increased at both dose levels and kidney weights at 1000 mg/kg/day only. Based on the magnitude of increase in liver weight at 1000 mg/kg/day, it was doubted if this dose level should be tolerated in animals treated for a longer duration. Since signs of (mild) toxicity were observed at 500 mg/kg/day, this dose levels was selected to be the highest for the main study. Therefore, based on the results of this range finding study, dose levels for the main study were: 50, 150 and 500 mg/kg/day. Since no clinical signs were observed in the range finding study, clinical observations in the main st udy were conducted immediately after dosing.

- Rationale for animal assignment (if not random): randomized
Positive control:
no
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. Clinical observations were conducted after dosing at no specific time point, but within a similar time period after dosing on each day. On the day of start of treatment and at weekly intervals thereafter this was also performed outside the home cage in a standard arena.

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

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/ kg body weight/day: Yes
Weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on PND 1, 4, 7 and 13. Relative food consumption was calculated and reported.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Not applicable

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

HAEMATOLOGY
- Blood samples were collected at the end of the treatment period on the day of the scheduled necropsy from the selected 5 animals/sex/group under anesthesia using isoflurane between 7.00 and 10.30 a.m. The animals were deprived from food overnight (with a maximum of 24 hours) before blood sampling; but water was available. Blood samples were drawn from the retro-orbital sinus and collected into tubes with K3-EDTA for hematology parameters and with citrate for clotting tests.
- Parameters assessed: white blood cells, differential leukocyte counts, red blood cells, reticulocytes, red blood cell distribution width, haemoglobin, haematocrit, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, platelets, prothombin time, activated thromboplastin time

CLINICAL BIOCHEMISTRY
- Blood samples were collected at the end of the treatment period on the day of the scheduled necropsy from the selected 5 animals/sex/group under anaesthesia using isoflurane between 7.00 and 10.30 a.m. The animals were deprived of food overnight (with a maximum of 24 hours) before blood sampling, but water was available. Blood samples were drawn from the retro-orbital sinus and collected into tubes with Li-heparin.
- Parameters assessed: alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, total protein, albumin, total bilirubin, bile acids, urea, creatinine, glucose, cholesterol, sodium, potassium, chloride, calcium, inorganic phosphate
- Thyroid hormone analysis

FUNCTIONAL OBSERVATIONS
- Time schedule: Between 1 and 3 hours after dosing on the selected 5 animals/sex/group. Selected males were tested during week 4 of treatment and the selected females were tested once during the last week of lactation. These tests were performed after observation for clinical signs (incl. arena observation if applicable)
- Parameters: hearing ability, pupillary reflex, static righting reflex, fore- and hindlimb grip strength recorded as the mean of three measurements per animal, locomotor activity
Oestrous cyclicity (parental animals):
Daily vaginal lavage was performed to determine the stage of estrous beginning 14 days prior to treatment (pretest), the first 14 days of treatment and during mating until evidence of copulation was observed. Vaginal lavage continued for those females with no evidence of copulation until termination of the mating period. During pretest, this was done for 48 females. On the day of 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
- Performed on day 4 postpartum: No. All pups were randomized per litter and individually identified by means of subcutaneous injection of Indian ink on post-natal day 1 (= day the litter was found completed)
- Maximum of 8 pups/litter (4/sex/litter as nearly as possible) were selected for culling on PND 4; blood samples were collected from two of the surplus pups.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
- Mortality / Viability: The numbers of live and dead pups were determined on PND 1 and daily thereafter. If possible, defects or cause of death were evaluated.
- Clinical signs: At least once daily, detailed clinical observations were made for all animals. Only days on which clinical signs were present between first and last litter check are presented in the respective table of the study report.
- Body weights: Live pups were weighed on PND 1, 4, 7 and 13.
- Sex: Sex was determined for all pups on PND 1 and 14.
- Anogenital distance: Anogenital distance (AGD) was measured for all live pups on PND 1. The AGD was normalized to the cube root of body weight.
- Areola/nipple retention: On PND 13, all males in each litter were examined for the number of areola/nipples.

GROSS EXAMINATION OF DEAD PUPS:
Yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead if possible.

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: no

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: no
Postmortem examinations (parental animals):
SACRIFICE:
- Male animals: All surviving animals, following completion of the mating period (a minimum of 28 days of dose administration).
- Female animals: All surviving animals, on PND 14-16 (females that delivered) or Female with total litter loss - Within 24 hours of litter loss.

GROSS PATHOLOGY: Yes
- All animals surviving to the end of the observation period were deeply anaesthetized using isoflurane and subsequently exsanguinated. After sacrifice, all animals were subjected to a full post-mortem examination, with special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities were recorded.
- The number of former implantation sites were recorded for all paired females.
- Samples of the following tissues and organs of the selected 5 animals/sex/group were collected and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution):
Adrenal glands (M/F), (Aorta) (M/F), Brain - cerebellum, mid-brain, cortex (7 levels) (M/F), Caecum (M/ F), Cervix (F), Clitoral gland (F), Colon (M/F), Coagulation gland (M), (Cowper’s gland) (M), Duodenum (M/F), Epididymides (M), Eyes (with optic nerve (if detectable) and Harderian gland) (M/F), Mammary gland area (M/F), Femur including joint (M/F), (Glans penis) (M), (Levator ani plus bulbocavernosus muscle complex (LABC)) (M), Heart (M/F), Ileum (M/F), Jejunum (M/F), Kidneys (M/F), (Lacrimal gland, exorbital) (M/F), (Larynx) (M/F), Liver (M/F), Lung, infused with formalin (M/F), Lymph nodes - mandibular, mesenteric (M/F), (Nasopharynx) (M/F) (Esophagus) (M/F), Ovaries (F), (Pancreas) (M/F), Peyer's patches [jejunum, ileum] if detectable (M/F), Pituitary gland (M/F), Preputial gland (M), Prostate gland (M), Rectum (M/F), (Salivary glands - mandibular, sublingual) (M/F), Sciatic nerve (M/F), Seminal vesicles (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), Clitoral gland (F), Coagulation gland (M), Cowper’s glands (M), Epididymides (M), Glans penis (M), Levator ani plus bulbocavernosus muscle complex (LABC) (M), Mammary gland area (M/F), Ovaries (F), Preputial gland (M), Prostate gland (M), Seminal vesicles (M), Testes (M), Thyroid including parathyroid if detectable (M/F), Uterus (F), Vagina (F), All gross lesions (M/F)

HISTOPATHOLOGY: Yes
- All organ and tissue samples were processed, embedded and cut at a thickness of 2-4 micrometers. These slides were stained with haematoxylin and eosin. The additional slides of the testes (to examine staging of spermatogenesis) were stained with PAS/haematoxylin.
- The following slides were examined by a pathologist: The preserved organs and tissues of the selected 5 animals/sex of Groups 1 and 4; The additional slides of the testes of the selected 5 males of Groups 1 and 4 to examine staging of spermatogenesis; All gross lesions of all animals (all dose groups); Liver of all selected 5 animals of Groups 2 and 3 (females and males), thymus of all selected 5 males of Groups 2 and 3, based on (possible) treatment-related changes in these organs in Group 4; The reproductive organs and mammary gland of female no. 65 (Group 3) that had a total litter loss.
- All abnormalities were described and included in the study report. An attempt was made to correlate gross observations with microscopic findings.
- A peer review on the histopathology data was performed by a second pathologist.
Postmortem examinations (offspring):
SACRIFICE
- Pups, younger than 7 days were killed by decapitation
- The F1 offspring were sacrificed at PND 4 (by decapitation between 7.00 and 10.30 a.m.), and at PND 7-15 (using Euthasol 20% by intraperitoneal injection).

GROSS NECROPSY
- All pups were sexed by both external and internal examination. Descriptions of all abnormalities were recorded.
- At terminal sacrifice (PND 13-15), the thyroid from 2 pups per litter, i.e. the same pups as selected for blood sampling, was preserved in 10% buffered formalin.
- The stomach of pups not surviving to the scheduled necropsy date was examined for the presence of milk, if possible. If possible, defects or cause of death were evaluated.

HISTOPATHOLOGY / ORGAN 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 (many-to-one ttest) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
• The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
• The Fisher Exact-test was applied to frequency data.
• The Kruskal-Wallis nonparametric ANOVA test was applied to motor activity data to determine intergroup differences.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance.
Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.
Reproductive indices:
For each group, the following calculations were performed:
Mating index (%) = (Number of females mated/Number of females paired) x 100
Fertility index (%) = (Number of pregnant females/Number of females paired) x 100
Gestation index (%) = (Number of females bearing live pups/Number of pregnant females) x 100
Duration of gestation = Number of days between confirmation of mating and the beginning of parturition
Offspring viability indices:
Survival indices:
Post-implantation survival index (%) = (Total number of offspring born/Total number of uterine implantation sites) x 100
Post-implantation survival index was expressed as 100% when the number of offspring exceeded the number of implantation sites recorded.
Live birth index (%) = (Number of live offspring on Day 1 after littering/Total number of offspring born) x 100
Viability index (%) = (Number of live offspring on Day 4 before culling/Number live offspring on Day 1 after littering) x 100
Lactation index (%) = Number of live offspring on Day 13 after littering/Number live offspring on Day 4 (after culling) x 100
Group mean values were calculated from individual litter values.
Sex ratio (percentage males) = (Number of males in litter/Total number of offspring in litter) x 100
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 rales, alopecia, scabs on the cheek or in the neck and red discolouration of the ear. 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.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
No mortality occurred during the study period.

Female no.65 (at 150 mg/kg/day) was early terminated on PND 1, because of total litter loss.
Body weight and weight changes:
effects observed, non-treatment-related
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, body weights and body weight gain over the premating period were similar in all groups. During the post-coitum phase, slightly lower body weights and body weight gain were observed in pregnant females in all three test groups when compared to controls, reaching levels of statistical significance for body weights on Day 14 at 150 mg/kg/day and on Day 17 at 150 and 500 mg/kg/day, and for body weight gain on Days 7, 11, 14 and 17 post-coitum at 500 mg/kg/day. In comparison with historical background data for body weight gain during the post-coitum period, the growth of the control animals was slightly higher than expected. Since body weight gain was similar in all three test groups and also comparable with the normal growth of pregnant rats of this strain and age, the statistically significant differences apparent during the post-coitum phase were considered not indicative of toxicological significant changes.

The difference in body weights on Day 20 post-coitum and Day 1 of lactation, indicative of the weight and size of the litter, showed a mean body weight loss of 85-90 grams in controls and high dose females and of approximately 75 grams in the low and mid dose females. As a result, the difference in mean body weights between controls and females at 50 and 150 mg/kg/day had diminished (to less than 3%), whereas that between controls and females at 500 mg/kg/day was maintained (at approx. 7%) and reaching a level of statistically significance. Levels of statistical significance were also observed for the mean body weights of these high dose females on Days 7 and 13 of lactation in comparison with controls. Body weight gain during lactation, however, showed that growth in controls and high dose females was normal and ran parallel.

In summary, the differences in body weights and body weight gain between treated females and controls were considered to have occurred as a result of a slightly higher than normal body weight gain in controls over the post coitum period, rather than a treatment-related effect. Therefore, no toxicological significance was attached to the changes in growth between the dose groups in this study.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Food consumption before or after allowance for body weight was similar between treated and control males.

In females, food consumption over the pre-mating period was similar in all groups. During the postcoitum phase, slightly higher than normal food consumption was observed in control females, in line
with the slightly higher body weight gains in these animals over the same period. Therefore, the statistically significant differences apparent during the post-coitum phase between treated females, i.e. over Days 4-7 in females at 150 and 500 mg/kg/day and over Days 11-14 in females at 500 mg/kg/day, were considered not indicative of changes in food consumption in treated females. During the lactation phase, the food consumption was similar in all groups, except for a slightly lower food consumption (not statistically significant) in the females at 500 mg/kg/day over Days 7-13.

In summary, the food consumption in females was considered not to have been affected by treatment in this study.
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. No dose related response was observed for any haematology parameter in males and all values were within normal limits and the test group mean values were close to that of the historical background values.

In a single female at 500 mg/kg/day an increase of neutrophil counts with concurrently reduced lymphocyte counts was noted. In addition, a relatively low value for red blood cell count, and relatively high values for red cells distribution width (RDW) and mean corpuscular volume (MCV) were observed in this female. The changes in these parameters might be indicative of a secondary non-specific response to stress and were possibly related to the reddish discolouration of the thymus observed at macroscopic examination and the congestion and lymphoid atrophy of the thymus of this female. Therefore, the findings in female no.78 were considered to have occurred by chance and of no toxicological significance. The statistical significance for the RDW in high dose females was considered to be the result of the deviating value apparent in the single female, rather than an indication of a treatment related effect.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
No toxicologically relevant changes occurred in clinical biochemistry parameters of treated rats.

Statistically significant decreases in ASAT and bile acids were observed in males at 500 mg/kg/day. The changes in these parameters might be related to the histopathological (non-adverse) alterations observed in the liver, but since decreases in these parameters are considered of limited toxicological significance, these findings were considered to be non-adverse.

Plasma levels of chloride were dose-dependently increased in treated animals, achieving levels of statistical significance in males at 150 and 500 mg/kg/day. Chloride was measured using an Ion Selective Electrode (ISE). It is known that bromide, a constituent of the test substance, can interfere with this measurement, resulting in falsely increased chloride results. Therefore, the apparent dose related increase in chloride in males and females was considered to be an artifact resulting from interference by test substance-derived bromide with the ISE and of no toxicological relevance.

Any statistically significant changes apparent for calcium in males at 500 mg/kg/day, for potassium and calcium in females at 150 mg/kg/day and for inorganic phosphate in females at 50 and 150 mg/kg/day were considered not to be toxicologically significant as they occurred in the absence of a treatment-related distribution and remained within the range considered normal for rats of this age and strain.

Thyroid hormone analyses: Serum levels of T4 in F0 males were considered not to be affected by treatment. The statistical significances found in all three test groups were considered to the result of a fortuitous difference in T4 values between controls and test groups, rather than an indication of a treatment related effect. In the absence of a clear dose response relationship and since the T4 values in all males were within the normal limits for rats of this strain and age and no toxicological significance was attached to this finding.
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.
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):
THYMUS: At 500 mg/kg/day, lymphoid atrophy of the thymus was recorded in 3 females at a minimal degree.

LIVER: At 500 mg/kg/day, centrilobular hepatocellular hypertrophy was recorded in the liver of all males and females at minimal or slight degree.

KIDNEYS: An increased incidence and severity of hyaline droplet accumulation was recorded in males at 500 mg/kg/day. This was recorded in all males at minimal-moderate degree. In the remaining dose groups this was seen in 3/5 males at minimal or slight degree.

There were no other test item-related histologic changes. The remainder of the recorded microscopic findings, including the follicular cell hypertrophy in the male thyroid gland of all dose groups, 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: oestrous cycle:
no effects observed
Description (incidence and severity):
Length and regularity of the estrous cycle were considered not to have been affected by treatment. All females had regular cycles of 4 days.
For one female confirmation of mating was overlooked. Since delivery was recorded 21 days after start mating, the recordings of extended di-estrus during the mating phase were a physiological consequence of its pregnancy.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
The spermatogenic staging profiles were normal.
Reproductive performance:
effects observed, non-treatment-related
Description (incidence and severity):
REPRODUCTION DATA
No reproduction toxicity was observed up to the highest dose level tested (500 mg/kg/day). Mating index, fertility and conception index, precoital time and number of implantation sites were considered not to be affected by treatment. All females showed evidence of mating; all mated females were pregnant. One female at 150 mg/kg/day showed total litter loss on post-natal Day 1. Histopathology did not reveal any changes in the reproductive organs that could explain this. There were no morphological findings in the reproductive organs of either sex which could be attributed to the test item.

DEVELOPMENTAL DATA
- Gestation index and duration of gestation were considered not to be affected by treatment. All females had living pups on PND 1, except for one female at 150 mg/kg/day. This latter female had one dead female pup at first litter check and was regarded to have total litter loss. This single event of a delivery of a small, non-viable litter in a mid-dose female was considered to have occurred by chance and was not related to 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.
- 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. A female at 150 mg/kg/day delivered one dead pup, which was regarded as total litter loss. Based on the single incidence and the absence of a dose response relationship no toxicological significance was attached to this finding.
- Viability index: The number of live offspring on Day 4 before culling compared to the number of offspring on Day 1 was considered not affected by treatment. A total of eleven pups went missing between Day 2 and Day 4 (before culling). The incidence of missing pups over the dose groups was 3 (from 2 litters), 2 (from 2 litters), 2 (from 2 litters) and 4 (from 3 litters), in controls and groups dosed at 50, 150 and 500 mg/kg/day, respectively. These pups were most likely cannibalised. 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.
- 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.

Parental results:
No treatment related changes in body weights and food consumption were observed in the parental males treated up to 500 mg/kg/day over the entire study period. In females, normal growth was observed over the pre-mating period, but differences in body weights and food consumption between treated and control animals were observed over the post-coitum period which persisted during lactation. As the body weight gain and food consumption in treated females were within the normal range, these differences were
considered the result of a somewhat higher growth in controls over the post-coitum period than was expected from historical control data. A relation to treatment of these changes in body weight gain and food consumption in females was not suspected and were therefore considered of no toxicological significance. In high dose animals at 500 mg/kg/day, treatment related changes were observed in the liver (both sexes), thymus (females) and kidneys (males)

Hepatocellular hypertrophy was recorded in males and females at 500 mg/kg/day and the severity varied from minimal to slight and without evidence of any degenerative, inflammatory or proliferative finding of the liver. The lymphoid atrophy of the thymus in females at 500 mg/kg/day correlated the lower thymic weights. Although lymphoid atrophy can be seen in (non-treated) lactating females, this finding was considered to be a test item related because it was not recorded in control females in the current study. The hyaline droplet accumulation recorded in the kidneys of males at 500 mg/kg/day likely represents alpha2uglobulin, a protein in male rats which undergoes re-absorption in the proximal cortical tubules. Mainly based on the incidences and low severities observed at histopathology these findings in liver, thymus and kidney were considered non-adverse.

No treatment-related changes were noted after treatment at 500 mg/kg/day in the other parental parameters investigated in this study (i.e. clinical appearance, functional observations and clinical laboratory investigations).

Any changes in the various study parameters after treatment at 50 and 150 mg/kg/day were considered no signs of parental toxicity.

Reproductive results:
No reproduction toxicity was observed up to the highest dose level tested (500 mg/kg/day).
Key result
Dose descriptor:
NOAEL
Remarks:
Parental
Effect level:
>= 500 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
organ weights and organ / body weight ratios
Remarks on result:
not determinable due to absence of adverse toxic effects
Remarks:
Treatment related changes in the thymus in females, in the kidneys in males and in the liver in both sexes at 500 mg/kg/day were considered non-adverse.
Key result
Dose descriptor:
NOAEL
Remarks:
Reproduction
Effect level:
>= 500 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse changes were noted in any of the parameters examined in this study.
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Critical effects observed:
no
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.

For one pup at 50 mg/kg/day that went missing on Day 2 of lactation, a wound on its back and neck was observed at first litter check. This injury was considered to be related to the cause why it went missing. For two other missing pups, a lean appearance and less milk in the stomach was recorded shortly after birth or from Day 3 (to Day 5) onwards at 500 mg/kg/day.

Incidental clinical symptoms in surviving pups consisted of a wound on the head, ear, front paw, abdomen or neck, a bend hind leg, scabs on the front paw, back or neck, missing tail apex, missing right foot, alopecia, (temporary) less milk in the stomach, a lean appearance, a pale appearance. The nature and incidence of these 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 affected by treatment. A total of eleven pups went missing between Day 2 and Day 4 (before culling). The incidence of missing pups over the dose groups was 3 (from 2 litters), 2 (from 2 litters), 2 (from 2 litters) and 4 (from 3 litters), in controls and groups dosed at 50, 150 and 500 mg/kg/day, respectively. These pups were most likely cannibalised. 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. One pup at 500 mg/kg/day was accidentally separated from its littermates on Day 3 of lactation, but could not be returned because of hygiene conditions. It was therefore prematurely sacrificed (on Day 3 of lactation).
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The body weights of pups in litters of the test groups at first litter check on PND 1 were in the same range as controls. During lactation, a trend towards a dose related retardation of body weight gain of the male and female pups was observed. On PND 13, the mean body weights in pups at 500 mg/kg/day were approximately 16% and 19% lower in respectively male and female pups compared to controls and achieved levels of statistical significance for both sexes. In comparison with the historical control data, the male and female body weight values on PND 13 were close to the lower limit of the historical control range. The retardation in growth in pups at 150 mg/kg/day was approximately 3% and 8% lower in respectively male and female pups compared to controls and achieved a level of statistical significance for female pups only. The body weight of pups in litters at 50 mg/kg/day remained in the same range as controls over the whole lactation period.
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.

The statistically significant difference observed for the mean T4 value in male pups at 50 mg/kg/day was considered to be a fortuitous finding and in the absence of a dose response relationship for this parameter of no toxicological significance.
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
No macroscopic findings were noted among pups that were considered to be related to treatment.

The nature and incidence of the incidental macroscopic findings remained within the range considered normal for pups of this age, and were therefore considered not to be toxicologically relevant.
Histopathological findings:
not examined
Other effects:
no effects observed
Description (incidence and severity):
Anogenital distance: Anogenital distance (absolute and normalized for body weight) in male and female pups was considered not to be affected by treatment.

Areola/nipple retention: Treatment up to 500 mg/kg/day had no effect on areola/nipple retention. For none of the examined male pups nipples were observed at PND 13.
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Developmental results:
- Although birth weights of pups were normal in all dose groups, a dose related trend towards retardation of the growth was observed during the lactation phase. Between 16-19% lower body weights were observed in the male and female pups at 500 mg/kg on PND 13. A minimal effect on growth was observed in pups at 150 mg/kg/day, resulting in 3-8% lower body weights in male and female PND 13 pups, respectively. The retardation in growth of the pups at 500 mg/kg was considered a indicative of developmental toxicity, based on the magnitude of the effect.
- No treatment-related changes were noted in 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, anogenital distance (PND 1), areola/nipple retention (PND 13 males), T4 thyroid hormone levels (PND 13-15) and macroscopy).
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
150 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: based on retardation of growth of the pups at 500 mg/kg/day during lactation
Remarks on result:
other: The retardation in growth of the pups at 500 mg/kg was considered a indicative of developmental toxicity, based on the magnitude of the effect.
Key result
Critical effects observed:
not specified
Key result
Reproductive effects observed:
yes
Lowest effective dose / conc.:
150 mg/kg bw/day (nominal)
Treatment related:
yes
Relation to other toxic effects:
not specified
Dose response relationship:
yes
Relevant for humans:
not specified
Conclusions:
In conclusion, treatment with JNJ-1597622-AAA (T000625) by oral gavage in male and female Wistar Han rats at dose levels of 50, 150 and 500 mg/kg/day revealed non-adverse, treatment related changes at 500 mg/kg/day, expressed as an effect in the thymus in females and effects in the liver in both sexes. No effects on the reproduction parameters were observed, whereas the magnitude of retardation in growth of the pups at 500 mg/kg/day during the lactation phase were indicative of signs of developmental toxicity.
Based on these results, the following No Observed Adverse Effect Levels (NOAEL) were derived:
Parental NOAEL: at least 500 mg/kg/day
Reproduction NOAEL: at least 500 mg/kg/day
Developmental NOAEL: 150 mg/kg/day (based on retardation of growth of the pups at 500 mg/kg/day during lactation)
Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
500 mg/kg bw/day
Study duration:
subacute
Species:
rat
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

In a combined repeated dose toxicity study with reproduction/developmental toxicity screening, the test substance was administered daily to male and female rats (10 rats/sex/dose level) at the dose level of 50, 150, and 500 mg/kg bw/day (OECD 422; Pels Rijcken, WR, 2018). The vehicle used was propylene glycol. Accuracy and homogeneity of formulations were demonstrated to be acceptable by chemical analyses.

No reproduction toxicity was observed up to the highest dose level tested (500 mg/kg/day). Based on these results, the reproductive  No Observed Adverse Effect Levels (NOAEL) was at least 500 mg/kg/day. 

 

Effects on developmental toxicity

Description of key information

In a combined repeated dose toxicity study with reproduction/developmental toxicity screening, the test substance was administered daily to male and female rats (10 rats/sex/dose level) at the dose level of 50, 150, and 500 mg/kg bw/day (OECD 422; Pels Rijcken, WR, 2018). The NOAEL for developmental toxicity was set at 150 mg/kg bw/day due to the effect of retardation in growth of the pups at 500 mg/kg bw/day during the lactation phase, which are considered to be indicative of signs of developmental toxicity.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
150 mg/kg bw/day
Study duration:
subacute
Species:
rat
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on the results of the OECD 422 test, the following No Observed Adverse Effect levels (NOAEL) were derived: 

Parental NOAEL: at least 500 mg/kg/day
Reproduction NOAEL: at least 500 mg/kg/day
Developmental NOAEL: 150 mg/kg/day (based on retardation of growth of the pups at 500 mg/kg/day during lactation)

Therefore, the substance is classified as a reproductive toxicant category 2 according to the CLP regulation.

Additional information