Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

In a reproduction/developmental toxicity screening test, the test item was administered daily to rats at dose levels up to 750mg/kg bw/day (OECD422, van Ottendijk F., 2017). The parental and reproduction NOAEL were established as 750mg/kg bw/day.

The substance is not classified as a reproductive toxicant accoprding to CLP regulation.

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-12-27 to 2017-04-25
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA OPPTS 870.3650 (Combined Repeated Dose Toxicity Study with the Reproduction/Deve lopmental Toxicity Screening Test)
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
other: EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
other: EPA OPPTS 870.3550 (Reproduction/Developmental Toxicity Screening Test)
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
other: OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
other: EPA Guideline 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. certificate)
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: M16FB2273
- Expiration date of the lot/batch: 2017-06-14 (retest date)

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 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. 513680).


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

OTHER SPECIFICS: Yes, 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 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 weeks (at start F0-treatment); females approx. 10 weeks (at start pretest) and approx. 12 weeks (at start F0-treatment).
- Weight at study initiation: 289-293 g (males) and 224-230 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: 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: 2016-12-27 To: 2017-04-25
Route of administration:
oral: gavage
Vehicle:
propylene glycol
Remarks:
specific gravity 1.036
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily within 6 hours prior to dosing and were homogenized to a visually acceptable level. Adjustment was made for specific gravity of the vehicle. No correction was made for the purity/composition of the test item

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), 80 mg/mL (group 2), 250 mg/mL (group 3), 750 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:
- Length of cohabitation: Following a minimum of 14 days of treatment for the males and females, the animals will be cohabitated on a 1:1 basis within the same treatment group, avoiding sibling mating.
- 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.
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 (22 February 2017, Day 1 of treatment) according to a validated method (Test Facility Study No. 513680). 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%.
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. 513680).
Duration of treatment / exposure:
29 days (males); 49-62 days (females that delivered); 42 days (non preagnant females). Female nos. 45, 48 (Group 1), nos. 56, 59 (Group 2), nos. 67, 70 (Group 3) and nos. 77 and 80 (Group 4) 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.
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Group 1
Dose / conc.:
80 mg/kg bw/day (nominal)
Remarks:
Group 2
Dose / conc.:
250 mg/kg bw/day (nominal)
Remarks:
Group 3
Dose / conc.:
750 mg/kg bw/day (nominal)
Remarks:
Group 4
No. of animals per sex per dose:
10 animals per sex per 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. 513676) in which animals were dosed for 10 days at 80, 250 and 750 mg/kg/ day
- 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 (early in the morning and close to the end of the working day).

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 treatment. Once prior to start of treatment and at weekly intervals during the treatment period this was also performed outside the home cage in a standard arena.

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

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
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: Yes
Blood samples were collected at the end of the treatment period on the day of 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 prepared with K3- EDTA for haematological parameters (0.5 mL), with citrate for clotting tests (0.45 mL) and tubes treated with Li-heparin for clinical biochemistry parameters (0.5 mL). The remnant of the Li-heparin sample was stored at ≤-75°C for possible future clinical biochemical analysis.
An additional blood sample (0.25 mL) was collected into serum tubes for determination of
bile acids, and a further blood sample (0.5 mL) was collected into a serum tube for possible future clinical biochemical analysis. After clotting and centrifugation, serum samples were stored at ≤-75°C; these samples were discarded prior to report finalization.
- Parameters checked : 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
- 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 for clinical biochemistry parameters. An additional blood sample was collected into serum tubes for determination of bile acids.
- parameters checked: 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
The following functional observations tests were performed on each individual animal of the selected 5 animals/sex/group: hearing ability, pupillary reflex, static righting reflex, fore- and hind-limb grip strength, 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 [all/P/F1/F2] male parental generations:
Parameters examined in F0 male parental generations: additional slides of the testes (to examine staging of spermatogenesis), testis weight
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no
All pups were randomized per litter and idividually 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); Blood samples were collected from two of the surplus pups; excess pups were killed and discarded.

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 ther eafter. 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.
- 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; possible cause of death was determined for pups born or found dead if possible
Pups found dead during the weekend were necropsied on the same day.

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)
- Maternal animals: all surviving animals, on PND 14-16 (females which delivered), on days 25-27 (females which failed to deliver, with evidence of mating) or within 24 hours of litter loss (females with total litter loss)

GROSS NECROPSY
- All animals surviving to the end of the observation period were deeply anaesthetized using isoflurane and subsequently exsanguinated. After sacrifice, all animals were subjected to a full post mortem examination, with special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities were recorded.
- Necropsy was conducted as soon as possible after spontaneous death and always within 24 hours.
- Samples of the following tissues and organs of the selected 5 animals/sex/group were collected and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution): Adrenal glands (M/F), (Aorta) (M/F), Brain - cerebellum, mid-brain, cortex (7 levels) (M/F), Caecum (M/F), Cervix (F), Clitoral gland (F), Colon (M/F), Coagulation gland (M), (Cowper’s gland) (M), Duodenum (M/F), Epididymides (M), Eyes (with optic nerve (if detectable) and Harderian gland) (M/F), Mammary gland area (M/F), Femur including joint (M/F), (Glans penis) (M), (Levator ani plus bulbocavernosus muscle complex (LABC)) (M), Heart (M/F), Ileum (M/F), Jejunum (M/F), Kidneys (M/F), (Lacrimal gland, exorbital) (M/F), (Larynx) (M/F), Liver (M/F), Lung, infused with formalin (M/F), Lymph nodes mandibular, mesenteric (M/F), (Nasopharynx) (M/F), (Esophagus) (M/F), Ovaries (F), (Pancreas) (M/F), Peyer's patches [jejunum, ileum] if detectable (M/F), Pituitary gland (M/F), Preputialgland (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, males that fail to sire and females which fail 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 para thyroid if detectable (M/F), Uterus (F), Vagina (F), All gross lesions (M/F)

HISTOPATHOLOGY / ORGAN WEIGHTS
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.
• The preserved organs and tissues of the animals of all dose groups which died spontaneously or were euthanized in extremis.
• 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, i.e. non-pregnant couples 51/11 (Group 2), 65/25 and 69/29 (Group 3) and 79/39 (Group 4).
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 euthanized by decapitation.
All remaining pups (PND 7-15) were sacrificed using Euthasol® 20% by intraperitoneal (ip) injection. These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

GROSS NECROPSY
- All pups were sexed both externally and internally.
- At terminal sacrifice (13-15 days of age), 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 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.
• 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 were noted among surviving animals during the observation period that were considered to be related to treatment.
Observed clinical signs among surviving animals 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 related to treatment.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
No mortality occurred during the study period that was considered to be related to treatment. One female at 250 mg/kg (no. 61) was sacrificed in extremis on PND 1 (i.e. after 40 days of treatment) due to a severe vaginal prolapse. At necropsy, this animal also showed pale discolouration of the whole body, and reddish discolouration of the thymus. Secondary to vaginal prolapse, distension of both uterine horns and serosal inflammation in the uterus were observed histopathologically.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Body weights and body weight gain of treated animals were not considered affected by treatment. Incidental occurrences of statistically significantly lower mean body weights or body weight gain of females at 80 and 250 mg/kg during the treatment period occurred in the absence of a dose-related trend, and as such were not considered to be related to treatment.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Food consumption before or after correction for body weight was not considered affected by treatment. The lower mean food consumption of females at 80 mg/kg during lactation (statistically significant on Days 1-4 and 7-13) was ascribed to a low food intake of two females (nos. 53 and 54), one of which only delivered a single pup. This variation in food intake as well as other statistically significantly lower mean absolute and/or relative food consumption values on several occasions during post-coitum at 250 mg/kg occurred in the absence of a doserelated trend. Therefore, these variations were not considered to be related to treatment.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
At 750 mg/kg, activated partial thromboplastin time (APTT) was elongated for females (approximately 34% increase compared to the control mean); the mean remained within the normal range for rats of this age and strain.
Other statistically significant changes in haematological parameters were not considered related to treatment in the absence of a dose-related trend, and/or concurrent changes in other red blood cell parameters.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Clinical biochemistry parameters of treated rats were not considered to be affected by treatment.
Thyroid hormone analyses:
Serum levels of T4 in F0 males were not considered to be affected by treatment. The statistically significantly lower T4 values at 80 and 750 mg/kg occurred in the absence of a dose-related trend and means remained within the normal range for rats of this age and strain. As such, these variations in T4 were not considered to be related to treatment.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Functional observation parameters were not considered to be affected by treatment.
Hearing ability, pupillary reflex and static righting reflex were normal in all selected animals.
Fore- and hindlimb grip strength was not considered affected by treatment. The statistically significantly lower forelimb grip strength of females at 80 mg/kg occurred in the absence of a dose-related trend. The variation in motor activity did not indicate a relation with treatment.
All groups showed a similar habituation profile with high activity in the first interval with a decreasing trend in activity over the duration of the test period.
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. One male rat at 750 mg/kg (no. 34) showed moderate chronic progressive nephropathy. This is an unusual finding in rats of this age, but based on the presence in only one animal and the absence of test item-related findings in the kidneys of the other animals, the finding was considered to be incidental.
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Reproductive function: oestrous cycle:
effects observed, non-treatment-related
Description (incidence and severity):
Length and regularity of the estrous cycle were not considered to have been affected by treatment. All females had regular cycles of 4 days. Extended di-estrus occurred in one female at 250 mg/kg (no. 68) with a regular cycle. Given this incidental nature, absence of a dose-related incidence and absence of an apparent correlation to pregnancy status, this finding did not indicate a relation with treatment.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
Spermatogenic staging profiles were normal for all males examined
Reproductive performance:
effects observed, non-treatment-related
Description (incidence and severity):
REPRODUCTION DATA
There were 1/10 couples treated at 80 mg/kg, 2/10 couples treated at 250 mg/kg and 1/10 couples treated at 750 mg/kg that failed to deliver healthy pups. Histopathology did not reveal any changes in the reproductive organs that could explain this and stage aware evaluation of the testes did not show any indication for abnormal spermatogenesis.
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.
Estrous Cycle- All females had regular cycles of 4 days. Extended di-estrus occurred in one female at 250 mg/kg (no. 68) with a regular cycle. Given this incidental nature, absence of a dose-related incidence and absence of an apparent correlation to pregnancy status, this finding did not indicate a relation with treatment.
Fertility and Conception index- One female at 80 mg/kg (no. 51), two females at 250 mg/kg (nos. 65 and 69) and one female at 750 mg/kg (no. 79) were not pregnant. The number of pregnant females compared to the number of paired females remained within the range considered normal for rats of this age and strain. Also, since these cases of non-pregnancy showed no dose-related incidence across the dose groups, and given the absence of any reproductive/developmental toxicity, this was not considered to be related to treatment.
Mating index, precoital time, number of implantation sites were not considered to be affected by treatment.

DEVELOPMENTAL DATA
Post-implantation survival index - For female nos. 46 (control), 58 (80 mg/kg) and 61 (250 mg/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 14-15 days of lactation. No toxicological relevance was attached to this finding in the current study.
Other developmental parameters were not considered to have been affected by treatment. These included gestation index and duration of gestation, parturition/maternal care, sex ratio, anogenital distance and areola/nipple retention.
No adverse parental effects were observed up to the highest dose level tested (750 mg/kg). However, an elongated activated partial thromboplastin time was recorded for females at that dose.
The mean remained within the normal range for rats of this age and strain, and there were no concurrent changes in other (clotting) parameters in this study. Therefore, this change was not considered to be adverse.
No treatment-related changes were noted in any of the other parental parameters investigated in the study (i.e. clinical appearance, functional observations, body weight, food consumption, clinical biochemistry investigations, macroscopic examination, organ weights,
and microscopic examination). In contrast to the range finding study (Test Facility Study no. 513676) where intermittently occurring clinical signs were observed at 500 and 1000 mg/kg including hunched posture, uncoordinated movements and/or piloerection, no such clinical signs were recorded for animals in the main study at 750 mg/kg. This may be related to the time point on which these signs were observed in the main study (i.e. shortly after dosing). Although no clear peak effect of occurrence of clinical signs were noted in the range finding study for the 500 and 1000 mg/kg dose levels combined, clinical signs recorded at 500 mg/kg were noted at 1 hour post-dose.
Reproductive results:
No reproduction toxicity was observed up to the highest dose level tested (750 mg/kg).
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).
Dose descriptor:
NOAEL
Remarks:
Parental toxicity
Effect level:
>= 750 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Dose descriptor:
NOAEL
Remarks:
Reproduction toxicity
Effect level:
>= 750 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Clinical signs:
no effects observed
Description (incidence and severity):
No clinical signs occurred among pups that were considered to be related to treatment.
The nature and incidence of 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:
not specified
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Body weights of pups were not considered to be affected by treatment.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
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 not considered to be affected by treatment.
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Histopathological findings:
not examined
Other effects:
not examined
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Post-implantation survival index- For female nos. 46 (control), 58 (80 mg/kg) and 61 (250 mg/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 14-15 days of lactation. No toxicological relevance was attached to this finding in the current study.
Litter size- A statistically significantly lower mean number of living pups was recorded at 250 mg/kg. As a dose-related response was absent and the mean remained within the range considered normal for rats of this strain and age, this was not considered to be toxicologically relevant.
Live birth index- A total of 3 pups of the control group (litter nos. 44, 46 and 48), 2 pups at 250 mg/kg (litter nos. 67 and 68) and 1 pup at 750 mg/kg (litter no. 76) were found dead at first litter check.
Viability index- The lower viability index at 250 mg/kg (89% vs. 98% in the control group) was due to one female (no. 61) that was sacrificed in extremis on Day 1 of lactation; her pups (9 in total) were sacrificed accordingly. Additionally, a total of 3 pups of the control group (litter nos. 41, 42 and 44) and 2 pups at 80 mg/kg (litter nos. 56 and 60) were found dead or missing between Days 2 and 4 of lactation. 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.
Key result
Dose descriptor:
NOAEL
Remarks:
Developmental toxicity
Generation:
F1
Effect level:
>= 750 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
no
Conclusions:
In conclusion, treatment with JNJ-42808389- AAA (T003421) by oral gavage in male and female Wistar rats at dose levels of 80, 250 or 750 mg/kg/day revealed no parental, reproduction or developmental toxicity up to 750 mg/kg.
Based on these results, the following No Observed Adverse Effect Levels (NOAEL) were derived:
Parental NOAEL: 750 mg/kg
Reproduction NOAEL: 750 mg/kg
Developmental NOAEL: 750 mg/kg
Therefore, the substance is not classified as a reproductive toxicant, according to CLP Regulation.
Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
750 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 reproduction/developmental toxicity screening test, the test item was administered daily to rats at dose levels up to 750mg/kg bw/day (OECD422, van Ottendijk F., 2017). No adverse parental effects were observed up to the highest dose level tested (750 mg/kg).

There was no test item-related mortality in the study. Changes in haematology parameter such as an elongated activated partial thromboplastin time noted mainly in females at 750 mg/kg were considered to be related to the treatment The mean remained within the normal range for rats of this age and strain, and there were no concurrent changes in other (clotting) parameters in this study. Therefore, this change was not considered to be adverse. No treatment-related changes were noted in the remaining parental parameters investigated in the study (i.e. clinical appearance, functional observations, body weight, food consumption, clinical biochemistry investigations, macroscopic examination, organ weights, and microscopic examination). In contrast to the range finding study (Test Facility Study no. 513676) where intermittently occurring clinical signs were observed at 500 and 1000 mg/kg including hunched posture, uncoordinated movements and/or piloerection, no such clinical signs were recorded for animals in the main study at 750 mg/kg. This may be related to the time point on which these signs were observed in the main study (i.e. shortly after dosing). Although no clear peak effect of occurrence of clinical signs were noted in the range finding study for the 500 and 1000 mg/kg dose levels combined, clinical signs recorded at 500 mg/kg were noted at 1hour post-dose.

No reproduction toxicity was observed up to the highest dose level tested (750 mg/kg) either. No treatment-related changes were noted in any of the reproductive parameters investigated in the study (i.e. mating, fertility and conception indices, precoital time, and number of implantations, estrous cycle, spermatogenic profiling, and histopathological examination of reproductive organs).

The substance is not classified as a reproductive toxicant accoprding to CLP regulation.

Annex IX testing

A testing proposal for a prenatal development study in a first species is included.

Effects on developmental toxicity

Description of key information

In a reproduction/developmental toxicity screening test (OECD 422, van Ottendijk F., 2017), the test item was administered daily to rats at dose levels up to 750 mg/kg bw/day. The developmental NOAEL was established as being 750 mg/kg bw/day.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
750 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
Additional information

Annex IX testing

A testing proposal for a prenatal development study in a first species is included.

Justification for classification or non-classification

Based on the results of the OECD 422 test, the following NOAELs were derived:

Parental NOAEL: 750 mg/kg/d

Reproduction NOAEL: 750 mg/kg/d

Developmental NOAEL: 750 mg/kg/d

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