Registration Dossier

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Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.

The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.

Diss Factsheets

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 2021-07-12 to 2021-10-05
Reliability:
1 (reliable without restriction)
Cross-reference
Reason / purpose for cross-reference:
reference to other study
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
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/Deve lopmental Toxicity Screening Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 421 (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: EPA OPPTS 870.3550 (Reproduction/Developmental Toxicity Screening Test)
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 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. 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: 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 or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: males approx. 10 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 pregnant 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.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2022
Report date:
2022

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
June 2018
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Version / remarks:
May 2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Version / remarks:
August 1998
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
[(2R,3R,4S,5R,6R)-6-bromo-3,4,5-tris[(2,2-dimethylpropanoyl)oxy]oxan-2-yl]methyl 2,2-dimethylpropanoate
EC Number:
686-241-8
Cas Number:
81058-27-7
Molecular formula:
C26H43BrO9
IUPAC Name:
[(2R,3R,4S,5R,6R)-6-bromo-3,4,5-tris[(2,2-dimethylpropanoyl)oxy]oxan-2-yl]methyl 2,2-dimethylpropanoate
Test material form:
solid: crystalline
Details on test material:
- Name of test material (as cited in study reports): JNJ-42808389-AAA (T003421)
- Physical state: crystalline powder
- Appearance: white powder
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: M21AB0732
- Chemical name: 2, 3, 4, 6-TETRAKIS-O-(2,2-DIMETHYLPROPANOYL)-ALPHA-D-GLUCOPYRANOSYL BROMIDE
- CAS number: 81058-27-7
- Physical appearance: white crystals
- Expiration date of the lot/batch: 2022-01-29
- Purity: 100% (w/w)
- Molecular weight: 579.53

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature container flushed with argon desiccated
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g.in the exposure medium) and during storage: Stability for at least 6 hours at room temperature under normal laboratory conditions was confirmed over the concentration range 1 to 200 mg/mL, Test Facility Study No. 513680.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING: no

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

OTHER SPECIFICS
- pH: 8 – 8.3 at concentration of 0.02 mg/L
- correction factor: 1.00

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Crl: WI(Han)
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: 88 time-mated females from Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: 11-15 weeks old
- Weight at study initiation: 172 - 271 g
- Fasting period before study: No
- Housing: Animals were individually housed in Polycarbonate cages (Makrolon type MIII, height 18 cm) containing sterilized wooden fibers as bedding material (Lignocel S 8-15, JRS-J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) equipped with water bottles. For psychological/environmental enrichment and nesting material, animals were provided
with paper and with aspen wooden sticks, except when interrupted by study procedures/activities
It is considered that there are no known contaminants that would interfere with the objectives of the study.
- Diet (e.g. ad libitum): Ad libitum, except during designated procedures. Pellets of SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany. It is considered that there were no known contaminants in the feed that would interfere with the objectives of the study.
- Water (e.g. ad libitum): Municipal tap water, freely available to each animal via water bottles.
Periodic analysis of the water is performed, and it is considered that there are no known contaminants in the water that could interfere with the outcome of the study.
- Acclimation period: 5 days prior to commencement of dosing.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18°C - 24°C . The actual daily mean temperature during the study was 19°C with an actual daily mean relative humidity of 59 to 75%. The values that were outside the targeted range occurred for 3 days with a maximum of 75% and were without a noticeable effect on the clinical condition of the animals or on the outcome of the study.
- Humidity (%): 40-70 % (actual 59-75%)
- Air changes (per hr): At least 10 air changes per hour
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 2021-07-28 To: 2021-08-20

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
propylene glycol
Remarks:
specific gravity 1.036
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
- Formulations (w/w) were prepared daily within 6 hours prior to dosing and were homogenized to a visually acceptable level. Test item dosing formulations were kept at room temperature until dosing. The dosing formulations and vehicle were continuously stirred until and during dosing. Adjustment was made for specific gravity of the vehicle (a correction factor of 1.036) was used. A correction was made for the purity/composition of the test item. A correction factor of 1 was used.
- the dose volume for each animal will be based on the most recent body weight measurement
- the dose will be given using a plastic feeding tube

VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on trial formulations performed at Charles River Den Bosch during preparation of Test Facility Study Nos. 513675 and 513680.
- Dose concentrations: 0, 22, 66, 200 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dose formulation samples were collected for analysis on week 1 of treatment.
- concentration (middle): all groups, 6x approx 500 mg
- homogeneity (top, middle, bottom): groups 2 and 4, 6x approx 500 mg
- sampling from dosing container
All samples to be analysed were transferred (at room temperature) to the analytical laboratory for same day analysis.
Analyses were performed using a validated analytical procedure (Test Facility Study No. 513680).
- concentration and homogeneity analysis:
- storage conditions: temperature set to maintain 18-22 °C
- Acceptance Criteria: For concentration, mean sample concentration results within or equal to
± 15% of theoretical concentration. For homogeneity, relative standard deviation (RSD) of concentrations of <=10% for each group.
- stability analysis: Stability analyses performed previously in conjunction with the method development and validation study demonstrated that the test item formulations at low target level were unstable when stored in the refrigerator for at least 15 days, and formulation at high target level (200 mg/mL) were stable when stored in the refrigerator for at least 15 days. Formulations at low and high target level were stable when stored at room temperature under normal laboratory light conditions for at least 6 hours. Homogeneity was confirmed in Study No. 513675.
Details on mating procedure:
- M/F ratio per cage: not indicated
- Length of cohabitation: not indicated
- Target Age at Mating: approximately 10-14 weeks.
Untreated females were mated at the Supplier and Day 0 was the day of successful mating.
- number of fetuses expected: approx. 1056 fetuses (88 litters x 12 fetuses)
Duration of treatment / exposure:
Day 6 to Day 20 post coitum
Frequency of treatment:
Once daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Group 1 (vehicle only)
Dose / conc.:
110 mg/kg bw/day (nominal)
Remarks:
Group 2 (Low dose)
Dose / conc.:
330 mg/kg bw/day (nominal)
Remarks:
Group 3 (mid dose)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
Group 4 (High dose)
No. of animals per sex per dose:
22 females per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose levels were selected based on information provided by the results of a (Combined 28-Day Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test of JNJ-42808389-AAA (T003421) in rats, Test Facility Study No. 513675), and in an attempt to produce graded responses to the test item. The high-dose level should produce some toxic effects, but not excessive lethality that would prevent meaningful evaluation. The mid-dose level is expected to produce minimal to moderate toxic effects. The low-dose level should produce no observable indications of toxicity.

Examinations

Maternal examinations:
MORTALITY: Yes
-Time schedule: At least twice daily beginning upon arrival through termination/release. Except on days of receipt and necropsy where frequency was be at least once daily.
- all parental animals
- Procedure: Animals were observed within their cage unless necessary for identification or confirmation of possible findings.

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least once daily; starting on Day 6 post-coitum up to the day prior to necropsy. 0-1 hour post-dose.
- all parental animals
- Procedure: Animals were observed within their cage unless necessary for identification or confirmation of possible findings. Cage debris were examined to detect premature birth, if applicable.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: On Days 2, 6, 15 and 21 post-coitum
- all parental animals
- Procedure: Animals are removed from the cage

BODY WEIGHT: Yes
- Time schedule for examinations: On Days 2, 6, 9, 12, 15, 18 and 21 post-coitum
- all parental animals
- Procedure: In order to monitor the health status animals might have been weighed more often (documented in the raw data).
- body weight gain was calculated for the following intervals: days 6 to 9, 9 to 12, 12 to 15, 15 to 18, 18 to 21
- corrected body weight gain: body weight on day 21 post-coitum - body weight on day 6 post-coitum - gravid uterus weight

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Time schedule: Over Days 2-6, 6-9, 9-12, 12-15, 15-18 and 18-21 post-coitum
- all parental animals
- Procedure: quantitatively measured.
- overall food consumption: calculated between each scheduled interval (individual data only) and as specified above for body weight gains

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No
- Time schedule for examinations: Regular basis throughout the study.
- all parental animals
- Procedure: Water consumption was monitored by visual inspection of the water bottles. Data were used for health monitoring of the animals only and therefore were not be reported.

CLINICAL PATHOLOGY: Yes
- Time schedule for collection of blood: on day 21 post-coitum, Sampled (1.0mL) between 07.00 and 09.00 from the jugular vein.
- Animals fasted: no
- Anaesthetic used for blood collection: not indicated
- anticoagulant: not applicable for serum
- How many animals: All F0 animals
- Thyroid hormone analysis: Triiodothyronine (T3), Thyroid-Stimulating Hormone (TSH), Thyroxine (T4).
The serum for T3, T4 and TSH analysis was divided in two aliquots. One aliquot was used for measurement of thyroid hormones using the IMMULITE® 1000 analyser (TSH). These samples were stored in an ultra-low freezer (≤ -75°C) until analysis.
The other aliquot was used for measurement of T3 and T4 using LC-MS. The aliquots for T3 and T4 was collected in uniquely labelled clear 1.4 mL V-bottom Micronic polypropylene tubes and stored in a freezer (≤ -15°C) until analysis. Measurements were performed according to the bioanalytical method validated in Test Facility Study No. 20213516

POST MORTEM EXAMINATION: Yes
- Sacrifice (day 21 post coitum):
Unscheduled euthanasia: Animals were euthanized using the carbon dioxide inhalation (gradual fill) procedure. If necessary, the animal was refrigerated to minimize autolysis.
Scheduled euthanasia: Animals surviving until scheduled euthanasia on Day 21 post-coitum were euthanized using the carbon dioxide inhalation (gradual fill) procedure. No body weight was recorded at necropsy
- Necropsy:
All animals (including animals found dead or sacrificed before planned necropsy and females with delivery prior to necropsy) were subjected to an external, thoracic and abdominal examination, with special attention being paid to the reproductive organs. All macroscopic abnormalities were recorded, collected and fixed in the appropriate fixative, together with the animal identification; collection of specific macroscopic abnormalities may be omitted only at the discretion of the Study Director.
- Organ weight (F0- Generation)
The thyroid gland was weighed at necropsy for all scheduled euthanasia animals, except for females that delivered their offspring prior to necropsy. Organ weights were also not recorded for animals found dead, euthanized in poor condition or in extremis or that delivered their offspring prior to necropsy. Paired organs were weighed together. Organ weight relative to body weight (using the body weight on Day 21 post-coitum) was calculated.
- Histology
The thyroid gland and macroscopic abnormalities are collected from all animals and preserved in 10% buffered formalin. Thyroid gland of all animals were embedded in paraffin, sectioned at a thickness of 2-4 micrometers, mounted on glass slides, and stained with hematoxylin and eosin.
-Microscopic evaluation
Thyroid gland of all animals were examined by a board-certified toxicological pathologist with training and experience in laboratory animal pathology.
Target tissues identified by the study pathologist during microscopic evaluation were evaluated and reported.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes, each ovary and uterine horn of all animals was dissected and examined as quickly as possible to determine the following:
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Number and distribution of live and dead fetuses: Yes
- Individual fetal weight
- the sex of each fetus based on anogenital distance, if possible

In case no macroscopically visible implantation sites are present, nongravid uteri were stained using the Salewski technique in order to detect any former implantation sites.
Blood sampling:
- Plasma: No
- Serum: Yes, for thyroid hormone analysis
- Volume collected: 1.0 mL
Fetal examinations:
- Sacrifice:
Live fetuses were euthanized by administration of sodium pentobarbital into the oral cavity using a small metal feeding tube. In case this was not possible due to a malformation, the fetus was euthanized by decapitation or by interscapular injection of sodium pentobarbital.
Recognizable live fetuses of females found dead or sacrificed before planned necropsy or pups from females that delivered prior to necropsy, was euthanized by decapitation.
Malformed late resorptions were collected and fixed in 10% buffered formalin. Late resorptions without malformations were discarded.
The following examinations were performed on viable and non-viable fetus of dam surviving until scheduled necropsy on day 21 post-coitum:
- internal sex confirmation: 100% of the litter
- visceral body examinations: Yes, approx. half the litter. abnormalities may be collected and fixed at the discretion of the Study Director. Fetuses were eviscerated, fixed in 96% aqueous ethanol, macerated in potassium hydroxide and stained (Alizarin Red S)
- Skeletal examinations: Yes, approx. half the litter with head. Fetuses were eviscerated, fixed in 96% aqueous ethanol, macerated in potassium hydroxide and stained (Alizarin Red S). Specimens will be archived in glycerin with bronopol as preservative.
- visceral head examinations: Yes, half fetuses per litter viscerally screened. Fixed in Bouin's solution, tissues were stored until finalisation of the study

In the case of (suspected) visceral malformations in fetuses that are selected for skeletal examination, this fetus will only be examined for visceral abnormalities in first instance. In the case of (suspected) skeletal malformations in fetuses that are selected for visceral examination, this fetus will only be examined for skeletal abnormalities in first instance.
Statistics:
see section: Any other information on materials and methods incl. tables
Indices:
Pregnancy Rate (%) = (No. of pregnant females / No. of mated females) x 100
Male Fetuses (%) = (No. of male fetuses / No. of fetuses) x 100
Female Fetuses (%)= (No. of female fetuses / No. of fetuses) x 100
Pre-Implantation Loss (%) = ((No. of corpora lutea – No. of implantations) / No. of corpora lutea) x 100
Post-Implantation Loss (%) = ((No. of implantations – No. of live fetuses) / No. of implantations) x 100
Litter % of Fetuses with Abnormalities = (No. of fetuses in litter with a given finding /
No. of fetuses in litter examined) x 100
Historical control data:
Historical control data regarding thyroid hormone levels, fetal pathology is available on record.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Hunched posture was observed in one female at 330 mg/kg/day (No. 47) and in three females at 1000 mg/kg/day (Nos. 72, 73 and 76) and erected fur in one female at 110 and 330 mg/kg/day each (Nos. 36 and 65, respectively). Both clinical signs were only observed in test item treated animals and were therefore considered test item-related.
Salivation seen after dosing among two females at 330 and 1000 mg/kg/day each (Nos. 47 and 56 at 330 mg/kg/day and Nos. 72 and 80 at 1000 mg/kg/day) was considered not toxicologically relevant taking into account the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). This sign was considered to be a physiological response rather than a sign of systemic toxicity.
Any other clinical signs noted during the treatment period (i.e., abnormal breathing sounds, fur loss and skin scabs) 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 and did not show any apparent dose-related trend. At the incidence observed, these were considered to be unrelated to treatment with the test item
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
No unscheduled mortality occurred during the study period.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
No toxicological relevant effects were observed on mean body weight, mean body weight gain and weight gain corrected for gravid up to 1000 mg/kg/day.
Any statistically significant changes in body weight gain were considered to be unrelated to treatment with the test item as no trend was apparent regarding dose. At 110 mg/kg/day, body weight gain was lower between Days 15-18 post-coitum and over Days 6-21 post-coitum when compared to the control group. This was most likely caused by the statistically significant lower gravid uterus weight (16% lower compared to control) on Day 21 postcoitum, as body weight gain corrected for gravid uterus weight was comparable to control.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption was similar to the control level over the study period in animals treated up to 1000 mg/kg/day.
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:
not examined
Endocrine findings:
effects observed, non-treatment-related
Description (incidence and severity):
Note: Lower limit of quantification were 0.008 µIU/mL, 0.1 ng/mL and 5.00 ng/mL for thyroid stimulating hormone (TSH), triiodothyronine (Total T3) and total thyroxine (Total T4), respectively.

Serum levels of Total T3 and Total T4 were considered to be unaffected by treatment with the test item up to 1000 mg/kg/day.

Slightly increased serum levels of TSH were noted at all test item-treated groups (0.3106, 0.3126 and 0.3939 µIU/mL at 110, 330 and 1000 mg/kg/day, respectively, compared to 0.2504 µ IU/mL in the control group, not reaching statistical significance). However, as the concurrent mean value of TSH is at the low end of the available historical control data and all mean values of test item-treated groups are well within the historical control data, this slight increase was considered unrelated to treatment with the test item.
The increased mean value at 1000 mg/kg/day was mainly caused by Female No. 69, for which a TSH serum level of 2.020 µIU/mL (8.1x of the mean control level) was measured.
For this individual animal, T3 level was below LLOQ and its T4 concentration was 7.2 ng/mL (0.33x of the mean control level).
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Thyroid weights and thyroid body weight ratios of animals treated up to 1000 mg/kg/day were considered to be similar to those of control animals.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Macroscopic observations at necropsy did not reveal any alterations that were considered to have arisen as a result of treatment with the test item.
Findings that were noted among control and/or treated animals were considered to be of no toxicological significance, since they remained within the range of biological variation for rats of this age and strain
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related microscopic observations in the thyroid glands up to 1000 mg/kg/day.
The recorded microscopic findings in the thyroid gland were within the range of background pathology encountered in rats of this age and strain.

Histopathological findings: neoplastic:
not examined
Other effects:
not specified

Maternal developmental toxicity

Number of abortions:
no effects observed
Description (incidence and severity):
All females were gravid and had litters with viable fetuses.
Pre- and post-implantation loss:
effects observed, non-treatment-related
Description (incidence and severity):
The numbers corpora lutea and implantation sites, and pre- and post-implantation loss in the control and test groups were comparable and in the range of normal biological variation.
The slightly higher percentage of pre-implantation loss observed at 110 mg/kg/day was caused by two individual females (Nos. 39 and 43), which had a pre-implantation loss of 83.3% and 85.7%, respectively. As this was observed in the low dose group only, and as treatment was only initiated after implantation was completed, this was unrelated to treatment with the test item.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
All females were gravid and had litters with viable fetuses.
Early or late resorptions:
no effects observed
Description (incidence and severity):
All females were gravid and had litters with viable fetuses.
Dead fetuses:
no effects observed
Description (incidence and severity):
All females were gravid and had litters with viable fetuses.
Changes in pregnancy duration:
not specified
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
All females were gravid and had litters with viable fetuses.

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Remarks on result:
not determinable due to absence of adverse toxic effects

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
There were no test item-related effects on fetal body weights (both sexes) noted by treatment
with the test item up to 1000 mg/kg/day.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
There were no test item-related effects on litter size of any group.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
The male:female ratio was unaffected by treatment with the test item up to 1000 mg/kg/day.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
There were no test item-related effects on litter size of any group.There were no test item-related effects on fetal body weights (both sexes) noted by treatment with the test item up to 1000 mg/kg/day.
Anogenital distance of all rodent fetuses:
no effects observed
Description (incidence and severity):
There were no test item-related effects on fetal anogenital distance (both sexes) noted after
treatment up to 1000 mg/kg/day.
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Description (incidence and severity):
There were no external malformations or variations observed in any groups.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
No skeletal malformations or variations were observed up to 1000 mg/kg/day that were considered to be test item-related.
The only skeletal malformations noted were a bent humerus (Fetus No. 8-06, control) and supernumerary lumbar vertebra (Fetus No. 84-L8, 1000 mg/kg/day). These were considered chance findings due to single occurrence and/or occurrence in the control group only.
At 330 mg/kg/day, the incidence variation “misaligned sternebrae” was statistically significantly decreased (0 fetuses compared to 6(6) fetuses (litters) in the control group). The absence of fetuses presenting with misaligned sternebrae is not considered toxicologically relevant. Moreover, as dose-relationship was lacking, this decreased incidence was considered unrelated to treatment with the test item.
All other skeletal variations occurred in the absence of a dose-related incidence trend, infrequently and/or in control fetuses only. Therefore, they were considered not to be test item-related.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
No visceral malformations or variations were observed up to 1000 mg/kg/day that were considered to be test item-related.
At 110 mg/kg/day, Fetus No. 41-L4 was observed with the malformations “situs inversus” and “fused lung lobes”. Due to single occurrences of these malformations and as they occurred at the low dose group only, they were considered chance findings.
Visceral variations (such as absent renal papilla, supernumerary liver lobes, small spleen and convoluted and/or dilated ureter) were seen in single fetuses or observed in test item groups with incidences comparable to the control group. These findings did not suggest any association with the test item.
Other effects:
not specified

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
no

Any other information on results incl. tables

Dose formulation analyses


Accuracy of preparation and homogeneity of the test substance in formulations was determined. 


- accuracy: the concentrations analyzed in the formulations of groups 2, 3 and 4 were in agreement with target concentrations (ie, mean sample concentration results were within or equal to 85.00-115.00% of target concentration). No test item was detected in the Group 1 (vehicle control) formulation.


- homogeneity: the formulations of groups 2 and 4 were homogeneous (ie coefficient of variation <=10.00%)


 


Summary of Body Weight Gains (g): Gestation- Bodyweight Gain (Interval)






























































































Sex Female



Days relative to mating



6-9



9-12



12-15



15-18



18-21



6-21



0 mg/kg/day


22 animals



Mean



11.8



15.6



14.8



30.0



37.9



110



SD



4.2



3.2



5.3



5.7



6.5



13.1



110 mg/kg/day


22 animals



Mean



10.1



14.7



14.6



24.0**



32.0



95.5**



SD



4.3



3.8



5.3



7.5



8.3



19.0



330 mg/kg/day


22 animals



Mean



10.7



16.7



13.9



30.3



36.4



108.0



SD



2.1



4.7



3.8



5.9



7.0



14.0



1000 mg/kg/day


22 animals



Mean



10.9



14.7



14.0



27.6



34.8



102.1



SD



3.3



3.5



3.1



5.8



8.9



15.3



Anova & Dunnett: ** = p ≤ 0.01


 


Summary of Gravid Uterine Weights and Gravid Uterus Adjusted Body Weights: Gestation 





































































































Sex Female


Days relative to mating



0


mg/kg/day



110


mg/kg/day



330


mg/kg/day



1000


mg/kg/day



Bodyweight on Day 6 (g) [G]



Mean



212.6



210.1



211.7



209.2



SD



20.2



12.2



18.9



17.5



%diff



-



-1.2



-0.4



-1.6



Terminal Body Weight (g) [G]



Mean



322.6



305.6



319.7



311.3



SD



27.5



20.0



29.6



29.2



%diff



-



-5.3



-0.9



-3.5



Gravid Uterus Weight (g) [G]



Mean



80.18



67.33*



75.15



72.38



SD



11.66



18.58



14.44



13.50



%diff



-



-16.06



-6.28



-9.73



Adjusted BWG (6-abw) (g) [G]



Mean



29.86



28.17



32.85



29.71



SD



8.17



9.45



9.51



7.33



%diff



-



-5.68



10.02



-0.50



[G] - Anova & Dunnett: * = p ≤ 0.05


 


Summary of Thyroid Hormone Values - Day: 21 Relative to Mating













































































































Sex Female



Reporting Special Chemistry



T3


(ng/mL)


(G)



T4


(ng/mL)


(G)



TSH


(mU/L)


(G)



0 mg/kg/day


 



Mean



0.396



22.13



0.2504



SD



0.065



3.83



0.1098



N



22



22



22



110 mg/kg/day


 



Mean



0.445



23.41



0.3106



SD



0.110



5.77



0.1931



N



22



22



22



tCtrl



1.13



1.06



1.24



330 mg/kg/day


 



Mean



0.432



24.03



0.3126



SD



0.081



6.69



0.1658



N



22



22



22



tCtrl



1.09



1.09



1.25



1000 mg/kg/day


 



Mean



0.420



23.12



0.3939



SD



0.093



6.96



0.3885



N



21



22



22



tCtrl



1.06



1.04



1.57



 (G) - Anova & Dunnett 


 


Summary of Ovarian and Uterine Examinations and Litter Observations




















































































































Sex Female


Days relative to mating



0


mg/kg/day



110


mg/kg/day



330


mg/kg/day



1000


mg/kg/day



Female with live fetuses



N+ve



22



22



22



22



%



100.0



100.0



100.0



100.0



Number of Corpora Lutea [k]



Mean



12.5



12.3



11.6



12.0



SD



1.2



2.5



2.0



2.0



%diff



-



-2.2



-7.2



-4.7



Number of Implantations [k]



Mean



12.0



10.0



11.1



10.9



SD



1.7



3.2



2.4



2.2



%diff



-



-16.0



-7.2



-8.7



Pre-implantation Loss (%) [k]



Mean



5.02



16.66



5.20



8.18



SD



7.38



24.54



8.34



13.41



%diff



-



231.56



3.48



62.91



Total Number of Fetuses [k]



Mean



11.8



9.7



10.6



10.5



SD



1.9



3.0



2.4



2.3



%diff



-



-17.8



-10.0



-11.2



[k] - Kruskal-Wallis & Dunn

Applicant's summary and conclusion

Conclusions:
In conclusion, based on the results of this prenatal developmental toxicity study in time-mated female Wistar Han rats, the maternal and developmental No Observed Adverse Effect Levels (NOAELs) for JNJ-42808389-AAA (T003421) were established as being at least 1000 mg/kg/day.