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Diss Factsheets

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

In a combined 28 -day repeated dose toxicity study with the reproduction/developmental toxicity screening test, the test substance was administered daily to rats at dose levels up to 1000 mg/kg bodyweight/day (OECD 422; Peter, 2017). The parental and reproduction NOAEL was established as at least 1000 mg/kg/day.


The test item is not to be classified as reproductive toxicant according to CLP Regulation.

Link to relevant study records

Referenceopen allclose all

Endpoint:
extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
the extended one-generation reproductive toxicity study does not need to be conducted because there are no results from available repeated dose toxicity studies that indicate adverse effects on reproductive organs or tissues, or reveal other concerns in relation with reproductive toxicity
Reason / purpose for cross-reference:
data waiving: supporting information
Reason / purpose for cross-reference:
data waiving: supporting information
Reason / purpose for cross-reference:
data waiving: supporting information
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-08-29 to 2016-12-20
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: OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developm ental Toxicity Screening Test
Deviations:
no
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: I15AB0305
- Expiration date of the lot/batch: 2017-01-23


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature


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. 11 weeks (at start F0-treatment); females approx. 11 weeks (at start pretest) and approx. 13 weeks (at start F0-treatment).
- Weight at study initiation: 311-315 grams
- 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 (MI II 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).
Route of administration:
oral: gavage
Vehicle:
propylene glycol
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily within 6 hours prior to dosing and were homogenized to a visually acceptable level. No adjustment was made for specific gravity/density of the test item.
Adjustment was made for specific gravity of the vehicle (1.036). A correction was made for the purity/composition of the test item. A correction factor of 1 was used.


VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on trial formulations performed at Charles River Den Bosch.
- Concentration in vehicle: 5 mL/kg body weight
- Amount of vehicle (if gavage): 0, 110, 330, 1000 mg/kg
Details on mating procedure:
- M/F ratio per cage: 1 animal/sex/cage
- Length of cohabitation: Following a minimum of 14 days of treatment for the males and females, one female was cohabitated with one male of the same treatment group, avoiding sibling mating, until detection of mating was confirmed.
- Proof of pregnancy: Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating was confirmed, the males and females were separated.
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): Females were individually housed in Macrolon plastic cages (MIII type, height 18 cm)
- Any other deviations from standard protocol: no
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were conducted on a single occasion during the treatment phase (26 October 2016, Day 2 of treatment) according to a validated method (Test Facility Study No. 512709).
Sextuplicate samples (i.e. 3 sets of duplicate samples) were collected. Two sets of duplicate samples were stored in the refrigerator as reserve samples. Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations).
In addition to the criteria mentioned in the validated analytical method, each calibration curve was accepted if the average of the retention times and response factors of the data points used to construct the calibration line were within a range of ±10.00% compared to those obtained during the method validation. The accuracy of preparation was considered acceptable if the mean measured concentrations were 85.00-115.00% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10.00%. Once analytical results were approved in the raw data by the Principal Scientist, the reserve samples were destroyed.
Duration of treatment / exposure:
Males-29 days ; 50-56 days (females that delivered); 39 days (females with total litter loss). 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:
Control
Dose / conc.:
110 mg/kg bw/day (nominal)
Remarks:
Group 2
Dose / conc.:
330 mg/kg bw/day (nominal)
Remarks:
Group 3
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
Group 4
No. of animals per sex per dose:
10 animals/sex/dose level
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were selected based on the results of the acute oral
toxicity study (LD50>2000 mg/kg; data on file at Sponsor site)
- Rationale for animal assignment (if not random): randomized
Positive control:
no
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
Once prior to start of treatment and at weekly intervals during the treatment period


DETAILED CLINICAL OBSERVATIONS: Yes
At least once daily from start of treatment onwards up to the day prior to necropsy, detailed clinical observations were made for all animals, at least 1 hour ( ± 15 minutes) after treatment (on the peak period of anticipated effects after treatment).


BODY WEIGHT: Yes
Males and females were weighed on the first day of treatment (prior to first dosing) and weekly ther eafter. 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. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on PND 1, 4, 7 and 13.
Relative food consumption was calculated and reported.


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


HAEMATOLOGY: Yes
- blood samples were collected at the end of the treatment period on the day of the scheduled necropsy from the selected 5 animals/sex/group under anaesthesia using isoflurane. The animals were deprived of food overnight (with a maximum of 24 hours) before blood sampling, but water was available.
Blood samples were drawn from the retro-orbital sinus and collected into tubes with K2-EDTA for hematology parameters, and with citrate for clotting tests
- parameters assessed: white blood cells, differential white blood counts, red blood cells, reticulocytes, red blood cell distribution width, haemoglobin, haematocrit, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, platelets, prothrombin time, activated partial thromboplastin time


CLINICAL CHEMISTRY: Yes
- blood samples were collected at the end of the treatment period on the day of the scheduled necropsy from the selected 5 animals/sex/group under anaesthesia using isoflurane. The animals were deprived of food overnight (with a maximum of 24 hours) before blood sampling, but water was available. Blood samples were drawn from the retro-orbital sinus and collected into tubes with Li-heparin 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, and static righting reflex, fore- and hind-limb grip strength, locomotor activity. Total movements and ambulations are reported. The selected males were tested during Week 4 of treatment and the selected females were tested once during the last week of lactation (e.g. PND 6-13). These tests were performed after observation for clinical signs.
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. During pretest, this was done for 48 females.
On the day of scheduled necropsy, a vaginal lavage was taken to determine the stage of estrous.
Sperm parameters (parental animals):
Parameters examined in F0 male parental generation: additional slides of the testes to examine staging of spermatogenesis; testis weight
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: No. All pups were randomized per litter and individually identified by means of subcutaneous injection of Indian ink on post-natal day 1 (= day the litter was found completed)
- Maximum of 8 pups/litter (4/sex/litter as nearly as possible) were selected for culling on PND4; 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 PND1 and daily thereafter . If possible, defects or cause of death were evaluated.
- Clinical signs: At least once daily, detailed clinical observations were made for all animals. Only days on which clinical signs were present between first and last litter check are presented in the respective table in the study report.
- Body weights: Live pups were weighed on PND 1, 4, 7 and 13.
- Sex: Sex was determined for all pups on PND1 and 14.
- Anogenital distance: Anogenital distance (AGD) was measured for all live pups on PND1. The AGD was normalized to the cube root of body weight.
- Areola/nipple retention: On PND13, all males in each litter were examined for the number of areola/nipples.


GROSS EXAMINATION OF DEAD PUPS:
Yes, 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
The animals were deprived of food overnight (with a maximum of 24 hours) before blood sampling, but water was available.
All animals surviving to the end of the observation period were deeply anaesthetized using isoflurane and subsequently exsanguinated and subjected to a full post mortem necropsy, with special attention being paid to the reproductive organs.

GROSS NECROPSY
Necropsy was conducted according to the following schedule:
• Males: following completion of the mating period (after 28 days of dose administration).
• Females which delivered: on PND 14-16
• Female No 59 with total litter loss: within 24 hours of litter loss.

ORGAN WEIGHTS
- Absolute organ weights and organ to body weight ratios were reported.
- The following organ weights and terminal body weight were recorded from the selected 5 animals/ sex/ group on the scheduled day of necropsy: Adrenal glands, Brain, Epididymides, Heart, Kidneys, Liver, Ovaries, Prostate, Seminal vesicles including coagulating glands, Spleen, Testes, Thymus, Thyroid, including parathyroid if detectable, Uterus, including cervix
- The following organ weights and terminal body weight were recorded from all remaining animals on the scheduled day of necropsy: Epididymides, Prostate, Seminal vesicles, including coagulation glands, Testes, Thyroid


HISTOPATHOLOGY
The following slides were examined by a pathologist:
- The preserved organs and tissues of the selected 5 animals/sex of Groups 1 and 4
- Additional slides of the testes of the selected 5 males of Groups 1 and 4.
- The mammary gland area of female no. 59 with total litter loss.
- All gross lesions of all animals (all dose groups).
- The reproductive organs of the male that failed to sire and the female that failed to deliver healthy pups( cervix, clitoral gland, coagulation gland, epididymides, ovaries, preputial gland, prostate gland, seminal vesicles, testes, uterus, and vagina)
Postmortem examinations (offspring):
SACRIFICE
On PND 4 (at culling), from 2 pups per litter, blood samples (0.4 mL in total) were collected by decapitation between 7.00 and 10.30 a.m. Blood samples from the 2 pups per litter were collected into one serum tube. All remaining pups (PND 13-15) were sacrificed using Euthasol® 20% by intraperitoneal (ip) injection.
On PND 13-15, from 2 pups per litter10 (if possible from one male and one female) blood samples were collected at planned necropsy. As part of the necropsy procedure, blood samples (0.9 mL) were drawn by aorta puncture under anaesthesia using isoflurane, between 7.00 and 10.30 a.m., followed by exsanguination. Blood was collected into serum tubes.

GROSS NECROPSY
Culling - PND 4.
Terminal sacrifice - PND 13-15.
All pups were sexed by both external as well as internal examination. Descriptions of all abnormalities were recorded.
At terminal sacrifice (PND 13-15), the thyroid from 2 pups per litter10, i.e. the same pups as selected for blood sampling, was preserved in 10% buffered formalin.

HISTOPATHOLOGY / ORGAN WEIGTHS
not examined
Statistics:
The following statistical methods were used to analyse the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one ttest)
based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test 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.
In case intergroup differences were seen, the Wilcoxon test was applied to compare the treated groups to the control group.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.
Reproductive indices:
For each group, the following calculations were performed:
Mating index (%) = (Number of females mated/Number of females paired) x 100
Fertility index (%) = (Number of pregnant females/Number of females mated) x 100
Gestation index (%) = (Number of females bearing live pups/Number of pregnant females) x 100
Duration of gestation = Number of days between confirmation of mating and the beginning of parturition
Offspring viability indices:
Survival indices:
Post-implantation survival index (%) = (Total number of offspring born/Total number of uterine implantation sites) x 100
Post-implantation survival index was expressed as 100% when the number of offspring exceeded the number of implantation sites recorded.
Live birth index (%) = (Number of live offspring on Day 1 after littering/Total number of offspring born) x 100
Viability index (%) = (Number of live offspring on Day 4 before culling/Number live offspring on Day 1 after littering) x 100
Lactation index (%) = Number of live offspring on Day 13 after littering/Number live offspring on Day 4 (after culling) x 100
Group mean values were calculated from individual litter values.
Sex ratio (percentage males) = (Number of males in litter/Total number of offspring in litter) x 100
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No clinical signs of toxicity were noted during the observation period.
Incidental findings that were noted included rales, scabs, alopecia, salivation and enophthalmos. As these 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, were seen in control animals only or did not show any apparent dose-related trend, they were considered to be unrelated to treatment.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One female at 110 mg/kg (no. 59) was sacrificed on Day 2 of the lactation phase due to total litter loss.
All remaining animals survived until scheduled necropsy.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Body weights and body weight gain of treated animals remained in the same range as controls over the treatment period.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption before or after allowance for body weight was similar between treated and control animals.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Haematological parameters of treated rats were not considered to be affected by treatment.
Any statistically significant changes were not considered to be toxicologically relevant as they occurred in the absence of a treatment-related distribution and remained within the range considered normal for rats of this age and strain.
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.
The statistically significantly lower creatinine level in males at 110 mg/kg was not considered to be toxicologically relevant as they occurred in the absence of a treatment-related distribution and remained within the range considered normal for rats of this age and strain.
One female at 110 mg/kg (no. 55) showed a notably high bile acid level of 384 umol/L. All other parameters for liver function and liver morphology were normal in this female. This together with the fact that other individual bile acid values of this group remained in the range considered normal for rats of this age and strain and as no dose-response relationship was observed, it was 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, static righting reflex and grip strength were normal in all selected animals. The variation in motor activity did not indicate a relation with treatment. All groups showed a similar habituation profile with a decreasing trend in activity over the duration of the test period. At 330 mg/kg, statistically significantly increased grip strength of the foreleg for males (1543 vs. 1316 gram for controls) and total ambulations for females (1077 vs. 611 counts for controls) were noted.
These changes were not considered to be toxicologically relevant as they occurred in absence of a dose-related trend.
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. The recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain.
Reproductive performance
There was 1/10 couples (male no. 19 and female no. 59) of the 110 mg/kg group with total litter loss.
At histopathology inflammation of the uterine endometrium was noted. This finding was considered to be related to the early post-natal phase at the time of total litter loss and not test item-related. All other tissues (including mammary gland) were normal.
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Description (incidence and severity):
Parental results:
No parental toxicity was observed up to 1000 mg/kg.
No treatment-related effects were noted in any of the parental parameters investigated in the study (i.e. clinical appearance, functional observations, body weight, food consumption,clinical laboratory investigations, macroscopic examination, organ weights, and microscopic
examination).
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
Length and regularity of the estrous cycle were not affected by treatment. All females had regular cycles of 4-5 days.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
Spermatogenic staging profiles were normal (at least unilateral) for all males examined
Reproductive performance:
effects observed, non-treatment-related
Description (incidence and severity):
Reproduction Data
Estrous Cycle- Length and regularity of the estrous cycle were not affected by treatment. All females had regular cycles of 4-5 days.
Mating index- Mating index was not affected by treatment. All females showed evidence of mating.
Precoital time- Precoital time was not considered to be affected by treatment. All females were mated within 4 days.
Number of implantation sites - Number of implantation sites was not considered to be affected by treatment. For female nos. 49 (Group 1), 55, 57 (Group 2), 63 (Group 3), 71, 73 and 78 (Group 4) the number of pups born was slightly higher than the number of implantations. This was considered to be due to normal resorption of these areas as the enumeration was performed on Days 14 or 16 of lactation.
Fertility index - Fertility index was not affected by treatment. All mated females were pregnant
Developmental Data
Gestation index and duration - Gestation index and duration of gestation were not considered to be affected by treatment. All pregnant females delivered live pups after 21-22 days of gestation.
Parturition/maternal care - No signs of difficult or prolonged parturition were noted among the pregnant females. Examination of cage debris of pregnant females revealed no signs of premature birth. No treatment-related deficiencies in maternal care were observed. Deficiencies in maternal care were observed for one female at 330 mg/kg (no. 61). Three pups were noted cold on PND 2 and two pups had a lean appearance on one or more days during PND 1-10. In addition, 2/10 pups were found missing on PND 2-4. The remaining pups survived until scheduled necropsy on PND 13. As this was observed for a single female of the mid dose group only, it was considered unrelated to treatment.
Post-implantation survival index - The total number of offspring born compared to the total number of uterine implantations was not considered to be affected by treatment.
Litter size - Litter size was not affected by treatment.
Live birth index - The number of live offspring on Day 1 after littering compared to the total number of offspring born was not considered to be affected by treatment. Five pups at 110 mg/kg (litter nos. 54, 57 and 59) and two pups at 330 mg/kg (litter no. 64) were found dead at first litter check. No toxicological relevance was attributed to these dead pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.
Viability index - The number of live offspring on Day 4 before culling compared to the number of offspring on Day 1 was not considered to be affected by treatment. One pup of the control group (litter no. 44), seven pups at 110 mg/kg (litter no. 59; total litter loss on PND 2) and seven pups at 330 mg/kg (litter nos. 61, 62, 64 and 65) were missing on PND 2-4. Pups missing were most likely cannibalised. No toxicological relevance was attributed to these missing pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.
Lactation index - The number of live offspring on Day 13 after littering compared to the number of live offspring on Day 4 (after culling) was not affected by treatment. One pup at 330 mg/kg (litter no. 69) was found dead on PND 6. As this was a single pup in the mid dose group, no toxicological relevance was attributed to this finding.

Reproductive results:
No reproduction toxicity was observed up to 1000 mg/kg. No treatment-related effects were noted in any of the reproductive parameters investigated in this study (i.e. mating and fertility indices, precoital time, and number of implantations, estrous cycle, spermatogenic profiling, and histopathological examination of reproductive organs).
Key result
Dose descriptor:
NOAEL
Remarks:
parental toxicity
Effect level:
>= 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse changes were noted in any of the parameters examined in this study.
Key result
Dose descriptor:
NOAEL
Remarks:
reproduction toxicity
Effect level:
>= 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse changes were noted in any of the parameters examined in this study.
Key result
Critical effects observed:
no
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No clinical signs occurred among pups that were considered to be related to treatment. 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:
mortality observed, non-treatment-related
Description (incidence and severity):
The number of live offspring on Day 4 before culling compared to the number of offspring on Day 1 was not considered to be affected by treatment.
One pup of the control group (litter no. 44), seven pups at 110 mg/kg (litter no. 59; total litter loss on PND 2) and seven pups at 330 mg/kg (litter nos. 61, 62, 64 and 65) were missing on PND 2-4. Pups missing were most likely cannibalised. No toxicological relevance was attributed to these missing pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.
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:
effects observed, non-treatment-related
Description (incidence and severity):
No toxicologically relevant effects on serum T4 levels in male and female PND 13-15 pups were noted.
The statistically significantly lower T4 levels in male pups of all treated groups compared to controls was not considered to be toxicologically relevant as the changes were only slight, occurred in the absence of a dose-related trend and remained within the range of available historical control data.
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:
no effects observed
Description (incidence and severity):
Anogenital distance
Anogenital distance (absolute and normalized for body weight) in male and female pups was not considered to be affected by treatment.
Areola/nipple retention
Treatment up to 1000 mg/kg had no effect on areola/nipple retention. For none of the examined male pups nipples were observed at PND 13.
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Developmental results:
No developmental toxicity was observed up to 1000 mg/kg.
No treatment-related effects were noted in any of the developmental parameters investigated in this study (i.e. gestation, viability and lactation indices, duration of gestation, parturition, sex ratio, maternal care and early postnatal pup development consisting of mortality, clinical signs, body weight, anogenital distance (PND 1), areola/nipple retention (PND 13 males), T4 thyroid hormone levels (PND 13-15) and macroscopy.
Dose descriptor:
NOAEL
Remarks:
developmental toxicity
Generation:
F1
Effect level:
>= 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse changes were noted in any of the parameters examined in this study.
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
no
Conclusions:
In conclusion, treatment with JNJ-42808415-AAA (T003422) by oral gavage in male and female Wistar Han rats at dose levels of 110, 330 and 1000 mg/kg revealed no parental, reproduction and developmental toxicity up to 1000 mg/kg.
Based on these results, the No Observed Adverse Effect Level (NOAEL) was concluded to be of at least 1000 mg/kg.
Therefore, the substance is not classified as a repeated dose toxicant (STOT RE) according to the CLP Regulation.
Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
GLP-compliant study, performed according to OECD guidelines
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

Toxicity to reproduction


A combined repeated dose toxicity study with the reproduction/developmental toxicity screening test was performed in rats, in which male and female rats were exposed to 0 (vehicle), 110, 330, and 1000 mg/kg bw/day via gavage (OECD 422; Peter, 2017).


The vehicle used was propylene glycol and the test solutions were prepared daily. There were no adverse parental, reproduction and developmental toxicity up to 1000 mg/kg bw/day.


No reproduction toxicity was observed up to 1000 mg/kg/d.


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


No developmental toxicity was observed up to 1000 mg/kg/d.


No treatment-related effects were noted in any of the developmental parameters investigated in this study (i.e. gestation, viability and lactation indices, duration of gestation, parturition, sex ratio, maternal care and early postnatal pup development consisting of mortality, clinical signs, body weight, anogenital distance (PND 1), areola/nipple retention (PND 13 males), T4 thyroid hormone levels (PND 13-15) and macroscopy.


The Parental, Reproduction and Developmental NOAEL were established as at least 1000 mg/kg bw/ day.


 


 


 

Effects on developmental toxicity

Description of key information

In a key prenatal developmental toxicity study in time-mated female Wistar Han rats, the test item was administrated from Day 6 to 20 post-coitum inclusive, up to the dose level of 1000 mg/kg bw/day. The maternal and developmental NOAELs of at least 1000 mg/kg/day were established (OECD 414; Bressers, 2022).


In the combined 28 -day repeated dose toxicity study with the reproduction/developmental toxicity screening test (OECD 422; Peter, 2017), a developmental NOAEL of 1000 mg/kg bw/day was established.


The test substance is not to be classified as reproductive toxicant.


 

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 2021-06-14 to 2022-03-09
Reliability:
1 (reliable without restriction)
Reason / purpose for cross-reference:
reference to other study
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
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: I20IB2949
- Chemical name: (1S)-1,5-ANHYDRO-2,3,4,6-TETRAKIS-O-(2,2-DIMETHYLPROPANOYL)-1-(3-([5-(4-FLUOROPHENYL)THIOPHEN-2-YL]METHYL)-4-METHYLPHENYL)-D-GLUCITOL
- CAS number: 1283129-18-9
- Physical appearance: white powder
- Expiration date of the lot/batch: 2022-09-19 (retest date)
- Purity, including information on contaminants, isomers, etc.: 98.9 %
- Molecular weight: 781.0

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions and during storage: stability for at least 6 hours at room temperature under normal laboratory light conditions, and for 26 days in the refrigerator was confirmed over the concentration range 1 to 200 mg/mL.

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

OTHER SPECIFICS
- pH: 7.7 at concentration of <0.01 g/L
- correction factor: 1.00
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 or Charles River Laboratories France, L'Arbresle Cedex, France.
- Age at study initiation: 11-15 weeks old
- Weight at study initiation: 192 - 254 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 period was 20 to 21 °C with an actual daily mean relative humidity of 50 to 77%. The values that were outside the targeted range (40-70%) occurred for 2 days with a maximum of 77% and were without a noticeable effect on the clinical condition of the animals or on the outcome of the study
- Humidity (%): 40-70 % (actual 50-77%)
- Air changes (per hr): At least 10 air changes per hour
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 2021-06-21 To: 2021-07-09
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 weekly (storage condition 4 °C) and were homogenized to a visually acceptable level. At least 30 minutes before dosing, formulations will be removed from the refrigerator and are kept at room temperature until and during dosing. The dose formulations were be stirred continuously during dosing. No correction was made for the purity/composition of the test item (correction factor is 1.00). A correction factor of 1.036 was used to correct for the specific gravity of the vehicle.
- the dose volume for each animal was based on the most recent body weight measurement
- the doses are given using a plastic feeding tube


VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on trial formulations performed at the test facility (study Nos 512709 and 512704).
- Concentration in vehicle: 0 mg/mL (group 1- control), 22 mg/mL (group 2), 66 mg/mL (group 3) and 200 mg/mL (group4).
- 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 500 mg
- homogeneity (top, middle, bottom): groups 2 and 4, 6x 500 mg
- sampling from dosing container
All samples to be analysed were transferred (at room temperature) to the analytical laboratory for same day analysis within 6 hours after preparation, or stored for analysis within known formulation stability period.
Analyses were performed using a validated analytical procedure (Test Facility Study No. 512709).
- concentration and homogeneity analysis
- storage conditions: At room temperature set to maintain 21°C if analyzed within 6 hours
after preparation of the formulations, or in the refrigerator set to maintain 4°C, if stored for more than 6 hours after preparation of the formulations.
- Acceptance Criteria: for concentration, the 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 is stable in the vehicle when prepared and stored under the same conditions at concentrations bracketing those used in the present study. Stability for at least 6 hours at room temperature under normal laboratory light conditions , and for 26 days in the refrigerator set to maintain at 4 °C was confirmed over the concentration range 1 to 200 mg/mL. Stability data have been retained in the study records of Test Facility Study No. 512709. Homogeneity was confirmed in Test Facility Study No. 512704.
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 litter x 12 fetuses)
Duration of treatment / exposure:
Day 6 to Day 20 post-coitum.
Frequency of treatment:
once daily
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 (28-day repeated dose toxicity study with the Reproduction/Developmental Toxicity Screening Test with oral exposure of JNJ-42808415-AAA (T003422) in rats, Test Facility Study No. 512704), 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.
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.

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.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: Regular basis throughout the study.
- all parental animals
- Procedure: Water consumption was monitored by visual inspection of the water bottles. If inter group differences were noted, consumption might have be assessed by weight. 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 (1.0 mL) 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 be 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 were 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 as a percent of 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 data
- 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:
- external examinations: see 'Any other information on materials and methods'
- internal sex confirmation: yes
- 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.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Salvation was observed for Female No. 45 (330 mg/kg/day) on Day 19 post-coitum. Furthermore, for Female No. 28 (110 mg/kg/day) abnormal breathing sounds were heard on Day 17 post-coitum. Based on the absence of a dose response and the low incidences, these clinical observations were considered as not test item-related. Other clinical signs noted during the treatment period (i.e. scabs and fur loss) 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:
no effects observed
Description (incidence and severity):
Up to 1000 mg/kg/day, mean body weights, body weight gain and weight gain corrected for
gravid uterus of pregnant animals remained in the same range as control.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Up to 1000 mg/kg/day, food consumption was comparable to the control level over the study period.
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:
no effects observed
Description (incidence and severity):
Serum levels of Total TSH, T3 and Total T4 were considered to be unaffected by treatment
with the test item up to 1000 mg/kg/day.
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 test item-treated pregnant animals were
considered to be similar to those of control animals up to 1000 mg/kg/day
Gross pathological findings:
no effects observed
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 up to 1000 mg/kg/day.
There were no test item-related gross observations in the thyroid glands.
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:
no effects observed
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
Number of abortions:
no effects observed
Description (incidence and severity):
All females, except No. 3 (control), Nos. 53 and 63 (330 mg/kg/day) and No. 76
(1000 mg/kg/day), were gravid and had litters with live fetuses.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
The numbers of 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.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
All females, except No. 3 (control), Nos. 53 and 63 (330 mg/kg/day) and No. 76
(1000 mg/kg/day), were gravid and had litters with live fetuses.
Early or late resorptions:
no effects observed
Description (incidence and severity):
All females, except No. 3 (control), Nos. 53 and 63 (330 mg/kg/day) and No. 76
(1000 mg/kg/day), were gravid and had litters with live fetuses.
Dead fetuses:
no effects observed
Description (incidence and severity):
All females, except No. 3 (control), Nos. 53 and 63 (330 mg/kg/day) and No. 76
(1000 mg/kg/day), were gravid and had litters with live fetuses.
Changes in pregnancy duration:
not specified
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
All females, except No. 3 (control), Nos. 53 and 63 (330 mg/kg/day) and No. 76
(1000 mg/kg/day), were gravid and had litters with live fetuses.
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
Fetal body weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Mean fetal weights (male, female and combined) were higher at 1000 mg/kg/day compared to the control animals, reaching statistical significance for females (5.051 grams vs 4.866 grams) only. However, the mean fetal weight of male and female of the control group was lower than the historical data1, which resulted in a significant difference in the high dose group. The fetal weights of the high dose group are within the range of this data, and therefore the higher fetal weight was considered not to be test item-related. At 100 and 300 mg/kg/day, fetal body weights were comparable to concurrent control.
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.
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:
effects observed, non-treatment-related
Description (incidence and severity):
There were no external malformations or variations observed in fetuses of any group. Only
gastroschisis occurred in a late resorption at the high dose and this isolated case was
considered a chance finding.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were no skeletal malformation or variations attributed to the treatment with test item up
to 1000 mg/kg/day.
The only malformations noted were bending of limb bones (i.e. humerus and/or radius),
which affected two littermates at each 110 and 330 mg/kg/day. This does not indicate a test
item-relationship and were therefore considered as chance findings.
Of the skeletal variations, four control fetuses showed incomplete ossification of the right
squamosal bone, whereas none were observed in test item groups. This caused statistical
significance at 110, 330 and 1000 mg/kg/day. The females treated with the test item had
fetuses with squamosal bones that were incompletely ossified at the left or both skull sides,
contrary to the control group. As the general ossification was similar between control and test
item-treated animals, this variation was considered not test item-related.
All other skeletal variations occurred in the absence of a dose-related incidence trend and/or
infrequently. Therefore, they were considered not related to treatment with the test item.
Visceral malformations:
no effects observed
Description (incidence and severity):
There were no visceral malformation or variations attributed to the treatment with test item up to 1000 mg/kg/day.
Malformations were not observed in this study and the observed variations affected the liver
(supernumerary lobes) and ureters (convoluted and dilatation) at low incidences or in isolated cases, which did not indicate any test item relationship.
Other effects:
not specified
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
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no

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 accuracies between 85.00% and 115.00%). No test item was detected in the group 1 formulation.


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


 


Study mean fetal body weight













































































































 



0 mg/kg/day



110 mg/kg/day



330 mg/kg/day



1000 mg/kg/day



Mean Fetal Weight males (g) [G]



Mean



5.152



5.172



5.201



5.308



 



SD



0.277



0.319



0.242



0.261



 



N



21



22



20



21



 



% Diff



-



0.395



0.946



3.021



Mean Fetal Weight females (g) [G]



Mean



4.866



4.858



4.906



5.051*



 



SD



0.230



0.264



0.261



0.220



 



N



21



22



20



21



 



% Diff



-



-0.169



0.814



3.795



Mean Fetal Weight all (g) [G]



Mean



5.022



5.019



5.055



5.178



 



SD



0.232



0.295



0.222



0.216



 



N



21



22



20



21



 



% Diff



-



-0.063



0.651



3.101



[G] - Anova & Dunnett: * = p ≤ 0.05


 


Historical Control Data Rat: Crl:WI(Han) (outbred, SPF-Quality) – mean Fetal body weight














































Endpoint



mean



SD



Median



Min



Max



P5



P95



Mean Fetal body weight (g)



5.3



0.12



5.3



4.9



5.4



5.0



5.4



Mean male body weight (g)



5.4



0.11



5.4



5.0



5.5



5.2



5.5



Mean female body weight (g)



5.1



0.13



5.2



4.7



5.3



4.9



5.3


Conclusions:
In conclusion, based on the absence of adverse effects in this prenatal developmental toxicity study in time-mated female Wistar Han rats the maternal and developmental No Observed Adverse Effect Levels (NOAELs) for JNJ-42808415-110 (T003422) were established as being at least 1000 mg/kg/day.
Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
GLP-compliant study, performed according to OECD guideline
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

Prenatal development toxicity study in rat


The potential of the test substance to induce developmental toxicity after maternal exposure during the critical period of organogenesis was investigated, and maternal toxicity was characterised. Time-mated female Wistar Han rats were treated with JNJ-42808415-110 (T003422) from Day 6 to 20 post-coitum, inclusive, by daily oral gavage at dose levels of 110, 330 and 1000 mg/kg/day (OECD 414; Bressers, 2022). The dose levels were selected based on the results of a 28-day repeated dose toxicity study (reproduction/developmental toxicity screening test) with oral exposure of the test item, and in an attempt to produce graded responses to the test item. The rats of the control group received the vehicle, propylene glycol, alone. Chemical analyses of formulations were conducted once during the study to assess accuracy, homogeneity. Test formulations prepared were considered homogeneous at the concentrations tested and analysis of the accuracy revealed acceptable levels.
No test item-related changes were noted in any of the maternal parameters investigated in this study (i.e. mortality/moribundity, clinical signs, body weight, food consumption, thyroid hormone levels (triiodothyronine (T3), thyroxine (T4), thyroid-stimulating hormone (TSH)), macroscopic evaluation, thyroid gland weights, uterine contents, microscopic evaluation of the thyroid gland, corpora lutea, implantation sites and pre- and post-implantation loss). No maternal toxicity was observed in the 110, 330 and 1000 mg/kg/day groups.
No test item-related changes were noted in any of the remaining developmental parameters investigated in this study (i.e. litter size, sex ratio, fetal weight, anogenital distance, external, visceral and skeletal malformations and developmental variations). No developmental toxicity was observed in the 110, 330 and 1000 mg/kg/day groups. Based on the results in this study, a maternal and developmental NOAEL of at least 1000 mg/kg/day were established.

Justification for classification or non-classification

 


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


Parental NOAEL: at least 1000 mg/kg/d.


Reproduction NOAEL: at least 1000 mg/kg/d.


Developmental NOAEL: at least 1000 mg/kg/d.


 


Based on the results of the OECD 414 test, the following NOAELs were derived:


Maternal NOAEL: 1000 mg/kg/d


Developmental NOAEL: 1000 mg/kg/d


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

Additional information