Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

In a reproduction/developmental toxicity screening test, the test item was administered daily to rats at dose levels up to 1000 mg/kg body weight/day (OECD 421; van de Ven, 2017). The parental NOAEL was established as 200 mg/kg body weight/day and the reproduction NOAEL as at least 1000 mg/kg body weight/day. The substance is not classified as a reproductive toxicant according to the CLP Regulation.

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 2016-11-16 to 2017-01-31
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA OPPTS 870.3550 (Reproduction/Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No. of test material: M16BC0342
- Expiration date of the lot/batch: 2019-02-01
- Purity test date: 2015-12-11


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature protected from light.
- Solubility and stability of the test substance in the solvent/vehicle: Stability for at least 6 hours at room temperature protected from light and 15 days in the refrigerator is confirmed over the concentration range 1 to 200 mg/mL, Test Facility Study No. 514654.


FORM AS APPLIED IN THE TEST (if different from that of starting material): Suspension (Groups 2-4).

OTHER SPECIFICS: correction factor is 1
Species:
rat
Strain:
Wistar
Details on species / strain selection:
This species and strain of rat has been recognized as appropriate for general and reproduction toxicity studies. Charles River Den Bosch has general and reproduction/developmental historical data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants.
Sex:
male/female
Details on test animals 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. 11 weeks (at start pretest) and approx. 13 weeks (at start F0-treatment).
- Weight at study initiation: Males: 269-312 g; Females: 211-242 g
- Fasting period before study: no
- Housing:
Pretest: Females were housed in groups of 5 females/cage in Macrolon plastic cages (MIV type, height 18 cm).
Pre-mating: Animals were housed in groups of 5 animals/sex/cage in Macrolon plastic cages (MIV type, height 18 cm).
Mating: Females were caged together with males on a one-to-one basis in Macrolon plastic cages (MIII type, height 18 cm).
Post-mating: Males were housed in their home cage (Macrolon plastic cages, MIV type, height 18 cm) with a maximum of 5 animals/cage. Females were individually housed in Macrolon plastic cages (MIII type, height 18 cm).
- Diet (e.g. ad libitum): Free access to pelleted rodent diet.
- Water (e.g. ad libitum): Free access to tap-water.
- Acclimation period: At least 5 days prior to start of pretest (females) or treatment (males).


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


IN-LIFE DATES:
From: 2016-11-17 (start pretest); 2016-12-01 (start treatment); 2017-01-07/08/09/10/11/18 (delivery of litters)
To: 2016-12-30 (necropsy males); 2017-01-20/23/24/25/26/31 (necropsy females); 2017-01-19/22/23/24/25/30 (necropsy pups)
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 (1.036). A correction was made for the purity/composition of the test item. A correction factor of 1 was used. Formulations were placed on a magnetic stirrer during dosing


VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on trial formulations performed at Charles River Den Bosch.
- Concentration in vehicle: 0 mg/mL (group 1), 10 mg/mL (group 2), 40 mg/mL (group 3), 200 mg/mL (group 4)
- Amount of vehicle (if gavage): 5 mL/kg body weight. Actual dose volumes were calculated according to the latest body weight.
- Lot/batch no. (if required): no data
- Purity: no data
Details on mating procedure:
- M/F ratio per cage: 1:1 basis
- Length of cohabitation: Following a minimum of 14 days of treatment for the males and females, until detection of mating was confirmed.
- Proof of pregnancy: Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum.
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): Females were individually housed in Macrolon plastic cages (MIII type, height 18 cm)
- Any other deviations from standard protocol: no
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were conducted on a single occasion during the treatment phase (01 December 2016, Day 1 of treatment) according to a validated method (Test Facility Study No. 514654). 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%.
Duration of treatment / exposure:
29 days (males) (except male no. 37 who was treated for 28 days), 50-61 days (females that delivered), 53 days (one female that failed to deliver healthy offspring). Female nos. 53 (Group 2), 63 and 64 (Group 3) were left out from treatment for one day as they were littering at the moment of dosing. The omission of one day of dosing over a period of several weeks was not considered to affect the toxicological evaluation. 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 (control group)
Dose / conc.:
50 mg/kg bw/day (nominal)
Remarks:
Group 2
Dose / conc.:
200 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
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were selected based on the results of a 28-day repeated dose toxicity study, provided by the Sponsor (data on file at the Sponsor site). No toxicologically relevant clinical signs were noted and no effects on body weight, food consumption, clinical biochemistry parameters or macroscopy were observed by treatment up to 1000 mg/kg. Liver weights were increased in males and females treated at 1000 mg/kg, and spleen weights were increased in females at 1000 mg/kg. Therefore, dose levels of 50, 200 and 1000 mg/kg were selected for this study.

- Rationale for animal assignment (if not random): randomized
Positive control:
no
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Days 1-2: At least once daily, directly after dosing, detailed clinical observations were made for all animals. From Day 3 of the treatment phase onwards up to the day prior to necropsy: Detailed clinical observations were made for all animals at least once within 15 minutes after dosing, at approximately 1 hour after dosing, and at approximately 3 hours after dosing.

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

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

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

HAEMATOLOGY: No

CLINICAL CHEMISTRY: Limited to thyroid hormone analysis.
- End of study from all animals at planned necropsy; this included females on Day 14-16 of lactation, the female that failed to deliver healthy pups and all males after at least 4 weeks of treatment (including the male that failed to sire).
- Blood samples were collected under anaesthesia using isoflurane between 7.00 and 10.30 a.m. The animals were deprived of food overnight (with a maximum of approximately 24 hours) before blood sampling, but water was available. Blood samples (0.9 mL) were drawn from the retro-orbital sinus and collected into serum tubes.
- Males: 1 aliquot of 150 μL serum was used for measurement of thyroxine (T4), and the remaining volume of serum was stored for possible future measurement of thyroid stimulating hormone (TSH).
- Females: The serum was stored for possible future measurement of thyroxine (T4) and/or thyroid-stimulating hormone (TSH).
Oestrous cyclicity (parental animals):
Daily vaginal lavage was performed to determine the stage of estrous beginning 14 days prior to treatment (pretest), the first 14 days of treatment and during mating until evidence of copulation was observed. Vaginal lavage continued for those females with no evidence of copulation until termination of the mating period. During pretest, this was done for 48 females. On the day of scheduled necropsy, a vaginal lavage was taken to determine the stage of estrous.
Sperm parameters (parental animals):
Parameters examined in F0 male parental generation: additional slides of the testes to examine staging of spermatogenesis and testes weight.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no
On PND 1, all pups were randomized per litter and individually identified by means of subcutaneous injection of Indian ink.
- To reduce variability among the litters, on PND 4 eight pups from each litter of equal sex distribution (if possible) were selected. Blood samples were collected from two of the surplus pups. Selective elimination of pups, e.g. based upon body weight, or anogenital distance was not done. Whenever the number of male or female pups prevented having four of each sex per litter, partial adjustment (for example, five males and three females) was acceptable.

PARAMETERS EXAMINED
The following parameters were examined in F1:
- Mortality/viability: The numbers of live and dead pups were determined on PND1 and daily thereafter. If possible, defects or cause of death were evaluated.
- Clinical signs: At least once daily, detailed clinical observations were made for all animals. Only days on which clinical signs were present between first and last litter check were reported in the study report.
- Body weights: Live pups were weighed on PND 1, 4, 7 and 13.
- Sex: Sex was determined for all pups on PND 1 and 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:
All pups were sexed by both external as well as internal examination. Descriptions of all abnormalities were recorded.

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: no

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: no
Postmortem examinations (parental animals):
SACRIFICE
- 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 day 25 after the last day of the mating period (female which failed to deliver, without evidence of mating).
- Euthanized in extremis: Two animals (former male no. 37 and female no. 72) were euthanized when pain, distress or discomfort was considered not transient in nature or was likely to become more severe.

GROSS NECROPSY
- All animals surviving to the end of the observation period and all moribund animals were deeply anaesthetized using isoflurane and subsequently exsanguinated. After sacrifice, all animals were subjected to a full post-mortem examination, with special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities were recorded.
- The number of former implantation sites were recorded for all paired females. In case no macroscopically visible implantation sites were present, nongravid uteri were stained using the Salewski technique in order to detect any former implantation sites.
- Samples of the following tissues and organs were collected and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution): Cervix (F), Clitoral gland (F), Coagulation gland (M), (Cowper’s gland) (M), Epididymides (M), Mammary gland area (M/F), (Glans penis) (M), (Levator ani plus bulbocavernosus muscle complex (LABC)) (M), Ovaries (F), Preputial gland (M), Pituitary gland (M/F), Prostate gland (M), Seminal vesicles (M), Testes (M), Thyroid including parathyroid if detectable (M/F), Uterus (F), Vagina (F), All gross lesions (M/F), Liver (M/F), Spleen (M/F). Tissues/organs mentioned in parentheses were not examined by the pathologist, since no signs of toxicity were noted at macroscopic examination.
- Reproductive organs were only examined by the pathologist for males that failed to sire and all females that failed to deliver healthy pups. Female mammary gland area was only examined by the pathologist for females with total 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 all animals on the scheduled day of necropsy: Epididymides, Testes, Thyroid (including parathyroid if detectable), Prostate gland, Seminal vesicles (including coagulation gland),liver, spleen.

HISTOPATHOLOGY
- All organ and tissue samples selected for histopathology were processed, embedded and cut at a thickness of 2-4 micrometers. These slides were stained with haematoxylin and eosin. The additional slides of the testes (to examine staging of spermatogenesis) were stained with PAS/haematoxylin.
- The following slides were examined by a pathologist: The ovaries, testes, epididymides and thyroids of the animals of Groups 1 and 4; Additional slides of the testes of all males of Groups 1 and 4 and all males that failed to sire to examine staging of spermatogenesis; All gross lesions of all animals (all dose groups); The preserved organs and tissues of female no. 72 which was euthanized in extremis; The reproductive organs of all male no. 7 that failed to sire and female nos. 47 and 72 that failed to deliver healthy pups.
- All abnormalities were described and included in the study report. An attempt was made to correlate gross observations with microscopic findings.
- A peer review on the histopathology data was performed by a second pathologist.
Postmortem examinations (offspring):
SACRIFICE
- On PND 4 (at culling), 2 pups per litter were killed by decapitation for blood collection.
- On PND 13-15, 2 pups per litter were anaesthetized using isoflurane for blood collection by aorta puncture followed by exsanguination.
- All remaining pups (PND 13-15) were sacrificed using Euthasol 20% by intraperitoneal injection.

GROSS NECROPSY
- All pups were sexed both externally and internally. Descriptions of all abnormalities were recorded.
- At terminal sacrifice (PND 13-15), the thyroid from 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.

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

Live birth index (%) = (Number of live offspring on Day 1 after littering/Total number of offspring born) x 100

Viability index (%) = (Number of live offspring on Day 4 before culling/Number live offspring on Day 1 after littering) x 100

Lactation index (%) = Number of live offspring on Day 13 after littering/Number live offspring on Day 4 (after culling)) x 100

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

Percentage of live males at first litter check (%)

Percentage of live females at first litter check (%)

- Group mean values were calculated from individual litter values.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
During the pre-mating period, treatment-related clinical signs were noted at 1000 mg/kg in both sexes (about one and/or three hours after dosing). Main findings were calm behaviour (most animals on Day 5) and hunched posture and piloerection (about half of the males on Day 4, a few females on Days 2, 5 or 9). A flat posture was noted in a few males and females. Rales (slight) were noted after dosing in about half of the animals at 1000 mg/kg (mostly males) and a few animals at 200 mg/kg. Two females additionally showed lethargy (severe), ptosis (moderate), shallow respiration (moderate or severe) and uncoordinated movements on Day 2. One female further showed severe rales on Days 5-7 and was sacrificed moribund on Day 9.

Salivation was noted after dosing, starting after a few days of treatment, at 200 and 1000 mg/kg with a dose-related trend in frequency. Incidentally, salivation was also noted at 50 mg/kg. This salivation was considered to be a physiological response rather than a sign of systemic toxicity considering its slight severity and the time of occurrence (i.e. immediately after dosing).
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, treatment-related
Description (incidence):
There were two unscheduled deaths in the high dose group (1000 mg/kg). One high dose male was sacrificed for humane reasons on the first day of the treatment period as it showed a hunched posture, lethargy and piloerection after receiving 1000 mg/kg once.

One high dose female was euthanized for ethical reasons on Day 9 of the treatment period. On the day of sacrifice, this animal showed paresis of the right leg, a flat posture, piloerection, rales (severe) and gasping. No clear cause of moribundity could be established from the tissue sections examined microscopically, but was possibly related to the macroscopic finding of gelatinous skeletal muscle of the right flank (microscopic hemorrhages were present in this tissue). These macroscopic and microscopic findings were unlikely to be related to treatment with the test item. Based on the observed clinical signs (except for paresis), which were comparable to the findings noted for several of the remaining high dose males and females, a relationship to treatment cannot be excluded.

All remaining animals survived until scheduled necropsy.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weight gain was reduced in females at 1000 mg/kg during the post-coitum period (differences from controls were statistically significant on Days 11-20 post-coitum). Mean body weights of these females were statistically significantly lower at Days 17 and 20 postcoitum and from Day 4 of the lactation period onwards (up to 10% relative difference from controls).

Males at 1000 mg/kg gained slightly less weight than controls during the first week of the pre-mating period.

No treatment-related changes in body weight or body weight gain were observed in animals treated up to 200 mg/kg.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food consumption before and after allowance for body weight was decreased at 1000 mg/kg in both sexes in the first week of the pre-mating period.
In 1000 mg/kg females, food consumption was also decreased (statistically significant) in the second week of the gestation period (absolute and relative to body weight) and from Day 4 of the lactation period (absolute only).

No treatment-related changes in food consumption before or after allowance for body weight were observed in animals treated up to 200 mg/kg.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Serum levels of T4 in P0-males were statistically significantly decreased at 200 and 1000 mg/kg (relative differences from controls: -35 and -70%, respectively). Values at 200 mg/kg were at the lower end of the historical control range, whereas those at 1000 mg/kg were considerably below the normal range (7/10 values at 1000 mg/kg were below the limit of detection (1.0 ug/dL)).
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
There were test item-related organ weight changes consisting of increased weight of the liver at 200 mg/kg in females (relative to body weight) and at 1000 mg/kg in both sexes (absolute and relative to body weight) and increased weight of the spleen at 1000 mg/kg in males (absolute and relative to body weight) and females (relative to body weight).

There were no test item-related organ weight changes in the remaining organs, including thyroid glands.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Test item-related liver enlargement was noted in 4/10 males and 2/10 females at 1000 mg/kg.

There were no test item-related gross observations in the reproductive organs.

The remainder of the recorded macroscopic findings in the animals surviving until scheduled sacrifice, including the findings in single animals at 50, 200 and 1000 mg/kg (enlarged, reduced in size and pale) in the thyroid glands, were within the range of background gross observations encountered in rats of this age and strain. These necropsy findings were therefore considered to be unrelated to treatment.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Minimal centrilobular hepatocellular hypertrophy was noted in the macroscopically enlarged livers of 4 males and 2 females treated at 1000 mg/kg (the liver of the remaining high-dose animals were not examined microscopically). This finding was attributed to treatment with the test item.

The remainder of the recorded microscopic findings, including the minimal hypertrophy of follicular cells in the thyroid glands, in the animals surviving the scheduled study period, were within the range of background pathology encountered in rats of this age and strain.There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

There were no histopathological findings in the reproductive organs of either sex which could be attributed to the test item.

There was one couple of the control group with no offspring (no evidence of mating). Histopathology did not reveal any changes in the reproductive organs that could explain this.

One female at 1000 mg/kg had no offspring as she was euthanized before the mating period.
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 affected by treatment.

Most females had regular cycles of 4-5 days. Extended di-estrus during the mating period occurred in two females of the control group and irregular cycles occurred in two females at 200 mg/kg and one female at 1000 mg/kg. Except for a control female which had no evidence of mating, these females had normal litters. These findings were considered to be unrelated to treatment because their incidence was within normal limits and showed no dose-related trend.
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
As one female (no. 72 in the 1000 mg/kg group) was sacrificed in the pre-mating period, reproduction data is available for 9/10 females in the 1000 mg/kg group.
- No reproduction toxicity was observed up to 1000 mg/kg.
- Mating index, Precoital time, Number of implantation sites and Fertility index were not affected by treatment. The mean number of implantation sites at 1000 mg/kg was slightly lower than that in the control group. As all other 1000 mg/kg females had normal numbers of implantation sites, and the difference from the control group was only slight and not statistically significant, this finding was not considered to be toxicologically relevant.

DEVELOPMENTAL DATA
- Gestation index and duration: All pregnant females delivered live offspring after 21 or 22 days of gestation.
- Parturition/maternal care: Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No deficiencies in maternal care were observed.
- Post-implantation survival index: The total number of offspring born compared to the total number of uterine implantation sites was not considered to be affected by treatment. The post-implantation survival indices across the groups ranged from 88 to 96%.
- Litter size: The mean number of living pups at first litter check in the 1000 mg/kg group was slightly lower compared to that in the control group. The difference was not statistically significant and could partly be explained by the small size of a single litter at 1000 mg/kg consisting of five healthy pups which correlated with the low number of implantation sites in this dam. Such a small litter size occasionally occurs in untreated controls. All other litters at 1000 mg/kg had normal sizes. Therefore, the apparently lower litter size at 1000 mg/kg was considered not to reflect an adverse effect of the test item.
- Live birth index: The number of live offspring on Day 1 after littering compared to the total number of offspring born was not affected by treatment. The live birth indices across the groups ranged from 98 to 100%. Four females of the control group had one dead pup at first litter check at 200 mg/kg, and one at 1000 mg/kg. This incidental pup mortality was within normal limits and unrelated to treatment with the test item.
- Viability index: The viability indices the across the groups ranged from 98 to 100%. Two pups of the control group and one pup at 200 mg/kg were missing on PND 2-4, which were most likely cannibalised. One pup at 50 mg/kg was found dead on PND 3. No toxicological relevance was attributed to these dead/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: Breeding loss between lactation Days 5 and 13 was limited to one pup at 50 mg/kg which went missing (presumably cannibalized) on PND 10. This incidental breeding loss was unrelated to treatment with the test item. The lactation index was 99% in the 50 mg/kg group and 100% in the other groups.



Parental results:
Treatment at 1000 mg/kg resulted in two unscheduled deaths. One high dose male was sacrificed for humane reasons on the first day of treatment as it showed a hunched posture, lethargy and piloerection. At necropsy, a reddish foci on the glandular mucosa of the stomach was noted. One high dose female was euthanized for ethical reasons on Day 9 of dosing due to paresis of the right leg, a flat posture, piloerection, rales and gasping. The moribundity was considered related to the macroscopic finding of gelatinous skeletal muscle of the right flank. Treatment-related clinical signs were noted at 1000 mg/kg in both sexes, mainly on Days 4-5 of dosing and consisted of a calm behaviour, hunched posture and piloerection in the majority of the rats; a flat posture was noted in a few animals. Rales were noted after dosing in half of the animals at 1000 mg/kg and a few animals at 200 mg/kg. Two females additionally showed lethargy, ptosis, shallow respiration and uncoordinated movements on Day 2 of dosing. Females treated at 1000 mg/kg showed reduced body weight gain during the gestation period, accompanied by reduced food consumption in the second week of gestation. Mean body weights were about 10% lower compared to controls towards the end of the gestation period and during the lactation period. Serum level of thyroid hormone T4 was markedly decreased in males treated at 1000 mg/kg (only 3/10 males had values above the limit of detection; the mean of these values was 70% lower compared to controls). However, there were no treatment-related histopathological changes or changes in the weight of the thyroid (in either sex) observed to correlate with the changes in hormonal values. The decrease in T4 may be due to increased T4 turnover resulting from metabolic enzyme induction in de liver (hepatocellular hypertrophy). Reduced serum T4 levels were also observed in males treated at 200 mg/kg but these levels remained within the range of available historical control data (although they were at the lower end). Liver weights were increased at 1000 mg/kg in both sexes (liver to body weight ratios were about 40% higher relative to controls) and at 200 mg/kg in females (ratio was 9% higher relative to controls). Minimal centrilobular hepatocellular hypertrophy at a minimal degree was noted in the liver of males and females at 1000 mg/kg, which correlated with increased liver weight and enlarged livers observed at macroscopy. These hepatic changes were regarded as non-adverse, adaptive responses. Spleen weights were increased at 1000 mg/kg in both sexes (spleen to body weight ratio was about 20% higher relative to controls). In this study, no data on possible associated findings (spleen morphology or haematology) was obtained and an adverse effect cannot be excluded.

Reproductive results:
No reproduction toxicity was observed up to 1000 mg/kg.

Analysis of dose preparations:
- Accuracy of preparation: The concentrations analysed in Group 2, 3 and 4 formulations were in agreement with target concentrations (i.e. mean accuracies between 85.00% and 115.00%). No test item was detected in the Group 1 formulation.
- Homogeneity: The Group 2 and 4 formulations were homogeneous (i.e. coefficient of variation ≤ 10.00%).
Key result
Dose descriptor:
NOAEL
Remarks:
Parental toxicity
Effect level:
200 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
clinical biochemistry
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
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 evaluated in this study.
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day (nominal)
System:
haematopoietic
Organ:
spleen
other: Note that the hepatic changes observed in this study were regarded as non-adverse, adaptive responses.
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified
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 clinical signs observed incidentally remained within the range considered normal for pups of this age, and were therefore considered to be unrelated to treatment.
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 live offspring on Day 1 was not affected by treatment. The viability indices the across the groups ranged from 98 to 100%. Two pups of the control group and one pup at 200 mg/kg were missing on PND 2-4, which were most likely cannibalised. One pup at 50 mg/kg was found dead on PND 3. No toxicological relevance was attributed to these dead/missing pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Mean body weights of both male and female pups at 1000 mg/kg were statistically significantly decreased at PND 13, on average by about 15%. At PND 7, mean body weights of these pups were about 9% lower compared to controls but the difference was not statistically significant. Body weights of pups at birth (PND 1) and at PND 4 were similar across the groups.
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 affected by treatment.
Urinalysis findings:
not examined
Sexual maturation:
not examined
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No macroscopic findings were noted among pups that were considered to be related to treatment. The nature and incidence of the macroscopic findings noted incidentally remained within the range considered normal for pups of this age, and were therefore considered to be unrelated to treatment.
Histopathological findings:
not examined
Other effects:
no effects observed
Description (incidence and severity):
- Sex ratio: Sex ratio was not affected by treatment.
- 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:
- Male and female pups at 1000 mg/kg had lower body weights than controls at PND 7 and 13, which was statistically significant at PND 13. The relative differences from controls were about 15% and 9%, respectively. Pup body weights at PND 1 and 4 were similar across the groups, indicating that the lower weights at PND 7 and 13 resulted from reduced post-natal growth. This effect on post-natal growth was considered to be toxicologically relevant.
- There was no test item-related effect on post-natal growth at 50 or 200 mg/kg.
- No toxicologically significant changes were noted in any of the other developmental parameters investigated in this study (i.e. gestation, viability and lactation indices, duration of gestation, parturition, sex ratio, maternal care and early postnatal pup development consisting of mortality, clinical signs, anogenital distance (PND 1), areola/nipple retention (PND 13 males), T4 thyroid hormone levels (PND 13-15) and macroscopy).
Key result
Dose descriptor:
NOAEL
Remarks:
Developmental toxicity
Generation:
F1
Effect level:
200 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
other: The lower weights at PND 7 and 13 resulted from reduced post-natal growth.
Critical effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day (nominal)
System:
other: Lower weights at PND 7 and 13 resulted from reduced post-natal growth.
Organ:
not specified
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified
Reproductive effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day (nominal)
Treatment related:
yes
Relation to other toxic effects:
not specified
Dose response relationship:
yes
Relevant for humans:
not specified
Conclusions:
In conclusion, treatment with JNJ-27261429-AAA (T002251) by oral gavage in male and female Wistar Han rats at dose levels of 50, 200 and 1000 mg/kg revealed treatment related parental and developmental toxicity and no reproduction toxicity up to 1000 mg/kg.
Based on these results, the following No Observed Adverse Effect Levels (NOAELs) were derived:
Parental NOAEL: 200 mg/kg.
Reproduction NOAEL: at least 1000 mg/kg
Developmental NOAEL: 200 mg/kg
Therefore, the substance is not classified as a reproductive toxicant 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
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 reproduction/developmental toxicity screening test was performed in rats in which male and female rats were exposed to 0 (vehicle), 50, 200, 1000 mg/kg bw/day via gavage (OECD 421; van de Ven, 2017). The vehicle used was propylene glycol and the test solutions were prepared daily within 6 hours prior to dosing. Treatment at 1000 mg/kg resulted in two unscheduled deaths. One high dose male was sacrificed for humane reasons on the first day of treatment as it showed a hunched posture, lethargy and piloerection. At necropsy, a reddish foci on the glandular mucosa of the stomach was noted. One high dose female was euthanized for ethical reasons on Day 9 of dosing due to paresis of the right leg, a flat posture, piloerection, rales and gasping. The moribundity was considered related to the macroscopic finding of gelatinous skeletal muscle of the right flank. Treatment-related clinical signs were noted at 1000 mg/kg in both sexes, mainly on Days 4-5 of dosing and consisted of a calm behaviour, hunched posture and piloerection in the majority of the rats; a flat posture was noted in a few animals. Rales were noted after dosing in half of the animals at 1000 mg/kg and a few animals at 200 mg/kg. Two females additionally showed lethargy, ptosis, shallow respiration and uncoordinated movements on Day 2 of dosing. Females treated at 1000 mg/kg showed reduced body weight gain during the gestation period, accompanied by reduced food consumption in the second week of gestation. Serum level of thyroid hormone T4 was markedly decreased in males treated at 1000 mg/kg (only 3/10 males had values above the limit of detection; the mean of these values was 70% lower compared to controls). However, there were no treatment-related histopathological changes or changes in the weight of the thyroid (in either sex) observed to correlate with the changes in hormonal values. Liver weights were increased at 1000 mg/kg in both sexes (liver to body weight ratios were about 40% higher relative to controls) and at 200 mg/kg in females (ratio was 9% higher relative to controls). Minimal centrilobular hepatocellular hypertrophy at a minimal degree was noted in the liver of males and females at 1000 mg/kg, which correlated with increased liver weight and enlarged livers observed at macroscopy. These hepatic changes were regarded as non-adverse, adaptive responses. Spleen weights were increased at 1000 mg/kg in both sexes (spleen to body weight ratio was about 20% higher relative to controls). In this study, no data on possible associated findings (spleen morphology or haematology) was obtained and an adverse effect cannot be excluded.

No toxicologically relevant effects on reproductive parameters were noted up to 1000 mg/kg.

Based on the abovementioned considerations, no adverse reproduction toxicity was revealed up to 1000 mg/kg, while parental toxicity was observed at 1000 mg/kg. The parental NOAEL was establised as 200 mg/kg bodyweight/day, while the reproduction NOAEL was established as being at least 1000 mg/kg bodyweight/day.

Effects on developmental toxicity

Description of key information

In a reproduction/developmental toxicity screening test (OECD 421; van de Ven, 2017), the test item was administered daily to rats at dose levels up to 1000 mg/kg body weight/day. The developmental NOAEL was established as 200 mg/kg body weight/day. The substance is not classified as a reproductive toxicant according to the CLP Regulation.

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

Justification for classification or non-classification

Based on the results of the OECD 421 test, the following No Observed Adverse Effect Levels (NOAEL) were derived:

Parental NOAEL: 200 mg/kg

Reproduction NOAEL: at least 1000 mg/kg

Developmental NOAEL: 200 mg/kg

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