cis-2-(bromomethyl)-2-(2,4-dichlorophenyl)-1,3-dioxolane-4-ylmethyl benzoate

Endpoint summary

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; Mounier R., 2017). The parental and reproduction NOAELs were established as at least 1000 mg/kg body weight/day. The test item 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:

2016-10-14 to 2017-01-18

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3650 (Combined Repeated Dose Toxicity Study with the Reproduction/Developm ental Toxicity Screening Test)

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

other: EPA OPPTS 870.3550 (Reproduction/Developmental Toxicity Screening Test)

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

other: OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

other: EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

other: EPA OPPTS 870.3050 (Repeated dose 28-day oral toxicity study in rodents)

Deviations:

no

Principles of method if other than guideline:

No testing guidelines were applicable for the pilot phase, as this part of the study was intended for dose level selection purposes only.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: I15DB1648
- Expiration date of the lot/batch: 2020-04-19 (retest date)
- Purity test date: 2016-04-27

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature (+15 to +25 °C)
- Stability in vehicle: At least 6 hours at room temperature and 14 days refrigerated at concentration of 1 to 200 mg/mL based on results of Stability Study No. AB21452.
- Stability under test conditions: Homogeneity and stability of the test item under test conditions we re demonstrated in the analytical method development and validation study (Test Facility Study No. AB21452).

FORM AS APPLIED IN THE TEST (if different from that of starting material): White homogeneous suspension (Groups 2, 3 and 4).

OTHER SPECIFICS: Correction factor was 1.03

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 Lyon 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 Laboratories France, Domaine des Oncins, 69210 Saint-Germain-Nuelles, France.
- 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: 261.6-373 g (males); 209-252.6 g (females)
- Fasting period before study: no
- Housing: Group housed males and females and individual housed females, including females during mating and with litters, were housed in plastic cages meeting European directive 2010/63/EU requirements.
Pretest: Animals were housed in groups of 5 sex/cage.
Pre-mating: Animals were housed in groups of 5 sex/cage.
Mating: Males and females were cohabitated on a 1:1 basis.
Post-mating: Males were housed in their home cage with a maximum of 5 animals/cage. Females were individually housed.
- Diet (e.g. ad libitum):Free access to pelleted complete rodent diet.
- Water (e.g. ad libitum): Free access to softened and filtered (0.2 μm) mains drinking water
- Acclimation period: 8 days prior to start of pretest (females) or treatment (males).

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25 °C
- Humidity (%): >35 %
- Air changes (per hr): 10 room air changes/hour
- Photoperiod (hrs dark / hrs light): 12-hour light/12-hour dark cycle

IN-LIFE DATES
From: 2016-11-01 (start pretest, females); 2016-11-15 (start treatment, males); 2016-12-22 through 2017-01-04 (delivery of litters)
To: 2016-12-16 (necropsy males); 2017-01-05 through 2017-01-18 (necropsy females); 2017-01-04 through 2017-01-17 (necropsy pups)

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Formulations (w/v) were prepared weekly and were homogenized to a visually acceptable level. No adjustment was made for specific gravity/density of the test item, vehicle and/or formulation. A correction factor of 1.03 was applied for the purity/composition of the test item. All formulations were placed at ambient temperature for at least 15 minutes before the dosing procedure and test item form ulations were placed on a magnetic stirrer for at least 15 minutes before and throughout the dosing procedure.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on trial formulations performed at Charles River Lyon.
- Concentration in vehicle: 0 mg/mL (group 1); 22 mg/mL (group 2); 66 mg/mL (group 3); 200 mg/ mL (group 4)
- Amount of vehicle (if gavage): 5 mL/kg body weight/day (Actual dose volumes were calculated acco rding to the latest body weight)
- Lot/batch no. (if required): MKBZ1609V and MKBV0644V
- Purity: no data

Details on mating procedure:

- M/F ratio per cage: 1 animal/sex/cage
- Length of cohabitation: Following a minimum of 14 days of treatment for the males and females, one female was cohabitated with one male of the same treatment group 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 plastic cages meeting European directive 2010/63/EU requirements.
- 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 formulations used on the first day of treatment during the main phase (15 November 2016), according to a validated method (Test Facility Study no. AB21452). Three sets of duplicate samples (2 x 1 g) were collected. Two sets of duplicate samples were stored in a refrigerator (+2 to +8 °C) as reserve samples. Once analytical results were approved in the raw data by the Principal Scientist for analytical chemistry, the reserve samples were destroyed. Samples of formulations were analyzed for homogeneity (highest and lowest concentrations) 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% compared to those obtained during the method validation. The accuracy of preparation was considered acceptable if the mean measured concentrations were 85-115 % of the target concentration for suspensions. Homogeneity was demonstrated if the coefficient of variation was ≤10 %.

Duration of treatment / exposure:

31 days (males); 51-64 days (females that delivered); 42-44 days (females which failed to deliver).

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 less than 5 hours difference between the earliest and latest dose.

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Group 1 (control group)

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 results of a dose range finding study. During the DRF phase, oral (gavage) administration of T001095 to the female Han Wistar rat for 10 days at doses of 500 and 1000 mg/kg/day was not associated with any systemic effects. Based on these results, dose levels of 110, 330 and 1000 mg/kg/day were selected for the main study in consultation with the Sponsor.
- 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 (i.e. at the beginning and at the end of the working day, including weekends and public holidays).

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical observations were made for all animals before and at least once after dosing (between 1 and 3 hours), from start of treatment onwards up to the day prior to necropsy. Animals were observed once daily on non-treatment days. A full clinical examination was performed on each weighing day. The time of onset, grade (where applicable) and duration of any observed sign was recorded.


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. 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): No
- Time schedule for examinations: Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no treatment related effect was suspected.


HAEMATOLOGY: Yes
- Blood samples were collected at the end of the treatment period on the day of 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 K2EDTA 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 taken from the abdominal aorta as part of the necropsy procedure in tubes without anticoagulant for clinical biochemistry parameters.
- 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
- Time schedule: Between 1.5 and 2 hours after dosing for males and females, respectively, on each individual animal of the selected 5 animals/sex/group. The selected males were tested once during week 5 of treatment and the selected females were tested once during the last week of lactation, on PND 12 for group 3 female no. 136 and on PND 13 for the other females. These tests were performed after observation for clinical signs.
- Parameters: Hearing ability, pupillary reflex and static righting reflex, fore- and hind-limb grip strength, recorded as the mean of three measurements per animal, locomotor activity in an open field test (recording period: 3 minutes under normal laboratory light conditions, using a validated video image analysis system).

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 any 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; testis weight.

Litter observations:

STANDARDISATION OF LITTERS
- The pups were not identified individually. 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 each weighing day. Sex ratio (% male pups on PND 1) was calculated per group .
- Anogenital distance: Anogenital distance (AGD) was measured for all live pups on PND 1. The AGD was normalized to the cube root of body weight.
- Areola/nipple retention: On PND 13, all males in each litter were examined for the number of areola/ nipples.


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

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: no

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: no

Postmortem examinations (parental animals):

SACRIFICE:
Necropsy was conducted according to the following schedule:
- Males: following completion of the mating period (after 31 days of dose administration).
- Females which delivered: on PND 14.
- Females which failed to deliver: on post-coitum Day 26 (females with evidence of mating).
- Spontaneous death: as soon as possible after death (within 24 hours).
- Pups: on PND 4 or PND 13.
The 5 selected males and females for clinical pathology blood sampling were deeply anaesthetized using isoflurane and subsequently exsanguinated. Other adult animals surviving to the end of the observation period and all non-pregnant females were killed by carbon dioxide inhalation and exsanguination then necropsied. Any found dead adult animals were also submitted to necropsy procedures. On PND 4 and PND 13, pups were sacrificed by intraperitoneal injection of sodium pentobarbitone. Pups that died were necropsied and their stomach examined for the presence of milk, if possible. Defects or cause of death were evaluated, if possible.

GROSS NECROPSY
- 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.
- Samples of the following tissues and organs of the selected 5 animals/sex/group were collected a nd fixed in 10% buffered formalin: Adrenal glands (M/F), (Aorta) (M/F), Brain - cerebellum, mid-brain, cortex (7 levels) (M/F), Caecum (M/F), Cervix (F), Clitoral gland (F), Colon (M/F), Coagulation gland (M), (Cowper’s gland) (M), Duodenum (M/F), Epididymides (M), Eyes (with optic nerve (if detectable) and Harderian gland) (M/F), Mammary gland area (M/F), Femur including joint (M/F), (Glanspenis) (M), (Levator ani plus bulbocavernosus muscle complex (LABC)) (M), Heart (M/F), Ileum (M/F), Jejunum (M/F), Kidneys (M/F), (Lacrimal gland, exorbital) (M/F), (Larynx) (M/F), Liver (M/F), Lung, infused with formalin (M/F), Lymph nodes - mandibular, mesenteric (M/F), (Nasopharynx) (M/F), (Esophagus) (M/F), Ovaries (F), (Pancreas) (M/F), Peyer's patches [jejunum, ileum] if detectable (M/F), Pituitary gland (M/F), Preputial gland (M), Prostate gland (M), Rectum (M/F), (Salivary glands - mandibular, sublingual) (M/F), Sciatic nerve (M/F), Seminal vesicles (M), Skeletal muscle (M/F), (Skin) (M/F), Spinal cord -cervical, midthoracic, lumbar (M/F), Spleen (M/F), Sternum with bone marrow (M/F), Stomach (M/F), Testes (M), Thymus (M/F), Thyroid including parathyroid if detectable (M/F), (Tongue) (M/F), Trachea (M/F), Urinary bladder (M/F), Uterus (F), Vagina (F), All gross lesions (M/F) Tissues/organs mentioned in parentheses were not examined by the pathologist, since no signs of toxicity were noted at macroscopic examination.
- Samples of the following tissues and organs of all remaining animals, males that fail to sire and females which fail to deliver, were collected and fixed in 10% buffered formalin: Cervix (F), Clitoral gland (F), Coagulation gland (M), Epididymides (M), Mammary gland area (M/ F), Ovaries (F), Preputial gland (M), Prostate gland (M), Seminal vesicles (M), Testes (M), Thyroid including parathyroid if detectable (M/F), Uterus (F), Vagina (F), All gross lesions (M/F)

HISTOPATHOLOGY
- Organ and tissue samples were processed, embedded and cut at a thickness of 2-4 micrometers. These slides were stained with haematoxylin and eosin. The additional slides of the testes (to examine staging of spermatogenesis) were stained with PAS/haematoxylin.
- The following slides were examined by a pathologist: The preserved organs and tissues of the selected 5 animals/sex of Groups 1 and 4; Additional slides of the testes of the selected 5 males of Groups 1 and 4 and all males that failed to sire to examine staging of spermatogenesis; The preserved organs and tissues of group 1 male no. 161 which died spontaneously; All gross lesions of all animals (all dose groups); The reproductive organs of all males that failed to sire and all females that failed to deliver healthy pups.
- All abnormalities were described and included in the report. An attempt was made to correlate gross observations with microscopic findings.
- A pathology peer review was conducted by an appropriately experienced pathologist nominated by Charles River Lyon.

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, Uterus (including cervix).
- The following organ weights and terminal body weight were recorded from all remaining animals on the scheduled day of necropsy: Epididymides, Testes, Thyroid (including parathyroid if detectable), Prostate, Seminal vesicles including coagulating glands.

Postmortem examinations (offspring):

SACRIFICE
- Pups, younger than 7 days were killed by decapitation
- The F1 offspring were sacrificed at PND 4 (by decapitation between 7.00 and 10.30 a.m.), and at PND 7-15 (using Euthasol 20% by intraperitoneal injection).

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

HISTOPATHOLOGY / ORGAN WEIGHTS
not examined

Statistics:

Statistical analyses were performed by the Provantis data acquisition system, where appropriate, as follows:
- The best transformation for the data (none, log or rank) was determined depending upon the kurtosis of the data, the probability of the Bartlett's test for homogeneity of the variances and an assessment of whether the size of the groups were approximately equal or not.
- Non- or log-transformed data were analysed by parametric methods.
- Rank transformed data were analysed using non-parametric methods.
- Data were then analysed to test for a dose-related trend to detect the lowest dose at which there was a significant effect, based on the Williams test for parametric data or the Shirley's test for nonpa rametric data.
- Homogeneity of means was assessed by analysis of variances (ANOVA) for parametric data or Kruskal-Wallis test for non-parametric data.
- If no trend was found and means were not homogeneous, the data were analysed by parametric or non-parametric Dunnett's test to look for significant differences from the control group.
- Selected incidence data were analysed using the Provantis data acquisition system and/or a SAS software package. A chi2 test was used for all groups followed by Fisher’s two-tailed test with Bonferroni correction for each treated group versus the control if the chi2 was significant.
- Functional test (locomotor activity in an open field test), oestrous cycle and pre-coital interval and anogenital distance data were analysed using a SAS software package. Levene’s test was used to test the equality of variance across groups and Shapiro-Wilk's test was used to assess the normality of the data distribution in each group. Data with homogenous variances and normal distribution in all groups were analysed using ANOVA followed by Dunnett’s test.
- Data showing non-homogenous variances or a non-normal distribution in at least one group were analysed using Kruskal-Wallis test followed by the Wilcoxon's rank sum test.

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 in any group for both sexes during the observation period. Incidental clinical signs that were noted included scabs, severed tail or broken tooth. These findings occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study. Localized hairloss were observed in all groups but with a higher incidence for females given 330 mg/kg/day. In the absence of a doserelationship, this increase was considered incidental.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

No mortality occurred during the study period that was considered to be related to treatment with the test item.

Male no. 161 given 1000 mg/kg/day was found dead just after dosing on day 28 of treatment. At necropsy, all lung lobes were dark (which correlated with incomplete exsanguination) and uncollapsed. There were no treatment-related changes and the cause of death could not be determined at pathological evaluation. This isolated death was considered as unrelated to treatment.

There were no other unscheduled deaths in any group.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

No toxicologically relevant changes in body weights and body weight gain were noted in both sexes during the study.

Males: There was an overall slightly lower mean body weight gain for males given 330 and 1000 mg/kg/day (64.5 and 65.3 g between Days 1 and 29, respectively) compared with 74.5 g and 70.0 in the control and 110 mg/kg/day groups, respectively. The mean final body weights were, however, only slightly lower (approximately -2 %) than controls. These minor differences were therefore not considered toxicologically relevant.

Females: During the pre-mating period, there was a lower mean body weight gain in treated females (9.7, 12.4 and 7.4g in the 110, 330 and 1000 mg/kg/day respectively) than in control females (14.9 g) between Days 1 and 15. Since the initial mean body weights were already slightly lower in the 110 and 1000 mg/kg/day groups than in the control and 330 mg/kg/day groups and in the absence of a dose-relationship, this change was considered incidental.

There were no obvious effects on mean body weights or body weight gains during the gestation or lactation periods. There was a slightly lower mean body weight gain during the lactation period for females given 110 mg/kg/day. The effect was not dose-related. Moreover, the mean body weights were slightly higher in this group on first day of lactation than in the control, 330 and 1000 mg/kg/day groups and there was no impact on the mean final body weights. This change was thus considered as incidental and unrelated to treatment.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

No toxicologically relevant changes in food consumption were noted.

Males: There was no obvious effect on food consumption in any group.

Females: During the premating period, consistent with the observations noted on body weights, there was a slightly lower mean food consumption from Days 1 to 15 in all treated females compared with controls. As for body weights, the effect was minor and not dose-related and therefore considered of no toxicological relevance. There were no obvious effects on mean food consumption in other groups during the gestation or lactation periods. Consistent with a lower mean body weight gain, there was also a slightly lower mean food consumption during the lactation period for females given 110 mg/kg/day (-10.4 % compared with controls). This effect was not considered as toxicologically relevant since it did not overtly affect mean body weights in this group.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

no effects observed

Description (incidence and severity):

There was no obvious test item-related effect on water consumption in either group.

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no differences noted in haematological parameters between control and treated rats of both sexes that were considered to be related to treatment with the test item.

Minor statistically significant differences arising between controls and animals receiving the test item (i.e. increased haemoglobin, PCV and MCV, lymphocytes and monocytes in females) were not considered to represent a change of biological relevance.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no differences noted in clinical biochemistry parameters between control and treated rats of both sexes that were considered to be related to treatment with the test item.

The following differences were observed and were not considered to be toxicologically significant as they occurred in the absence of a treatment-related distribution:
− Differences in mean bile acids values for males were due to a high intra-group variability. There were 3, 2, and 1 males in the control, 110 and 330 mg/kg/day groups, respectively, with high values, compared with none in the 1000 mg/kg/day group.
− High mean aspartate and alanine aminotransferase (ASAT and ALAT) levels in the control group were due to elevated values for male no. 105 (above the historical control range). Male no. 163 in the 1000 mg/kg/day also had, however less pronounced, elevated ASAT and ALAT values. In isolation, these changes were considered incidental.

Minor statistically significant differences (i.e. chloride levels in males) arising between controls and animals receiving the test item were not considered to represent a change of biological relevance and remained within the range considered normal of this age and strain.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

No toxicologically relevant effects on hearing ability, pupillary reflex, static righting reflex and grip strength were observed.

Differences in fore-limb mean values for males were due to a high intra-group variability. There were 1 and 2 males in the control and 110 mg/kg/day groups, respectively, with high values (>10 Newton), compared with none in the 330 and 1000 mg/kg/day group.

The variation in motor activity (open field) did not indicate a relation with treatment.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

All the microscopic findings were considered to be incidental and within the background histopathological variation in this species.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Reproductive function: oestrous cycle:

effects observed, non-treatment-related

Description (incidence and severity):

Length and regularity of the estrous cycle were not considered to have been affected by treatment.

An irregular cycle was noted for control female no. 116 before mating. Extended di-estrus (from Day 17 to Day 29) occurred from mating in female no. 152 given 330 mg/kg/day. These findings were not correlated to pregnancy status and were considered incidental.

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
No toxicologically relevant effects on reproductive parameters were noted.
- Mating index: Mating index was not affected by treatment. All females showed evidence of mating.
- Fertility index: Fertility index was not affected by treatment. There was one female in each of the control (no. 112), 110 (no. 135) and 1000 (no. 173) mg/kg/day groups that did not become pregnant. In the absence of a dose-related incidence of non-pregnancy, this was not considered to be related to treatment.
- Precoital time: Precoital time was not affected by treatment. Except female no.152 given 330 mg/kg/day with a pre-coital interval of 14 days, all mated females showed evidence of mating within the first 4 days of pairing (approximate duration of a normal oestrus cycle). The mean pre-coital interval was consequently comparable in all groups.
- Number of implantation sites: Number of implantation sites was not affected by treatment.

DEVELOPMENTAL DATA
No toxicologically relevant effects on developmental parameters were noted. There were 9, 9, 10 and 9 females that completed delivery in the control, 110, 330 and 1000 mg/kg/day groups, respectively.
- Gestation index and duration: The gestation index and duration were not affected by treatment in any group.
- Parturition/maternal care: No signs of difficult or prolonged parturition were noted among the pregnant females. No deficiencies in maternal care were observed.
- Post-implantation survival index: The total number of offspring born compared to the total number of uterine implantations was not considered affected by treatment. Pre-birth loss was slightly higher for females given 110 mg/kg/day (12.7%) compared to that of control (10.2%) but was exacerbated by one female (no. 137) with a post implantation loss of 60%. This was therefore not considered as treatment related
- Live birth index: The number of pups born alive compared to the total number of pups born was not considered affected by treatment. The mean number of pups delivered per litter was higher in the 330 and 1000 mg/kg/day groups (13.8 and 12.0, respectively) than in the control and 110 mg/kg/day groups (10.6 and 9.1, respectively). Three, one and three pups at 110, 330 and 1000 mg/kg/day were found dead or missing versus none in the control group at birth. 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.
- Viability index: The number of live offspring on Day 4 before culling compared to the number of offspring alive at birth was not considered affected by treatment. Three, three and one pups at 110, 330 and 1000 mg/kg/day were found dead or missing versus none in the control group between lactation day 0 and 4. 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: 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. No pups were found dead/missing between lactation Days 5 and 13.

Parental results:
No parental toxicity was observed up to the highest dose level tested (1000 mg/kg).

Reproductive results:
No reproduction toxicity was observed up to the highest dose level tested (1000 mg/kg). No treatment-related changes were noted in any of the reproductive parameters investigated in this study (i.e., mating and fertility indices, precoital time, number of implantations, estrous cycle, spermatogenic profiling, and histopathological examination of reproductive organs).


Analysis of Dose Preparations:
- Accuracy of preparation: All formulations at 22, 66 and 200 mg/mL of T001095 in vehicle (propylene glycol), including the vehicle, used on the first day of treatment of the main study, were in agreement with acceptance criteria. The accuracy expressed as the recovery from the nominal concentrations ranged from 100.1 % to 102.9 %, and the RSD was ≤0.6 %. No significant amount of test item was detected in the vehicle sample.
- Homogeneity: The formulations at 22 and 200 mg/mL were homogenous.

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 evaluated in this study.

Remarks on result:

not determinable due to absence of adverse toxic effects

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:

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.

Incidental clinical symptoms of pups consisted of occasional weak or thin pup, incomplete hair growth, sores, limp, and haematoma. The nature and incidence of these 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 alive at birth was not considered affected by treatment. Three, three and one pups at 110, 330 and 1000 mg/kg/day were found dead or missing versus none in the control group between lactation day 0 and 4. 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, non-treatment-related

Description (incidence and severity):

Body weights of pups were not considered affected by treatment.

The mean pup body weights (males and females) were slightly lower in the 330 and 1000 mg/kg/day groups than in the control and 110 mg/kg/day groups from Days 1 to 13 of lactation. There were, however, no obvious changes in body weight gains. These minor differences were considered unrelated to treatment with the test item but due to the slightly higher live litter size at birth in the two highest dose 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:

effects observed, non-treatment-related

Description (incidence and severity):

There was no adverse effect of treatment on the total T4 levels for the PND 13 pups of either sex.

Slightly lower mean values were noted for the male and female pups in the 330 and 1000 mg/kg/day groups compared with the concurrent control and generally attained statistical significance. However, values remained within the historical control range so the minor differences were considered to be of no toxicological significance.

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Anogenital distance (normalized for body weight) in male and female pups was not considered to be affected by treatment.

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 the highest dose level tested (1000 mg/kg).
- No treatment-related changes were noted in any of the developmental parameters investigated in this study (i.e. gestation, viability and lactation indices, gestation index and duration, 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) and macroscopy.

Key result

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 evaluated in this study.

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Conclusions:

In conclusion, treatment with JNJ-16479671-AAA (T001095) by oral gavage in male and female Wistar Han rats at dose levels of 110, 330 and 1000 mg/kg revealed no adverse parental, reproduction and developmental toxicity for treatment up to 1000 mg/kg.
Based on these results, the following No Observed Adverse Effect Levels (NOAEL) were derived:
Parental NOAEL: at least 1000 mg/kg
Reproduction NOAEL: at least 1000 mg/kg
Developmental NOAEL: at least 1000 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 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; Mounier R., 2017). The vehicle used was propylene glycol and the test solutions were prepared and administered daily. There was no adverse parental and reproduction toxicity up to 1000 mg/kg body weight/day.

No parental toxicity was observed up to 1000 mg/kg/day.

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

Based on the above mentioned considerations, no adverse parental and reproduction toxicity was revealed up to 1000 mg/kg bw/day. The parental and reproduction NOAELs were established as being at least 1000 mg/kg bodyweight/day.

Effects on developmental toxicity

Description of key information

In the combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test (OECD 422; Mounier R., 2017), the test item was administered daily to rats at dose levels up to 1000 mg/kg bodyweight/day.

The developmental NOAEL was established as being at least 1000 mg/kg bodyweight/day. The substance is not classified as a reproductive toxicant according to the CLP Regulation.

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

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Justification for classification or non-classification

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

Parental NOAEL: at least 1000 mg/kg.

Reproduction NOAEL: at least 1000 mg/kg.

Developmental NOAEL: at least 1000 mg/kg.

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