Registration Dossier

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2015-10-30 to 2016-02-16
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA OPPTS 870.3650 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
other: EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
other: OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
other: EPA, OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
other: 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. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
- Name of test material (as cited in study reports): JNJ-39453193-AAA (T003063)
- Physical state: solid (powder)
- Appearance: white to almost white powder
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: M14KB4863
- Expiration date of the lot/batch: 2017-11-10 (retest date)
- Purity test date: 2015-05-12

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature.
- Stability under test conditions: Over 6 hours at room temperature under normal laboratory light conditions.
- Solubility and stability of the test substance in the solvent/vehicle: Stability of formulations over 6 hours at room temperature under normal laboratory light conditions (concentration range 1-200 mg/mL) was confirmed as part of the analytical method development and validation study (Test Facility Study No. 509948).


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


OTHER SPECIFICS: No purity/composition correction factor applied

Test animals

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. The laboratory 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: Females: approximately 11 weeks (start of pretest) and approximately 13 weeks (start of F0-treatment); Males: approximately 11 weeks (start of F0-treatment).
- Weight at study initiation: 291-329 g (males); 204-235 g (females)
- Fasting period before study: No
- Housing:
Pretest: Females were housed in groups of 5 females/cage in Macrolon plastic cages (MIV type, height 18 cm).
Pre-mating: Animals were housed in groups of 5 animals/sex/cage in Macrolon plastic cages (MIV type, height 18 cm).
Mating: Females were caged together with males on a one-to-one-basis in Macrolon plastic cages (MIII type, height 18 cm).
Post-mating: Males were housed in their home cage (Macrolon plastic cages, MIV type, height 18 cm) with a maximum of 5 animals/cage. Females were individually housed in Macrolon plastic cages (MIII type, height 18 cm).
Lactation: Pups were kept with the dam until termination in Macrolon plastic cages (MIII type, height 18 cm).
- Use of restrainers for preventing ingestion (if dermal): no, not applicable
- 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): 18-24 °C
- Humidity (%): 40-70%
- Air changes (per hr): At least 10 room air changes/hr
- Photoperiod (hrs dark / hrs light): 12-hours light / 12-hours dark.

IN-LIFE DATES:
From: 2015-12-07 (start pretest, females); 2015-12-21 (start treatment, males); 2016-01-27/28/29/30 and 2016-02-02 (delivery of litters)
To: 2016-01-19 (necropsy males); 2016-02-09/10/11/14/15 (necropsy pups); 2016-02-10/11/12/15/16 (necropsy females)

Administration / exposure

Route of administration:
oral: gavage
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
- Formulations (w/w) were prepared daily within 6 hours prior to dosing and were homogenized to a visually acceptable level. Adjustment was made for specific gravity of the vehicle. No correction was made for the purity of the test item.
- 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; Control); 20 mg/mL (Group 2); 60 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
- Length of cohabitation: Following a minimum of 14 days of treatment, males and females were cohabited on a 1:1 basis within the same treatment group, avoiding sibling mating. Once mating was confirmed, the males and females were separated.
- 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 first week of the treatment phase (2015-12-23), according to a validated method (Test Facility Study No. 509948). Three sets of duplicate samples were collected. Two sets of duplicate samples were stored as reserve samples. Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Analytical results were approved by the Study Director and the reserve samples were destroyed.

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. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%.

Stability of the test item under test conditions was demonstrated in the method validation study (Test Facility Study No. 509948).

Density was determined from all formulations on a single day during the treatment phase to express analytical concentrations in mg/mL.
Duration of treatment / exposure:
Males: 29 days
Females that delivered: 51-57 days
Females which failed to deliver: 42 days
Pups: Pups were not dosed directly but could have potentially been exposed to the test item in utero, via maternal milk or from exposure to maternal urine/faeces.
Note: Female Nos. 51, 60 (Group 2) and 66, 70 (Group 3) were not dosed on Day 1 of lactation as these females were littering at the time of dosing. The omission of one day of dosing over a period of several weeks was not considered to affect the toxicological evaluation.
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.
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Group 1 (Control)
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
Group 2
Dose / conc.:
300 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 results of a dose range finding study (Test Facility Study No. 509945) in which animals were dosed for 10 days at 500 and 1000 mg/kg. In summary, no mortality occurred and there were no effects on clinical appearance, body weight, food consumption and macroscopic examination. Liver and kidney weights were considered normal. Based on the results of this range finding study, dose levels of 100, 300 and
1000 mg/kg/day were selected for the main study.
- Rationale for animal assignment (if not random): randomized
Positive control:
no

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least twice daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily from treatment onwards up to the day prior to necropsy, detailed clinical observations were made in all animals. Once prior to start of treatment and at weekly intervals during the treatment period this was also performed outside the home cage in a standard arena. These clinical observations were made after dosing (at no specific time point as there was no peak occurrence of clinical signs after dosing in the dose range finding study; Test Facility Study No. 509945).


BODY WEIGHT: Yes
- Time schedule for examinations: Males and females were weighed on the first day of treatment 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 and calculated body weight gain were 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. Both absolute food consumption and food consumption relative to body weight were reported.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Not applicable


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


HAEMATOLOGY: Yes
- Time schedule for collection of blood: At the end of the treatment period on the day of scheduled necropsy.
- Anaesthetic used for blood collection: Yes, Isoflurane.
- Animals fasted: Yes, the animals were deprived of food overnight (with a maximum of 24 hours) before blood sampling, but water was available.
- How many animals: The selected 5 animals/sex/group.
- Parameters examined: The following haematology parameters were determined in blood prepared with K3-EDTA as an anti-coagulant: White blood cells, Neutrophils, Lymphocytes, Monocytes, Eosinophils, Basophils, 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
- Time schedule for collection of blood: At the end of the treatment period on the day of scheduled necropsy.
- Animals fasted: Yes, the animals were deprived of food overnight (with a maximum of 24 hours) before blood sampling, but water was available.
- How many animals: The selected 5 animals/sex/group.
- Parameters examined: 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.
All parameters were determined in plasma, except for bile acids and thyroid hormone which were determined in serum.


FUNCTIONAL OBSERVATIONS:
- Time schedule for examinations: The selected males were tested during Week 4 of treatment and the selected females were tested once during the last week of lactation. These tests were performed after observation for clinical signs (incl. arena observation, if applicable).
- Dose groups that were examined: the selected 5 animals/sex/group
- Battery of functions tested: hearing ability, pupillary reflex, static righting reflex, fore- and hind-limb grip strength recorded as the mean of 3 measurements per animal, locomotor activity
Oestrous cyclicity (parental animals):
Daily vaginal lavage was performed to determine the stage of estrous beginning 14 days prior to treatment (pretest), the first 14 days of treatment and during mating until evidence of copulation was observed. Vaginal lavage continued for those females with no evidence of copulation until termination of the mating period. A total of 40 females was selected at random before initiation of the pretest phase. Any selected female without at least two regular estrous cycles at the end of the pretest phase was replaced at random by one of the 8 additional females having at least two regular estrous cycles. A total of 40 females with at least two regular estrous cycles continued in the study. The supernumerary females were then removed from the study, and their estrous cycle results were kept in the raw data but were not reported. On the day of necropsy, a vaginal lavage was taken to determine the stage of estrous.
Sperm parameters (parental animals):
Parameters examined in F0 male parental generation:
testis weight, additional slides of testes to examine staging of spermatogenesis
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: No
- On PND 1, all pups were individually identified by means of subcutaneous injection of Indian ink. When general hair growth blurs the identification, the pups were identified by tattoo on the feet.
- 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.
If there were insufficient pups in a litter to have two surplus pups, two of the pups that were typically retained were used for blood collection. When the litter size dropped below the culling target of eight pups/litter, preference was given to remove two female pups for PND 4 blood samples in order to retain more male pups for nipple retention on PND 13. However, retained female pups in each litter did not drop below 2.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
- Mortality/viability: The numbers of live and dead pups were determined on PND 1 and daily 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.
- Body weights: Live pups were weighed on PND 1, 4, 7 and 13.
- Sex: Sex was determined for all pups on PND 1 and 14.
- 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. 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.

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY:
Not examined

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY:
Not examined
Postmortem examinations (parental animals):
SACRIFICE:
Males: Following completion of the mating period (a minimum of 28 days of dose administration).
Females that delivered: On PND 14-16
Females which failed to deliver: On Post-coitum Days 25-27 (females with evidence of mating).
Spontaneous death: One animal (no. 68), necropsy as soon as possible after death and always within 24 hours.

GROSS NECROPSY
- After sacrifice, all animals were subjected to a full post mortem necropsy, with special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities were recorded.
- The number of implantation sites were recorded for all paired females.
- Samples of the following tissues and organs of the selected 5 animals/sex/group were collected and 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), (Glans penis) (M), (Levator ani plus bulbocavernosus muscle complex (LABC)) (M), Heart (M/F), Ileum (M/ F), Jejunum (M/F), Kidneys (M/F), (Lacrimal gland, exorbital) (M/F), (Larynx) (M/F), Liver (M/F), Lung , infused with formalin (M/F), Liver (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 (F), Skeletal muscle (M/F), (Skin) (M/F), Spinal cord -cervical, midthoracic, lumbar (M/F), Spleen (M/F), Sternum with bone marrow (M/F), Stomach (M/F), Testes ( M), Thymus (M/F), Thyroid including parathyroid if detectable (M/F), (Tongue) (M/F), Trachea (M/F), Urinary bladder (M/F), Uterus (F), Vagina (F), All gross lesions (M/F)
Tissues/organs mentioned in parentheses were not examined by the pathologist, since no signs of toxicity were noted at macroscopic examination.

-Samples of the following tissues and organs of all remaining animals, males that fail to sire and females which fail to deliver, were collected and fixed in 10% buffered formalin: Cervix (F), Clitoral gland (F), coagulation gland (M), Cowper’s glands (M), Epididymides (M), Glans penis (M), Levat or ani plus bulbocavernosus muscle complex (LABC) (M), Mammary gland area (M/F), Ovaries (F), Preputial gland (M), Prostate gland (M), Seminal vesicles (M), Testes (M), Thyroid including pa rathyroid if detectable (M/F), Uterus (F), Vagina (F), All gross lesions (M/F)

ORGAN WEIGHTS
- Absolute organ weights and organ to body weight ratios were reported.
- The following organ weights and terminal body weight were recorded for the selected 5 animals/sex/group on the scheduled day of necropsy: Adrenal glands, Brain, Cowper’s glands, Epididymides, Glans penis, Heart, Kidneys, Levator ani plus, bulbocavernosus muscle complex (LABC), 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 for all remaining animals on the scheduled day of necropsy: Cowper’s glands, Epididymides, Glans penis, Levator ani plus bulbocavernosus muscle complex (LABC), Testes, Thyroid


HISTOPATHOLOGY
- All organ and tissue samples were processed, embedded and cut at a thickness of 2-4 micrometers. These slides were stained with haematoxylin and eosin (Klinipath, Duiven, The Netherlands). The additional slides of the testes (to examine staging of spermatogenesis) were stained with PAS/haematoxylin.
- The following slides were examined by a pathologist:
1) The preserved organs and tissues of the selected 5 animals/sex of Groups 1 and 4.
2) The 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.
3) The preserved organs and tissues of the female (no. 68) of Group 3 which died spontaneously.
4) All gross lesions of all animals (all dose groups).
5) Thyroid gland of all selected 5 females of Groups 2 and 3, based on (possible) treatment-related changes in this organ in Group 4.
6) 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 peer review on the histopathology data was performed by a second pathologist.


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 both externally and internally. Descriptions of all abnormalities were recorded. Particular attention was paid to the external reproductive genitals to examine signs of altered development.
- At terminal sacrifice (PND 13-15), the thyroid from 1 male and 1 female pup per litter, i.e. the same pups as selected for blood sampling, was preserved in 10% buffered formalin.
- For pup 1 in litter 57 (Group 2) the testes were collected and fixed in 10% buffered formaldehyde due to abnormalities.
- 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 analyze the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-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 (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
- The Kruskal-Wallis nonparametric ANOVA test was applied to motor activity data to determine intergroup differences.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance.

Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.
Reproductive indices:
For each group, the following calculations were performed:
Mating index (%) = (Number of females mated/Number of females paired) x 100
Fertility index (%) = (Number of pregnant females/ Number of females paired) x 100
Gestation index (%) = (Number of females bearing live pups/Number of pregnant females) x 100
Duration of gestation = Number of days between confirmation of mating and the beginning of parturition
Offspring viability indices:
SURVIVAL INDICES:
Post-implantation survival index (%) = (Total number of offspring born/ Total number of uterine implantation sites) x 100

Post-implantation survival index was expressed as 100% when the number of offspring exceeded the number of implantation sites recorded.

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

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

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

Group mean values were calculated from individual litter values.

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

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
no effects observed
Description (incidence and severity):
No clinical signs of toxicity were noted up to the highest dose level (1000 mg/kg/day).
Any other clinical signs noted during the treatment period 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 and severity observed, these were considered signs of no toxicological relevance.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One female treated at 300 mg/kg/day (no. 68) was found dead after 6 treatment days. On the day prior to death, rales were noted at the clinical observations after dosing. The macroscopic findings (reddish/watery-clear fluid in the thoracic cavity and dark-red discoloration of the lungs) and microscopic findings (massive ulceration of the epithelium of the trachea) were suggestive of a gavage accident. Based on these findings and the absence of mortality in the high-dose group (1000 mg/kg/day), the death of female no. 68 was considered to be unrelated to treatment with the test item.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related changes in body weights and body weight gain were noted.
At Day 1 of the mating period, a statistically significantly higher body weight gain was noted in females at 1000 mg/kg/day. This incidental finding was considered to be unrelated to treatment because no remarkable differences in body weight gain occurred at other time points during the treatment period.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related changes in food consumption before or after allowance for body weight were noted.
In the second week of the lactation period (PND 7-13), mean food consumption at 1000 mg/kg/day was higher compared to controls (statistically significantly after allowance for body weight). This difference was considered to have arisen as a result of a slightly low mean control value. The lower control mean was particularly due to the low food consumption of one female (no. 41) which had only three pups of which one was missing on PND 2.
At the individual level, a relatively low food consumption (absolute and relative) was noted for one Group 3 female (no. 64) from Days 0-4 post-coitum, followed by complete recovery thereafter. No explanation for this finding could be found based on the available data. No clinical signs were noted for this female during the repro period, and she had a completely normal litter.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
No toxicologically relevant changes occurred in haematological parameters of treated rats.
Any statistically significant variations noted in haematology parameters were unrelated to treatment or not toxicologically relevant due to the slight magnitude of the change (values in treated rats remained within normal limits), low control values and/or absence of a dose-related response.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
No toxicologically relevant changes occurred in clinical biochemistry parameters of treated rats.
Any statistically significant variations noted in clinical biochemistry parameters were unrelated to treatment or not toxicologically relevant due to the slight magnitude of the change (values in treated rats remained within normal limits) and/or absence of a dose-related response
At the individual level, a relatively high value for bile acids was recorded for female no. 59 (Group 2). In the absence of any correlating findings, it was considered as a chance finding rather than to be related to treatment.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Hearing ability, pupillary reflex, static righting reflex and grip strength were not affected by treatment. The variation in motor activity did not indicate a relation with treatment. All groups showed a similar habituation profile with high activity in the first interval that decreased over the duration of the test period.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No treatment-related changes were noted in organ weights and organ to body weight ratios.
Any statistically significant variations noted in organ weights were unrelated to treatment due to the absence of a dose-related response.
The relative high liver organ weight (absolute and relative) recorded for male no. 11 (Group 2) was due to the incomplete exsanguination at necropsy and hence not test item related.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item related gross findings at necropsy.
In some females of all groups, including controls, reddish/dark red foci were noted in the glandular stomach. This was seen in 3 out of 10 females of the control group, 3 out of 10 females at 100 mg/kg, 4 out of 10 females at 300 mg/kg, and 2 out of 10 females at 1000 mg/kg. The microscopic correlates for this finding were congestion/hemorrhage, apoptosis and/or necrosis of the glandular mucosa. The incidence and severity of these findings in all dose groups of females including controls were above background levels, but as the findings didn’t show a dose relationship, they were regarded to be unrelated to treatment with the test item.
The remainder of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
A slightly increased incidence and severity of follicular cell hypertrophy was recorded in the thyroid gland of 4 out of 5 females at 100 mg/kg/day (3 minimal, 1 slight), 3 out of 6 females at 300 mg/kg/day (minimal) and 3 out of 5 females at 1000 mg/kg/day (2 minimal, 1 slight), compared to 1 out of 5 females of the control group (minimal).

There were no other test item-related histologic changes. Remaining histologic changes were considered to be incidental findings. There was no test item related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

There were two couples of the control group without offspring (male/female nos. 8/48 and 10/50). Female no. 48 had been pregnant based on the presence of placental tissue in the uterus (2 implantation sites were noted macroscopically at necropsy). No abnormalities were seen in the reproductive organs of any of these animals which could account for their lack of healthy offspring.

Female no. 68 (treated at 300 mg/kg) died before the start of the mating period. No abnormalities were seen in the reproductive organs of this female and the male (no. 28) intended for mating with this female.

There were no morphological findings in the reproductive organs of either sex which could be attributed to the test item and spermatogenic staging profiles were normal for all males examined.
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
effects observed, non-treatment-related
Description (incidence and severity):
Length and normality of the estrous cycle were not affected by treatment.
Most females had regular cycles of 4 days. Extended di-estrus during pairing occurred in two control females, and irregular cycles occurred in two other control females and one female at 100 mg/kg/day. The females showing estrous cycle abnormalities had normal litters, except for one control female (no. 41) which delivered only three pups. The incidence of these findings did not indicate a relation with treatment.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
Spermatogenic staging profiles were normal for all males examined.
Reproductive performance:
no effects observed
Description (incidence and severity):
REPRODUCTION DATA
No toxicologically relevant effects on reproductive parameters were noted.
- Mating and fertility indices, precoital time and number of implantation sites were not affected by treatment.
- All paired females had mated and, except for one control female (no. 50), all mated females were pregnant.
- The mean number of implantation sites at 1000 mg/kg/day was statistically significantly higher compared to that in controls. All females at 1000 mg/kg/day had normal numbers of implantation sites (between 11 and 16) whereas two control females had only four or two implantation sites (female nos. 41 and 48, respectively). Therefore, the higher mean value at 1000 mg/kg/day was attributed to a lower control group mean rather than to treatment with the test item.
- For female no. 59 (100 mg/kg/day), the number of pups was slightly higher than the number of implantations. This was considered to be due to the normal resorption of these areas as these enumerations were performed on PND 15.

DEVELOPMENTAL DATA
No toxicologically relevant effects on gestation index and duration, parturition, maternal care and early postnatal pup development were observed (postnatal endpoints evaluated are given below).

All pregnant females delivered live offspring, except for one control female (no. 48) which had implantations only. The number of females with living pups on PND 1 was 8, 10, 9 and 10 in the control, 100, 300 and 1000 mg/kg/day groups, respectively.

- Gestation: The gestation index and duration of gestation were unaffected by treatment up to 1000 mg/kg.
- 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 abortion or premature birth. No deficiencies in maternal care were observed.
- Post-implantation survival index: Post-implantation survival index was unaffected by treatment up to 1000 mg/kg/day.
- Early postnatal pup development: No treatment-related changes were noted in the live birth, viability and lactation indices, sex ratio, anogenital distance, areola/nipple retention, clinical signs, body weight, external macroscopy and serum concentration of the thyroid hormone T4 (PND 13-15).
- Mortality: At the first litter check, one pup of the control group, four pups at 100 mg/kg/day, two pups at 300 mg/kg/day and five pups at 1000 mg/kg/day were found dead. Early during lactation (PND 2 or 3), one pup of the control group, three pups at 100 mg/kg/day, six pups at 300 mg/kg/day and eight pups at 1000 mg/kg/day went missing, and one pup at 300 mg/kg/day was found dead. Pups missing were most likely cannibalised. No toxicological relevance was attributed to these missing/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. No breeding loss (i.e. between PND 5-13) occurred.
- The live birth, viability and lactation indices were not affected by treatment.

Details on results (P0)

Analysis of dose preparations: Formulation analysis showed that the formulations were prepared accurately and homogenously.

Parental results:
- No parental toxicity was observed up to the highest dose level tested (1000 mg/kg/day).
- Microscopic examination revealed a treatment-related increase in follicular cell hypertrophy in the thyroid of treated females starting at 100 mg/kg/day. The minor increase in incidence and severity (up to slight degree) of this change occurred in the absence of any degenerative changes and was considered to be an adaptive, nonadverse change.
- No treatment-related or toxicologically relevant changes were noted in any of the remaining parental parameters investigated in this study (i.e. clinical appearance, functional observations, body weight, food consumption, clinical laboratory investigations, serum concentration of the thyroid hormone T4 (measured in males only), macroscopic examination, organ weights, and microscopic examination of the remaining tissues and organs).

Reproductive results:
- No reproduction toxicity was observed up to the highest dose level tested (1000 mg/kg/day).
- 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).

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
Parental
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.
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Dose descriptor:
NOAEL
Remarks:
Reproduction
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.
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity (P0)

Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
- Clinical signs in pups that went missing included little or no milk in the stomach, scabs on the abdomen, and pale appearance. Incidental clinical signs in surviving pups consisted of lean appearance, swelling of the mouth, wound or scabs on the ear, scabs on the head, little or no milk in the stomach, and abnormal posture. The nature and incidence of these clinical signs remained within the range considered normal for pups of this age, and were therefore considered to be of no toxicological relevance.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
At the first litter check, one pup of the control group, four pups at 100 mg/kg/day, two pups at 300 mg/kg/day and five pups at 1000 mg/kg/day were found dead. Early during lactation (PND 2 or 3), one pup of the control group, three pups at 100 mg/kg/day, six pups at 300 mg/kg/day and eight pups at 1000 mg/kg/day went missing, and one pup at 300 mg/kg/day was found dead. Pups missing were most likely cannibalised. No toxicological relevance was attributed to these missing/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. No breeding loss (i.e. between PND 5-13) occurred.
The live birth, viability and lactation indices were not affected by treatment.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Body weights of pups were not affected by treatment.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
Serum T4 levels in male and female PND 13-15 pups were not affected by treatment.
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Incidental macroscopic findings in pups that were found dead included absence of milk in the stomach and beginning autolysis. Macroscopic findings among surviving pups were limited to isolated, reddish foci on the testes (bilateral) and lean appearance in one pup each at 100 mg/kg/day and 1000 mg/kg/day, respectively. No toxicological relevance was attached to these incidental findings.
Histopathological findings:
not examined
Other effects:
effects observed, non-treatment-related
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 affected by treatment.
In the absence of a dose-related response, the statistically significantly higher mean absolute anogenital distance noted in male pups at 100 mg/kg/day was not related to treatment.

AREOLA/NIPPLE RETENTION
Treatment up to 1000 mg/kg/day had no effect on areola/nipple retention. For none of the examined male pups nipples were observed at PND 13.

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Details on results (F1)

DEVELOPMENTAL RESULTS
- No developmental toxicity was observed up to the highest dose level tested 1000 mg/kg/day).
- No treatment-related changes were noted in any of the developmental parameters investigated in this study (i.e. gestation, live birth, 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), serum concentration of thyroid hormone T4 (PND 13-15) and macroscopy.

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Remarks:
Developmental
Generation:
F1
Effect level:
>= 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No treatment-related or toxicologically relevant changes in any of the parameters

Target system / organ toxicity (F1)

Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Applicant's summary and conclusion

Conclusions:
Based on the results, the following No Observed Adverse Effect Levels (NOAEL) were derived:
- Parental NOAEL: at least 1000 mg/kg/day
- Reproduction NOAEL: at least 1000 mg/kg/day
- Developmental NOAEL: at least 1000 mg/kg/day

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