Registration Dossier

Toxicological information

Toxicity to reproduction

Currently viewing:

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10. January to 4. April 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose:
reference to same study
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10. January to 4. April 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
July 2016
Deviations:
yes
Remarks:
food consumption was not measured in recovery males, clinical observation was not conducted on two days in females.
GLP compliance:
yes (incl. certificate)
Remarks:
Bayerisches Landesamt für Gesundheit- und Lebensmittelsicherheit
Limit test:
no
Species:
rat
Strain:
Wistar
Remarks:
Crl:WI(Han)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, 97633 Sulzfeld, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: approx. 13-15 weeks
- Weight at study initiation: males: 324 - 378 g, females: 203 - 250 g
- Fasting period before study: no
- Housing: 5 animals per sex per cage during pre-mating and post-mating period. During mating period males and females were housed together in ratio 1:1 (male to female).
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 55 ± 10
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: The test item was prepared at least once every 10 days which is with the stability frame.

VEHICLE
- Justification for use and choice of vehicle: The vehicle was selected in consultation with the sponsor based on the test item’s
- Concentration in vehicle: 0, 12.5, 37.5, 75 mg/mL
- Amount of vehicle: 4 mL/kg bw/day
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The determination of formulation concentrations of the test substance was based on a GC-FID method. Nominal concentrations were confirmed for all dose groups, as measured concentrations were within acceptance criterion of 10%.
Duration of treatment / exposure:
males: 28 days
females: up to 63 days
Frequency of treatment:
daily
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
150 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
main study: 10 animals per sex per dose
recovery group: 12 animals per sex per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The highest dose level of 300 mg/kg bw/day was chosen on the basis of a dose range finding study, where mortality was observed after repeated oral administration at a dose of 1000 mg/kg bw/day (later reduced to 600 mg/kg bw/day). Thereafter, a descending sequence of dose levels was selected with a view to demonstrate any dose-related response and a NOAEL.
- Fasting period before blood sampling for clinical biochemistry: no
Positive control:
no
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once a day
- Cage side observations: health condition, morbidity, moratility

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once before the first exposure and at least once a week thereafter

BODY WEIGHT: Yes
- Time schedule for examinations: once before the assignment to the experimental groups, on the first day of dosing and weekly thereafter as well as at the end of the study. During pregnancy, females were weighed on gestation days (GD) 0, 7, 14 and 20 and within 24 hours of parturition (day 0 post-partum), on PND 4, 9 and 13 along with pups. All animals were weighed directly before termination.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: in the week before the first treatment and during the last week of the treatment for the main group; for the recovery group additionally in the last week of the recovery period
- Dose groups that were examined: main study: in 5 randomly selected males and during the last week of the lactation period in 5 randomly selected females (only lactating females were evaluated); recovery study: all males and females

HAEMATOLOGY: Yes
- Time schedule for collection of blood: as part of the sacrifice: males: any time after the completion of the mating period (after a minimum dosing period of 28 days); females: on PND 13
- Anaesthetic used for blood collection: Yes (ketamine/xylazin)
- Animals fasted: No
- How many animals: 5 randomly selected males and females (only lactating females were evaluated) from each main group at the end of the treatment period and in all recovery group males and females at the end of the recovery period as part of the sacrifice of the animals
- Parameters checked: haematocrit value (HCT), haemoglobin content (HGB), red blood cell count (RBC), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), reticulocytes (Ret), platelet count (PLT), white blood cells (WBC), neutrophils (Neut), lymphocytes (Lym), monocytes (Mono), eosinophils (Eos), basophils (Baso), large unstained cells (Luc), prothrombin time (PT), activated partial thromboplastin time (aPTT)


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: as part of the sacrifice: males: any time after the completion of the mating period (after a minimum dosing period of 28 days); females: on PND 13
- Animals fasted: No
- How many animals: 5 randomly selected males and females (only lactating females were evaluated) from each main group at the end of the treatment period and in all recovery group males and females at the end of the recovery period as part of the sacrifice of the animals
- Parameters checked: alanine aminotransferase (ALAT); aspartate-aminotransferase (ASAT); alkaline phosphatase (AP); creatinine (Crea); total protein (TP); albumin (Alb); urea; total bile acids (TBA); total cholesterol (Chol); glucose (Gluc); sodium (Na); potassium (K)

URINALYSIS: Yes
- Time schedule for collection of urine: as part of the sacrifice: males: any time after the completion of the mating period (after a minimum dosing period of 28 days); females: on PND 13
- Metabolism cages used for collection of urine: No
- Animals fasted: No
- Parameters checked: specific gravity; nitrite; pH-value (pH); protein; glucose; ketone bodies (Ket); urobilinogen (UBG); bilirubin (BIL); erythrocytes (Ery); leukocytes (Leuc); urine colour and appearance

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: week before the first treatment and during the last week of the treatment for main study group; animals of the recovery groups were examined once before the first exposure, during the last week of treatment as well as in the last week of the recovery period.
- Dose groups that were examined: 5 randomly selected males and during the last week of the lactation period in 5 randomly selected females (only lactating females were evaluated) of each group outside the home cage using a functional observational battery of tests. For the recovery groups all males and females were examined.
- Battery of functions tested: sensory reactivity to different modalities, grip strength and motor activity assessments and other behavioural observations as well as rearing supported and not supported, urination, defecation, startle/ auditory response, equilibrium reflex, positional passivity, visual placing, fore and hind limb grip strength, tail pinch response, toe pinch reflex, extensor thrust/limb tone, hind limb reflex, righting reflex on the ground, air righting reflex, pupil response, body temperature

IMMUNOLOGY: No

OTHER: From 2 female pups/litter on day 4 after birth, from all dams and 2 pups/litter at termination on day 13, and from all adult males at termination, blood samples were collected. Blood samples from the day 13 pups and the adult males were assessed for serum levels for thyroid hormones (T4). Further assessment of T4 in blood samples from the dams and day 4 pups was not deemed necessary, based on the fact that no major histopathological finding was observed in thyroid/ parathyroid gland of selected male and female adult animals and no effect was observed on hormone levels of males and day 13 pups.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes: external surface of the body, all orifices and the cranial, thoracic and abdominal cavities and their contents (see table 1)

HISTOPATHOLOGY: Yes: see table 1








Other examinations:
Organ weights: testes (paired weight), uterus with cervix, epididymides (paired weight), ovaries (paired weight), prostate, seminal vesicles and coagulating glands (complete weight), thymus, thyroid/parathyroid glands (from 1 pup/sex/litter/group and from all adult males and females) - were weighed after fixation (complete weight), liver, kidneys (paired weight), spleen, adrenal (paired weight), brain, pituitary gland, heart
Statistics:
A statistical assessment of the results of body weight, food consumption and litter data was performed for each gender by comparing values of dosed with control animals using a one-way ANOVA and a post-hoc Dunnett Test. Results of absolute and relative organ weights, parameters of haematology, blood coagulation and clinical biochemistry was statistically analysed by comparing values of dosed with control animals using either a parametric one-way ANOVA and a post-hoc Dunnett Test or a non-parametric Kruskal-Wallis Test and a post-hoc Dunn’s Test, based on the results of homogeneity and normality tests. Statistical comparisons of data acquired during the recovery period were performed with a Dunn’s Test, Dunnett’s Test or Student’s T-Test. These statistics were performed with GraphPad Prism V.6.01 software or Ascentos 1.3.4 software (p<0.05 was considered as statistically significant).
Dose descriptor:
NOAEL
Remarks on result:
other: no final report available yet
Critical effects observed:
not specified

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
July 2016
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Bayerisches Landesamt für Gesundheit- und Lebensmittelsicherheit
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Crl: WI (Han)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, 97633 Sulzfeld, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: approx. 13-15 weeks
- Weight at study initiation: males: 324 - 378 g, females: 203 - 250 g
- Fasting period before study: no
- Housing: 5 animals per sex per cage during pre-mating and post-mating period. During mating period males and females were housed together in ratio 1:1 (male to female).
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 55 ± 10
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was prepared at least once every 10 days which is with the stability frame.

VEHICLE
- Justification for use and choice of vehicle: The vehicle was selected in consultation with the sponsor based on the test item’s characteristics and testing guideline.
- Concentration in vehicle: 0, 12.5, 37.5, 75 mg/mL
- Amount of vehicle: 4 mL/kg bw/day
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: not reported
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): 5 per cage
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The determination of formulation concentrations of the test substance was based on a GC-FID method. Nominal concentrations were confirmed for all dose groups, as measured concentrations were within acceptance criterion of 10%.
Duration of treatment / exposure:
males: 28 days
females: up to 63 days
Frequency of treatment:
daily
Details on study schedule:
- Age at mating of the mated animals in the study: 17 - 19 weeks
Doses / concentrationsopen allclose all
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
150 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
main study: 10 animals per sex per dose
recovery group: 12 animals per sex per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The highest dose level of 300 mg/kg bw/day was chosen on the basis of a dose range finding study, where mortality was observed after repeated oral administration at a dose of 1000 mg/kg bw/day (later reduced to 600 mg/kg bw/day). Thereafter, a descending sequence of dose levels was selected with a view to demonstrate any dose-related response and a NOAEL.
- Fasting period before blood sampling for clinical biochemistry: no
Positive control:
no

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once a day
- Cage side observations: health condition, morbidity, moratility

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once before the first exposure and at least once a week thereafter

BODY WEIGHT: Yes
- Time schedule for examinations: once before the assignment to the experimental groups, on the first day of dosing and weekly thereafter as well as at the end of the study. During pregnancy, females were weighed on gestation days (GD) 0, 7, 14 and 20 and within 24 hours of parturition (day 0 post-partum), on PND 4, 9 and 13 along with pups. All animals were weighed directly before termination.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE: No

OTHER: From 2 female pups/litter on day 4 after birth, from all dams and 2 pups/litter at termination on day 13, and from all adult males at termination, blood samples were collected. Blood samples from the day 13 pups and the adult males were assessed for serum levels for thyroid hormones (T4). Further assessment of T4 in blood samples from the dams and day 4 pups was not deemed necessary, based on the fact that no major histopathological finding was observed in thyroid/ parathyroid gland of selected male and female adult animals and no effect was observed on hormone levels of males and day 13 pups.
Oestrous cyclicity (parental animals):
Oestrous cycles were monitored using vaginal smears for 14 days before start of treatment to select the study females with regular cyclicity. Further on, vaginal smears were also examined daily from the beginning of the treatment period until evidence of mating. A vaginal smear was also examined on the day of necropsy.
Sperm parameters (parental animals):
Parameters examined in 5 randomly selected male parental generations: testis weight, epididymis weight
Litter observations:
PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), pup weight on the day of AGD, presence of nipples/areolae in male pups.

GROSS EXAMINATION OF DEAD PUPS: yes, for external and internal abnormalities; possible cause of death was/was not determined for pups born or found dead

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: no

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: no
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals of the main group were sacrificed any time after the completion of the mating period (after a minimum dosing period of 28 days).
- Maternal animals: All surviving animals were sacrificed on their respective PND 13.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY
The tissues indicated in Table 1 were prepared for microscopic examination.

ORGAN WEIGHTS
- testes (paired weight), uterus with cervix, epididymides (paired weight), ovaries (paired weight), prostate, seminal vesicles and coagulating glands (complete weight), thymus, thyroid/parathyroid glands (from 1 pup/sex/litter/group and from all adult males and females) - were weighed after fixation (complete weight), liver, kidneys (paired weight), spleen, adrenal (paired weight), brain, pituitary gland, heart
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring were sacrificed at 13 days of age.
- These animals were subjected to postmortem examinations (macroscopic examination) as follows: carefully examined externally for gross abnormalities
Statistics:
A statistical assessment of the results of body weight, food consumption and litter data was performed for each gender by comparing values of dosed with control animals using a one-way ANOVA and a post-hoc Dunnett Test. Results of absolute and relative organ weights, parameters of haematology, blood coagulation and clinical biochemistry was statistically analysed by comparing values of dosed with control animals using either a parametric one-way ANOVA and a post-hoc Dunnett Test or a non-parametric Kruskal-Wallis Test and a post-hoc Dunn’s Test, based on the results of homogeneity and normality tests. Statistical comparisons of data acquired during the recovery period were performed with a Dunn’s Test, Dunnett’s Test or Student’s T-Test. These statistics were performed with GraphPad Prism V.6.01 software or Ascentos 1.3.4 software (p<0.05 was considered as statistically significant).
Reproductive indices:
gestation length, pre-coital interval, number of live births, post-implanation loss, the number of implantations, corpora lutea
Offspring viability indices:
number and sex of pups, stillbirths, live births, runts

Results and discussion

Results: P0 (first parental generation)

Effect levels (P0)

Dose descriptor:
NOAEL
Remarks on result:
other: no final report available yet

Target system / organ toxicity (P0)

Critical effects observed:
not specified

Results: F1 generation

Effect levels (F1)

Dose descriptor:
NOAEL
Remarks on result:
other: no final report available yet

Target system / organ toxicity (F1)

Critical effects observed:
not specified

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Applicant's summary and conclusion