Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10 Jan - 06 Jul 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose:
reference to other study
Reference
Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
12 Mar - 03 Jun 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-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:
22 Mar 1996
Deviations:
yes
Remarks:
no data, if special emphasis on stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure was performed.
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Crl:CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Japan, Inc.
- Age at study initiation: 10 weeks
- Weight at study initiation: males: 350-436 g (mean: 391.9 g); females: 211-262 g (mean: 234.7 g)
- Housing: single housing in metal bracket-type cages (260 W x 380 D x 180 H, mm) with wire mesh floors
- Diet: pellet diet, CRF-1 (Oriental Yeast Co., Ltd.), ad libitum
- Water: Sapporo City tap water, ad libitum
- Acclimation period: 13 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 50 ± 20
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
water
Remarks:
Japanese Pharmacopoeia water for injection
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test substance was accurately weighed out. Water was added to obtain the specified concentration. A stirrer was used for dissolving the substance in the vehicle. The dosing solutions were prepared once every 2 to 8 days and stored refrigerated in an airtight and light protected container.

VEHICLE
- Concentration in vehicle: 10, 30 and 100 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg bw
- Lot/batch no.: 3C90
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: max. 5 days
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged: individually in metal bracket-type cages (260 W x 380 D x 180 H, mm) with wire mesh floors; for mated females (GD 17 - lactation day 4) the wire mesh floors of cages were replaced with small trays with bedding.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentration of the test substance in the first and last dosing solution preparations were analysed. The accuracies (% of the analysed values relative to the theoretical value defined as 100%) of the 10, 30 and 100 mg/L dosing solutions were 97.3, 93.5 and 99.3% for the first preparations and 103, 110 and 112% for the final preparations, respectively. Thus, the acceptance criteria (nominal concentration ± 15%) was met.
Stability of 8-day storage under refrigeration followed by 5 h storage at room temperature after preparation was analysed in the 1 and 100 mg/mL preparations. In the analysis results, the remaining rates of the 1 and 100 mg/mL preparations were 92.2% and 98.3%, respectively, which met the acceptance criteria (100% ± 10%).
Duration of treatment / exposure:
males: for 42 days, starting 14 days before mating
mated females: for 14 days before mating and during mating period until successful copulation, and during gestation through day 4 after parturition

Frequency of treatment:
once daily, 7 days/week
Details on study schedule:
not applicable for an OECD 422 study
Remarks:
Doses / Concentrations:
100, 300 and 1000 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
12 (main study)
5 males (control and high dose satellite group, selected from the main study group)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: A preliminary 14-day study was performed with dose levels of 100, 300 and 1000 mg/kg bw/day using 4 animals/sex/dose. The hemoglobin concentration and hematocrit value were low in males and triglyceride and renal weight were high in females in the 1000 mg/kg bw/day group. No changes related to the test substance administration were noted in the 100 or 300 mg/kg bw/d group.

- Other: Satellite groups:
Rationale for selecting satellite groups: five males with body weight around the central value of the population were selected from control and high dose main study group based on the study weight on administration day 28 to approximate the overall mean, after the end of mating period
Post-exposure recovery period in satellite groups: 14 days
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily before and after administration during the administration period, twice daily in the morning and afternoon during the recovery period, and once in the morning on the day of necropsy
- Cage side observations included: observations for mortality, external appearance and behaviour.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: before the start of administration and on administration days 7, 14, 21, 28, 35 and 42 and on recovery days 7 and 14
- Observations included:
1) from outside home cage: body position/posture, respiratory pattern, tremor/convulsion, stereotypes (rolling/repetitive circling) and bizarre behaviour (self-biting)
2) from out of cage: ease of removal, ease of handling, muscle tone, piloerection, fur conditions, appearance of skin, eyes, eyeballs, and mucous membranes, pupil size, lacrimation, salivation, and other secretions or excretions
3) from an open field: gait, co-ordination of movement, reactivity to environmental stimuli, searching (sniffing and standing), excretions (urination and defecation), stereotypes (excessive grooming and unusual head movement), bizarre behaviour (walking backwards and vocalization) and aggression

BODY WEIGHT: Yes
- Time schedule for examinations: all males and females in the satellite groups: before administration on administration days 1, 4, 7, 14, 21, 28, 35 and 42 and on recovery days 1, 7, and 14, and the day of necropsy; females in the main study group: before administration on administration days 1, 4, 7, and 14, and before administration on gestation days 0, 7, 14, and 20, and before administration on lactation days 0 and 4, and the day of necropsy (on the 5th day after parturition, except females without parturition which were necropsied on gestation day 26 (day of necropsy)).

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

OTHER: Hematology, clinical chemistry, urinalysis, neurobehavioural examination (for details refer to IUCLID section 7.5.1)
Oestrous cyclicity (parental animals):
Estrous cyclicity was determined in all females of the main study group from the first day of administration until mating. Vaginal smear specimens were prepared by staining with Giemsa solution, which were examined for the stages of the estrous cycle under a light microscope. Females showing repetition of a 4-, 5-, or 6-day estrous cycle (proestrous, estrous, metestrus, and diestrus stages) were judged normal.

Vaginal smear was collected from all females in the satellite groups before necropsy on the day of necropsy to determine the stage of estrous cycle. These data were used to support the evaluation of organ weights or histopathology.
Litter observations:
PARAMETERS EXAMINED
The following parameters were examined in offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals on the day after administration day 42 (main study groups) and on the day after recovery day 14 (satellite groups), respectively.
- Maternal animals: All surviving animals on the 5th day after parturition (the day after lactation day 4).

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

HISTOPATHOLOGY / ORGAN WEIGHTS
The following tissues/organs of all animals were fixed and preserved: brain (cerebrum, cerebellum, pons), spinal cord, pituitary gland, thymus, thyroids, parathyroids, adrenals, spleen, heart, esophagus, stomach, liver, pancreas, submandibular glands, duodenum, jejunum, ileum (with Peyer's patches), cecum, colon, rectum, trachea, lungs, kidneys, urinary bladder, testes, epididymides, prostate, seminal vesicles (with coagulating glands), ovaries, uterus (horn and cervix), vagina, eyeballs and harderian glands, mammary gland (female only, right abdominal region), femur (with bone marrow, right), mesenteric lymph nodes, mandibular lymph nodes, skeletal muscle (biceps femoris), sciatic nerve, and gross lesions (with border to the normal tissue)
Paraffin block specimens were prepared and those of all animals in the control and high dose groups were microscopically examined. Stomach was also examined in the low and mid dose group due to microscopic findings in the high dose group.
Organ weight was determined from the following tissues/organs: brain, heart, liver, kidneys, testes, epididymides, seminal vesicles, pituitary glands, thyroids (with parathyroids), spleen, thymus, adrenals, prostate, ovaries and uterus.
Postmortem examinations (offspring):
GROSS NECROPSY
- Gross necropsy consisted of observation of external surface (including the oral cavity) and after euthanization all organs and tissues were macroscopically observed.

HISTOPATHOLOGY / ORGAN WEIGTHS
not performed
Statistics:
Statistical analysis were performed using the toxicological data processing system (MiTOX): group means and standard deviations, Bartlett test, one-way analysis of variance, Kruskal-Wallis test, Dunett's test, Steel's test, Fisher's exact probability test, F-test, Student's t-test, Welch test, Wilcoxon rank-sum test; p=0.05
Reproductive indices:
Copulation index (%) = (number of animals with succesful copulation / number of animals used for mating) x 100
Fertility index (%) = (number of fertile animals / number of animals with successful copulation) x 100
Gestation index (%) = (number of animals that delivered normally / number of pregnant females) x 100
Implantation index (%) = (number of implantations / number of corpora lutea) x 100
Birth index (%) = number of live pups on postnatal day 0 / number of implantations) x 100
Offspring viability indices:
Incidence of pups with external anomalies (%) = (number of live pups with external anomalies/number of live pups examined) x 100
Incidence of litters with pups showing external anomalies = number of dams having pups with external anomalies/number of litters examined
Sex ratio (%) of all pups delivered on postnatal day 0 = number of male pups delivered / (number of male pups delivered + number of females pups delivered) x 100
Sex ratio (%) of live pups on postnatal day 0/4 = number of live male pups / (number of live male pups + number of live females pups) x 100
Nursing index (%) = (number of females having live pups on lactation day 4 / number of females delivered live pups) x 100
Viablity index (%) on postnatal day 0 = (number of live pups on postnatal day 0 / number of pups delivered) x 100
Viability index (%) on postnatal day 4 = (number of live pups on postnatal day 4 / number of live pups on postnatal day 0) x 100
Clinical signs:
no effects observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
1000 mg/kg bw/day, males (main study group, end of administration): hyperplasia of squamous cells at limiting ridge of the stomach (slight) in all 7 males with significantly high incidence, indicating slight irritability to the stomach mucosa.
Other effects:
not examined
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
not specified
Reproductive performance:
no effects observed
CLINICAL SIGNS AND MORTALITY
Subcutaneous mass was noted in one female each in the control and 1000 mg/kg bw/day group during the gestation and lactation periods. This observation was considered a spontaneous change. No other abnormalities were noted in any main study group or satellite group, respectively during clincal observations. The detailed clinical observations revealed no significant differences between the test substance groups and the control group.

BODY WEIGHT AND WEIGHT GAIN
No significant differences were noted in body weight or body weight gain between the test substance groups and the control group (main study groups and satellite groups).

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
No significant differences were noted in food consumption between the test substance groups and the control group (main study groups and satellite groups).

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
Observation for estrous cyclicity during 14 days before mating revealed no significant differences in the incidence of abnormal estrous cycles, estrous cycle length, or the number of estrus between the test stubstance groups and the control group.


REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
No statistically significant differences were noted in the copulation index, fertility index, or the number of days until copulation between the test substance groups and control group.
One pair each in the 100 and 1000 mg/kg bw/day groups was non-pregnant, which was considered spontaneous occurrence.
No signficant differences were noted in the number of corpora lutea or implantations, implantation index, gestation length, gestation index, the number of pups delivered, or live or dead pups, sex ratio of pups, or nursing index on lactation day 4 in any test substance group compared to the control group. The birth indices were significantly high in the 300 and 1000 mg/kg bw/day groups, which were considered incidental changes because an increase in birth index is meaningless (Table 1).

ORGAN WEIGHTS
Males, main study group [at the end of administration period]:
The relative kidney weight in all dose groups were significantly higher than in the control group. The absolute kidney weight was also significantly high in the 300 mg/kg bw/day group. These changes were not dose-related, the absolute weight was not changed in the 100 and 1000 mg/kg bw/day groups, and no effects of the test substance administration were found histopathologically; thus, these findings were considered incidental changes unrelated to the test substance administration. In the 1000 mg/kg bw/day group, the relative liver weight was significantly increased, which was considered an incidental range because the absolute weight was not changed and histopathological examination revealed no effects of the test substance administration.

Females, main study group [at the end of administration period]:
No significant differences were noted in organ weight measured in the 100 mg/kg bw/day group compared to the control group. In the 300 mg/kg bw/day group, the relative uterus weight was significantly increased, which was considered an incidental change because it was not dose-related. The absolute and relative liver and kidney weight was significantly increased in the 1000 mg/kg bw/day group.

Males, satellite group [at the end of the recovery period]:
In the 1000 mg/kg bw/day group, the absolute and relative kidney and adrenal weight was significantly increased and absolute and relative seminal vesicle weight was significantly decreased. These changes were not observed at the end of the administration period and no effects of the test substance administration were found histopathologically, therefore these changes were considered incidental.


GROSS PATHOLOGY
Males, main study group [at the end of administration period]:
No abnormal findings were noted in the control or 100 mg/kg bw/day group. Large size of the thymus was noted in one male in the 300 mg/kg bw/day group, which was considered incidental because no abnormal findings were noted in the 1000 mg/kg bw/day group.

Females, main study group [at the end of administration period]:
Grayish white subcutaneous mass was noted in one female in the 1000 mg/kg bw/day group, whtih was considered a spontaneous change because this was also observed in the control group. No abnormal findings were found in the 100 or 300 mg/kg bw/day groups.

Males, satellite group [at the end of recovery period]:
No abnormal findings were noted in then control or 1000 mg/kg bw/day group.


HISTOPATHOLOGY: NON-NEOPLASTIC
Males, main study group [at the end of the administration period]:
Hyperplasia of squamous cells at limiting ridge of the stomach (slight) was noted in all 7 males in the 1000 mg/kg bw/day group with significantly high incidence, indicating slight irritability to the stomach mucosa. This effect was considered an effect of the test substance administration. No abnormal findings were noted in the stomach in the 100 or 300 mg/kg bw/day group. Other findings were aggregation of alveolar macrophage in the lung, focal atrophy of the acinar cells in the pancreas, microgranuloma in the liver, atrophy of the seminiferous tubulus in the testis, interstitial inflammation in the prostate, ultimobranchial body in the thyroid, and focal atrophy of retina sporadically observed in the 1000 mg/kg bw/day group. These findings were observed also in the control group or historical control data, and there were no differences in their incidences or grades compared the control group. Therefore these changes were considered unrelated to the test substance administration. The male with macroscopic findings in the 300 mg/kg bw/day group showed also lymphoma in the thymus.

Females, main study group [at the end of the administration period]:
In the 1000 mg/kg bw/day group, aggregation of alveolar macrophage in the lung, microgranuloma in the liver, cyst, basophilic change of renal tubule and hyaline cast in the kidney, ultimobranchial body in the thyroid, retinal rosette, and adenocarcinoma in the mammary gland were sporadically observed. These findings were observed also in the control group or historical control data and there were no differences in their incidences or grades compared to the control group; therefore, they were considered unrelated to the test substance administration.

Males, satellite group [at the end of recovery period]:
In the 1000 mg/kg bw/day group, aggregation of alveolar macrophage in the lung, focal atrophy of the acinar cells in the pancreas, hyline cast, and basophilic change of renal tubule in the kidney, interstitial inflammation in the prostate, ultimobranchial body in the thyroid, and cyst of zona reticularis in the adrenal were sporadically observed. These findings were observed also in the control group or historical control data, and there were no differences in their incidences or grades compared to the control group; therefore, they were considered unrelated to the test substance administration.

OTHER:
For results on heamatology, clinical chemistry, urinalysis, neurobehavioural examination refer to IUCLID section 7.5.1.
Dose descriptor:
NOAEL
Remarks:
systemic and reproduction
Effect level:
>= 1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse systemic effects an no adverse effects on reproduction upto and including the highest dose tested
Clinical signs:
not specified
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined
VIABILITY (OFFSPRING)
Death or missing (possibly cannibalized by maternal rats) was sporadically observed in all study groups including the control group from postnatal days 0 to 4. These occurred at low incidence and no significant differences were noted in the viability index between the test substance groups and the control group (Table 1).

BODY WEIGHT (OFFSPRING)
No significant differences were noted in males or females in any test substance group compared to the control group.

GROSS PATHOLOGY (OFFSPRING)
Necropsy of offspring on postnatal day 4 revealed no abnormalities in any study group including the control. One pup found dead on postnatal day 3 in the control group showed dilatation of renal pelvis (bilateral). This was the only finding and no abnormalities were detected in the other pups found dead.

Dose descriptor:
NOAEL
Remarks:
developmental
Generation:
F1
Effect level:
>= 1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects on development of offspring upto and including the highest dose tested
Reproductive effects observed:
not specified

Table 1: Reproductive performance and delivery data*

Parameter

Dose group [mg/kg bw/day]

control

100

300

1000

Copulation index [%]

100

100

100

100

Fertility index [%]

100

91.7

100

91.7

No. of dams

12

11

12

11

Gestation period (day)

22.25 ± 0.45

22.27 ± 0.47

22.25 ± 0.45

22.45 ± 0.52

No. of implantation

15.3 ± 2.0

15.7 ± 1.2

16.3 ± 2.3

15.0 ± 3.9

Birth index (%)

84.09 ± 15.35

91.76 ± 5.24

93.82 ± 4.97#

95.74 ± 4.47#

No. of offspring (total)

154

159

184

157

No. of offspring (per dam)

12.9 ± 2.8

14.6 ± 1.6

15.4 ± 2.4

14.5 ± 3.6

No. of live newborns (per dam)

12.8 ± 2.7

14.5 ± 1.6

15.3 ± 2.3

14.3 ± 3.5

Sex ratio, all pups day 0 (%)

53.5

44.1

55.7

53.8

Sex ratio, live pups day 0 (%)

53.2

43.3

56.0

52.9

Sex ratio, live pups day 4 (%)

53.4

43.2

56.3

53.9

No. of dead newborn (dead +cannibalism)

0.1 ± 0.3

0.2 ± 0.6

0.1 ± 0.3

0.3 ± 0.5

Gestation index (%)

100

100

100

100

No. of corpora lutea

16.3 ± 1.5

16.0 ± 1.2

17.2 ± 2.2

15.8 ± 2.1

Implantation index (%)

93.87 ± 8.08

98.34 ± 2.85

95.21 ± 8.95

93.17 ± 18.97

Nursing index (%)

100

100

100

100

No. of dams with anomalous offspring (incidence%)

0

0

0

0

No. of offspring with any anomaly (incidence%)

0

0

0

0

# Significantly different from control group (p<0.05), Steel test (two-side)

* data provided by the sponsor in an extra sheet

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 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
adopted in 1998
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Version / remarks:
adopted in 2001
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Specific details on test material used for the study:
Name: RZETA (A mixture composed of (1,4-diazabicyclo[2.2.2]octane-2- yl)methanol (89.8%) and 1,5-diazabicyclo[3.2.2]nonan-3-ol (9.8%)
Supplier: Tosoh Corporation, Japan
Lot No.: 6Z02RZETACR
Analytical purity for 76950-43-1 (1,4-diazabicyclo[2.2.2]octane-2-yl)methanol): 89.8%
Appearance: pale yellow solid
Expiration date: December 2, 2018
Storage conditions: at room temperature in an airtight, light-protected, and N2 sealed container.
Certificate of Analysis of the test article provided: yes, attached to this report (Annex 1)

Test animals

Species:
rat
Strain:
other: Crl:CD(SD)
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Japan, Inc.
- Age at study initiation: males: 9 weeks, females: 11 weeks
- Weight at receipt: males: 362 - 427 g, females: 180 - 240 g
- Fasting period before study: no
- Housing: Animals were housed in metal bracket cages (260 W × 380 D × 180 H, mm) with wire mesh floors; two or less females per cage during quarantine and mating period, 2 males per cage during quarantine period; and 2 (1 male and 1 female) per cage during mating, 1 female per cage after group assignment
- Diet: pelleted feed CRF-1 (Oriental Yeast Co., Ltd.), ad libitum; analysis was performed
- Water: Sapporo-City tap water, ad libitum; analysis was performed
- Acclimation period: 5 days (from animal receipt to start of mating)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 20 - 50
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Remarks:
Otsuka distilled water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test article was accurately weighed out, to which the vehicle was added to achieve the prescribed concentration, and the test article was dissolved using a stirrer. Dosing solutions were stored refrigerated in an airtight and light protected container. Dosing solution was used within the verified stability period (within 8 days).

VEHICLE
- Concentration in vehicle: 0, 10, 30, 100 mg/mL
- Amount of vehicle: 10 mL/kg bw
- Lot/batch no.: 6G74
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
According to the study report, the stability of dosing solutions was investigated prior to the conduct of the present study (Report NCAS 14-012). Within this study, the stability of 1 and 100 mg/mL preparations of the test item during 8-day storage under refrigeration followed by 5-hr storage at room temperature after preparation was confirmed. With respect to the dose levels considered within the present study, the concentrations of test item in the dosing solutions were analysed at the first preparation, at mid-term of administration period and at the final preparation; in fact, all prepared concentrations were subjected to analysis. The analytic study report (study No. 16-181) is provided within the OECD 414 study report in Annex 2. In brief, the analytical results confirmed adequate concentrations of the test item in the dosing solutions with recoveries of 100%.
Details on mating procedure:
- Impregnation procedure: cohoused
- M/F ratio per cage: 1/1
- Length of cohabitation: mating was performed for 4 days until the required number (24 females/group) of successfully mated females was obtained.
- Further matings after two unsuccessful attempts: no
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
Duration of treatment / exposure:
Day 6 - 19 of gestation
Frequency of treatment:
daily, 7 days/week
Duration of test:
14 days
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
24 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dosage levels were selected based on the results of a Combined repeated dose toxicity study with the reproduction/developmental toxicity screening test (OECD422, Fuji, 2014) of the test article, in which 12 female rats/per group were dosed by oral gavage at 0, 100, 300, and 1000 mg/kg from pre-mating period to postnatal day 4. As a result, no effects of the test article administration were noted on female reproductive performance or offspring. Therefore, 1000 mg/kg/day was selected as the highest dose, and 300 and 100 mg/kg were selected by dividing the highest dose by a common ratio of 3.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: from gestation day 0 to the day of necropsy; twice daily (before and after administration) during the administration period, and once daily during the other periods

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: gestation days 0, 3, 6, 9, 12, 15, 18, and at necropsy on days 20

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

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: Incidence of anomalous placentas
Fetal examinations:
- External examinations: Yes: all live fetuses per litter
- Soft tissue examinations: Yes: half of the litter
- Skeletal examinations: Yes: half of the litter
- Head examinations: Yes: half of the litter
Statistics:
Statistical analyses were performed using the computer system (MiTOX). Fetal data was processed using the litter as the unit of analysis.
Group means and standard deviations were calculated for the maternal body weight, adjusted body weight, body weight gain, food consumption, gravid uterine weight, numbers of corpora lutea, implantations, live fetuses, and embryo-fetal deaths, body weight of live fetuses (by sex), and degree of ossification. These were analyzed for homogeneity of variances by the Bartlett test. When the variances were homogeneous (p ≥ 0.05), the One-way analysis of variance (ANOVA) was used, and when the variances were heterogeneous (p < 0.05), the Kruskal-Wallis test was used to detect significant differences among groups. When the One-way ANOVA indicates a significant difference (p < 0.10), Dunnett’s test was used for comparison with the control group. When the Kruskal-Wallis test indicates a significant difference (p < 0.10), Steel’s test was used for comparison with the control group. Data of females with no evidence of pregnancy were excluded from evaluation.
The preimplantation loss, implantation index, post-implantation loss, postimplantation loss in the early and late stages, incidences of fetal anomalies or variations, and incidence of anomalous placentas were analyzed by the Steel’s test to detect significant differences between the test article and control groups, using the litter as the unit of analysis.
Fetal sex rate, and the incidences of dams with fetal anomalies and variations and dams with anomalous placentas were analyzed by Fisher’s exact probability test. For comparative analysis with the control group, a p value of less than 5% was considered statistically significant.
Indices:
Preimplantation loss (%) = [(number of corpora lutea – number of implantations) / number of corpora lutea] × 100

Implantation index (%) = (number of implantations / number of corpora lutea) × 100

Postimplantation loss (%) = (number of embryofetal deaths / number of implantations) × 100

Postimplantation loss in the early stage (%) = (number of embryofetal deaths in the early stage / number of implantations) × 100

Postimplantation loss in the late stage (%) = (number of embryofetal deaths in the late stage / number of implantations) × 100

Sex rate (%) = (number of live male fetuses / total number of live fetuses) × 100

Incidence of anomalous placentas (%) = (number of live fetuses with anomalous placentas / total number of live fetuses examined) × 100

Incidence of dams with anomalous placentas = number of litters with fetuses with anomalous placentas / number of litters examined

Incidence of fetal anomalies (%) = (number of live fetuses with anomalies / total number of live fetuses examined) × 100

Incidence of dams with fetal anomalies = number of dams having live fetuses with external anomalies / total number of litters examined
Historical control data:
Historial control data (2008 to 2017) are on file at the testing laboratory.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Description (incidence and severity):
Clinical examinations revealed no signs indicative of toxicity.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
In all test groups including the control, none of the pregnant dams died.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No statistically significant differences were observed between all test groups including control with respect to body weight and body weight gain. Please refer to Table1 A and 1B for details.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Mean daily food consumption was similar for all test groups including control, with a concomitant increase in consumption in the course of the 20 day gestation period. Thus, the mean food consumption over all groups was measured to be in the range of 23 – 24 g/day on day 3 of gestation, reaching 28-29 g/day on day 20 of gestation.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
At necropsy, the pathological examination of the sacrificed dams revealed no abnormalities in the 100, 300, or 1000 mg/kg bw/day group or in the control group.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Maternal developmental toxicity

Number of abortions:
no effects observed
Description (incidence and severity):
In none of the test groups including control, abortions were noticed. In fact, live fetuses were obtained from all pregnant females.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
No statistically significant differences were observed with respect to pre-implantation losses. In fact, for the treated groups, the mean pre-implantation losses were 2.78, 4.48 and 4.95% for the low (100 mg/kg bw/day), the mid (300 mg/kg bw/day) and the high dose level (1000 mg/kg bw/day), respectively, versus 5.02% for the untreated control group (0 mg/kg bw/day). For details, please refer to Table 2A.
No statistically significant differences were observed with respect to post-implantation losses. In fact, for the treated groups, the mean total post-implantation losses were 4.14, 5.50 and 5.53% for the low (100 mg/kg bw/day), the mid (300 mg/kg bw/day) and the high dose level (1000 mg/kg bw/day), respectively, versus 4.13% for the untreated control group (0 mg/kg bw/day). For details, please refer to Table 2A.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
In none of the test groups including control, total litter losses by resorption were noticed.
Early or late resorptions:
no effects observed
Description (incidence and severity):
No statistically significant differences were noted between all test groups including control. For details, please refer to Table 2A.
Dead fetuses:
not specified
Description (incidence and severity):
No statistically significant differences were noted between all test groups including control. For details, please refer to Table 2B.
Changes in pregnancy duration:
not examined
Description (incidence and severity):
Not applicable since all pregnant female rats of all test groups including control were sacrificed on gestation day 20.
Changes in number of pregnant:
effects observed, non-treatment-related
Description (incidence and severity):
One of the 24 mated female rats in the mid dose group (i.e., 300 mg/kg bw/day) was non-pregnant. This single finding is incidental and not to be considered related to the treatment.
With respect to the control (0 mg/kg bw/day), the low (100 mg/kg bw/day) and the high dose group (1000 mg/kg bw/day), all mated females became pregnant.
Other effects:
no effects observed
Details on maternal toxic effects:
With respect to the dams, no mortality occurred and no clinical signs indicative of toxicity were noticed in the 100, 300, or 1000 mg/kg bw/day group or in the control group. Food consumption as well as body weight changes were similar for all test groups including control. At necropsy on day 20 of gestation, pathological examination of the dams revealed no abnormalities; no differences were noticed between the test groups including control with respect to the gravid uterine weight and the adjusted body weight (i.e., body weight on day 20 of gestation without gravid uterine weight; for details please refer to Table 3).

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: no signs of toxicity noted up to and including the highest dose tested

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
not examined
Description (incidence and severity):
Not applicable. In fact, fetal body weights were reported, and no statistically significant differences between all test groups including control were noticed; the mean fetal weight for each group was about 4.2 g and 3.9 g for for male and female fetuses, respectively; for more details please refer to Table 2B. Since no further fetal weighing data are available, fetal body weight changes could not be assessed.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
All of the treated groups (100, 300, 1000 mg/kg bw/day) as well as the control group showed similar numbers on live fetuses; for more details please refer to Table 2B.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
For all of the treated groups (100, 300, 1000 mg/kg bw/day) as well as the control group, no noticeable difference in sex rate was reported; for more details please refer to Table 2B.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
As based on the data available for gravid uterine weight and uterine contents, there is no indication for any changes or differences in litter size/weight for the treated groups (100, 300, 1000 mg/kg bw/day) when compared to the control group.
Changes in postnatal survival:
not examined
Description (incidence and severity):
Not applicable
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
The external examination of the live fetuses revealed only one runt fetus in the 300 mg/kg bw/day group; since no such findings were seen in all remaining groups (control, 100 and 1000 mg/kg bw/day), the finding is considered incidental.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Skeletal variations were noted as follows:
- Short thoracolumbar supernumerary rib in 18, 17, 9 and 20 fetuses at 0, 100, 300, and 1000 mg/kg bw/day, respectively;
- Bipartite ossification of thoracic centrum in 4, 2, 4 and 5 fetuses at 0, 100, 300, and 1000 mg/kg bw/day, respectively;
- Dumbbell ossification of thoracic centrum in 7, 5, 10 and 11 fetuses 0, 100, 300, and 1000 mg/kg bw/day, respectively;
- Short 13th rib in 2, 4, and 3 fetuses at 0, 100, and 300 mg/kg bw/day, respectively; no such finding seen at 1000 mg/kg bw/day;
- Asymmetric ossification of sternebra in one fetus; 25 presacral vertebrae in one fetus; unilateral ossification of thoracic centrum in one fetus at 300 mg/kg bw/day;
- Sacralization of lumbar vertebra in one fetus at 100 mg/kg bw/day;

In the fetal or litter incidences of these skeletal variations, no statistically significant differences were noted in any of the treated groups (100, 300, 1000 mg/kg bw/day) as compared to the control group. Furthermore, such skeletal variations are considered to be adverse effects.

Skeletal anomalies were noted as follows:
- Fused sternebra in 1 fetus at 300 and 1000 mg/kg bw/day, respectively:
- Dumbbell-shaped thoracic centrum in one and 2 fetuses at 0 and 300 mg/kg bw/day, respectively;
- Split thoracic centrum in 3 and 4 fetuses at 0 and 300 mg/kg bw/day, respectively;

Further to those findings described above, a combination of absent rib, fused rib, interrupted costal cartilage, absent cervical arch, fused cervical arch, absent thoracic arch, fused thoracic centrum, and fused lumbar centrum were seen in one fetus at 300 mg/kg bw/day.

No statistically significant differences were noted in the fetal or litter incidences of skeletal anomalies in any of the treated groups (100, 300, 1000 mg/kg bw/day) as compared to the control group; further, no statistically significant differences were noted in the mean numbers of ossifications in the sacro-caudal centrum, sternebra, metacarpus or metatarsus in fetuses.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Visceral anomalies were noted as follows:
- Thymic remnant in the neck in 10 , 6 , 2 and 2 fetuses at 0, 100, 300, and 1000 mg/kg bw/day, respectively;
- Left umbilical artery in 2, 1, 1 and 2 fetuses at 0, 100, 300, and 1000 mg/kg bw/day, respectively;
- Dilated renal pelvis in 1 and 2 fetuses at 100 and 300 mg/kg bw/day, respectively;
- Dilated ureter in 1 and 1 fetuses at 100 and 300 mg/kg bw/day, respectively;
- Dilated cerebral ventricle in 1 fetus; ventricular septum defect in one fetus at 300 mg/kg bw/day;

In the fetal or litter incidences of these visceral anomalies, no statistically significant differences were noted in any of the treated groups (100, 300, 1000 mg/kg bw/day) as compared to the control group.
Other effects:
not examined
Details on embryotoxic / teratogenic effects:
Examination of the fetuses obtained from treated pregnant female rats (100, 300 or 1000 mg/kg bw/day of the test item) sacrificed on day 20 of gestation revealed no obvious differences when compared to the fetuses obtained from the concomitant untreated control group. Thus, there was no indication for any treatment-related effects, and consequently no evidence for the test item to possess any embryo-fetotoxic properties.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse findings noted up to and including the highest dose tested.

Overall developmental toxicity

Key result
Developmental effects observed:
no

Any other information on results incl. tables

details.

Table 1A_Pregnant female rats_Body weight data (mean value ± SD):

Dose level

N

Day of Gestation

0

3

6

9

12

15

18

20

0 mg/kg bw/day

24

249.2±14.8

274.2±17.7

293.9±20.8

312.4±23.2

331.1±25.3

351.7±27.7

394.2±31.6

429.6±35.6

100 mg/kg bw/day

24

249.0±16.1

270.3±16.1

289.1±17.8

306.6±19.0

326.8±20.7

347.3±23.6

392.5±21.4

428.2±24.0

300 mg/kg bw/day

23

246.7±13.7

270.1±17.0

290.2±18.1

307.0±21.1

326.9±22.3

346.1±23.8

387.3±26.7

421.1±31.0

1000 mg/kg bw/day

24

248.8±14.1

273.3±15.3

293.0±16.5

309.5±20.9

327.4±21.5

346.7±24.0

387.8±25.0

422.5±28.4

N; Number of pregnant females

Table 1B_Pregnant female rats_Cumulative body weight gain data (mean value ± SD; base day day was day 6 of gestation):

Dose level

N

Day of Gestation

9

12

15

18

20

0 mg/kg bw/day

24

18.5±5.3

37.2±7.4

57.8±10.1

100.3±13.6

135.7±18.3

100 mg/kg bw/day

24

17.5±5.0

37.7±8.0

58.2±10.7

103.3±9.4

139.1±12.9

300 mg/kg bw/day

23

16.8±5.1

36.7±7.2

55.9±10.1

97.2±13.4

130.9±19.9

1000 mg/kg bw/day

24

16.4±5.9

34.4±6.1

53.6±9.7

94.8±12.2

129.5±15.7

N; Number of pregnant females

Table 2A_Pregnant female rats_Embryo- Fetotoxicity parameters (mean value ± SD):

Dose level

N

Number of corpora lutea (N)

Number of implantations

Pre-implantation loss (%)

Implantation Index (%)

Post-implantation loss (%)

Early

Late

Total

0 mg/kg bw/day

24

16.0±2.0

15.2±2.4

5.02±9.60

94.99±9.60

4.13±6.48

0.00

4.13±6.48

100 mg/kg bw/day

24

16.0±1.4

15.6±1.3

2.78±3.61

97.23±3.60

4.14±4.62

0.00

4.14±4.62

300 mg/kg bw/day

23

15.5±2.2

14.8±2.6

4.48±9.67

95.52±9.65

4.93±11.14

0.57±1.87

5.50±12.27

1000 mg/kg bw/day

24

16.2±2.0

15.5±2.8

4.95±12.69

95.05±12.69

5.02±7.82

0.51±1.74

5.53±7.67

N; Number of pregnant females

Table 2B_Pregnant female rats_Embryo- Fetotoxicity parameters (mean value ± SD):

Dose level

N

Total Number of dead fetuses

Number of live fetuses

Sex rate of the fetuses (%)

Fetal weights (g)

Males

Females

Total

Males

Females

Total

0 mg/kg bw/day

24

0.6±1.0

7.7±2.2

6.8±1.8

14.5±2.5

185/349 (53%)

4.2549±0.2437

3.9683±0.2306

4.1174±0.2237

100 mg/kg bw/day

24

0.7±0.8

7.7±2.4

7.3±2.0

14.9±1.2

184/358 (51.4%)

4.1927±0.2911

3.9828±0.2242

4.0928±0.2572

300 mg/kg bw/day

23

0.8±1.9

7.2±2.3

6.9±2.4

14.0±3.2

165/323 (51%)

4.1997±0.2179

3.9357±0.2510

4.0677±0.2334

1000 mg/kg bw/day

24

0.8±1.1

7.8±3.0

6.8±2.6

14.7±3.0

188/352 (53.4%)

4.2054±0.2703

3.9608±0.2005

4.0978±0.2162

N; Number of pregnant females

Table 3_Pregnant female rats_Adjusted final body weights and gravid uterine weights (mean value ± SD):

Dose level

N

Adjusted body weight (g)

Gravid uterine weight (g)

0 mg/kg bw/day

24

341.0±29.2

88.6±14.4

100 mg/kg bw/day

24

338.0±23.7

90.2±7.0

300 mg/kg bw/day

23

336.9±23.1

84.2±16.8

1000 mg/kg bw/day

24

335.2±22.1

87.4±16.6

N; Number of pregnant females

Applicant's summary and conclusion