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Toxicological information

Toxicity to reproduction

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Administrative data

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
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
other: study report (translation)
Title:
Unnamed
Year:
2014
Report Date:
2014

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:
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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): [trade name]
- Substance type: pale yellow solid
- Analytical purity: 90.7% (subsequent information by sponsor)
- Impurities (identity and concentrations): 1,5-diazabicyclo[3.2.2]nonane-3-ol 8.9% (subsequent information by sponsor)
- Expiration date of the lot/batch: 15 Oct 2015
- Storage condition of test material: at room temperature, light protected and under nitrogen seal

Test animals

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

Administration / exposure

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
Doses / concentrations
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

Examinations

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

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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 / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
not specified
Reproductive performance:
no effects observed

Details on results (P0)

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.

Effect levels (P0)

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

Results: F1 generation

General toxicity (F1)

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

Details on results (F1)

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.

Effect levels (F1)

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

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

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

Applicant's summary and conclusion