Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
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 guideline
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 11/0489-2
- Expiration date of the lot/batch: 17.12.2017
- Purity test date: 24.07.2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: ambient (room temperature)
- Stability under test conditions: The stability of the test substance under storage conditions over
the test period was guaranteed by the sponsor, and the sponsor
holds this responsibility. The test facility is organizationally
independent from the BASF SE sponsor division.
- Solubility and stability of the test substance in the solvent/vehicle:
Initially stability of 1,8-Diazabicyclo[5.4.0]undec-7-ene was proven over a period of 72 hours
at a concentration of 50 mg/100mL in drinking water (BASF SE 2017, Supplement). Due to
difficulties with the higher concentrations in preceding studies another stability analysis of 1,8-
Diazabicyclo[5.4.0]undec-7-ene at a concentration of 1200 mg/100mL in highly deionized
water was initiated before start of this study. Here the stability of 1,8-Diazabicyclo[5.4.0]undec-
7-ene in highly deionized water at room temperature over a period of 1.5 hours was proven
(BASF SE 2017, Supplement).

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: dilution in drinking water


OTHER SPECIFICS:
Physical state/appearance: liquid/colorless to yellow, clear
Batch identification: 43017568E0
Purity: >= 98 % (verified by Sponsor)
Content: 99.631 % (GC)
Homogeneity: given

Test animals

Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services
GmbH, Sulzfeld, Germany
- Females (if applicable): pregnant: [yes]
- Age at study initiation: 10-12 weeks
- Weight at study initiation: Females: 141.8 – 189.9
- Fasting period before study: no
- Housing: The animals were housed together (5 animals per cage) in H-Temp polysulfonate cages type
2000P supplied by TECNIPLAST, Hohenpeißenberg, Germany (floor area about 2065 cm2).
- Diet (e.g. ad libitum): yes
- Water (e.g. ad libitum): yes
- Acclimation period: 6 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): room temperature: a range of temperature of 20-24°C
- Humidity (%): relative humidity of
30-70%
- Air changes (per hr): 15 air changes per hour
- Photoperiod (hrs dark / hrs light): The day/night cycle was 12 hours (12 hours light from
06.00-18.00 h, 12 hours dark from 18.00-06.00 h).

IN-LIFE DATES: From: To:

Males Females Phase of study/ Examination Study day
- Study initiation date: 02 Mar 2016
- Experimental starting date: (arrival of 1st cohort of test animals) 02 Mar 2016
- Supply of animals/ Beginning of acclimatization (GD 0): 1st cohort 02 Mar 2016 / 2nd cohort 03 Mar 2016
- Beginning of treatment/ End of acclimatization (GD 6): 1st cohort 08 Mar 2016 / 2nd cohort 09 Mar 2016
- End of treatment (GD 19): 1st cohort 21 Mar 2016 / 2nd cohort 22 Mar 2016
- Sacrifice of the animals (GD 20): 1st cohort 22 Mar 2016 / 2nd cohort 23 Mar 2016

Experimental completion date: (draft to QAU*) 03 May 2017
* QAU = Quality Assurance Unit

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
VEHICLE
- Dose/Concentration/Volume in vehicle:
1. 0 [mg/kg BW/day] - 0 [g/100ml] - 10 [mL/kg]
2. 15 [mg/kg BW/day] - 0.15 [g/100ml] - 10 [mL/kg]
3. 50 [mg/kg BW/day] - 0.50 [g/100ml] - 10 [mL/kg]
4. 150 [mg/kg BW/day] - 1.50 [g/100ml] - 10 [mL/kg]
- Amount of vehicle (if gavage): please see above
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses of the test substance preparations
The analyses of the test substance preparations were carried out at the Analytical Chemistry Laboratory of Experimental Toxicology and Ecology of BASF SE, Ludwigshafen, Germany. The stability of the test substance in drinking water at room temperature over a period of 72 hours had been verified prior to the start of the study (Project No.: 01Y0489/11Y177, see PART III). Furthermore, the stability of the test substance over 3 days in a concentration range covering the high-concentration used in this study (1500 mg/100 mL) was initiated during the study (Project No. 01Y0489/11Y179, see PART III). Samples of the test substance preparations were sent to the analytical laboratory twice during the study period (at the beginning and towards the end of administration) for verification of the concentrations. Given that the test substance was completely miscible with drinking water, solutions were considered homogenous without further analysis.
Details on mating procedure:
- Impregnation procedure: purchased timed pregnant

EXPERIMENTAL PROCEDURE
The animals were paired by the breeder (“time-mated”); the day of evidence of mating (= detection
of vaginal plug/sperm) was referred to as GD 0. The animals arrived on the same day
(GD 0) at the experimental laboratory. The following day was designated as “GD 1”.
The animals were acclimated to the laboratory conditions between start of the study (beginning
of the experimental phase) and first administration (GD 6).
Duration of treatment / exposure:
The test substance preparations were administered to the animals once a day orally by gavage,
from implantation to one day prior to the expected day of parturition (GD 6 to GD 19), always
at approximately the same time in the morning. The animals of the control groups were treated
with the vehicle (drinking water) in the same way. The volume administered each day was
10 ml/kg body weight. The calculation of the administration volume was based on the most
recent individual body weight.
Frequency of treatment:
The test substance preparations were administered to the animals once a day orally by gavage,
from implantation to one day prior to the expected day of parturition (GD 6 to GD 19), always
at approximately the same time in the morning.
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
control
Dose / conc.:
15 mg/kg bw/day (actual dose received)
Remarks:
low dose
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Remarks:
mid dose
Dose / conc.:
150 mg/kg bw/day (actual dose received)
Remarks:
high dose
No. of animals per sex per dose:
25
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
Dose rational:
In an acute oral toxicity study (BASF Project No. 10A0064/901044), the LD (lethal dose) 50 of
1,8-Diazabicyclo-5,4,0-undecene-7 was found to be between 215 and 681 mg/kg bw/d.
A 14-day range-finding study (NOTOX Project 498334; BASF Project 01R0489/11X202) was
performed afterwards to select doses for an OECD 422 Combined Repeated Dose Toxicity
Study with the Reproduction/Developmental Toxicity Screening Test. Four groups of three
male and three female Wistar Han rats received the test substance by oral gavage at 0, 15, 40
and 100 mg/kg bw/d for 14 days. Since no in-life toxicity was seen at any dose level after the
14-day treatment period, group 2 animals (15 mg/kg bw/d) were retained for a second dosing
period of 14 days at 200 mg/kg bw/d. At 200 mg/kg bw/d, slight body weight loss and reduced
gains (- 3% from study day 0 to 14) were observed in females. Reddish foci on the stomach
glandular mucosa were seen for all animals during the macroscopic examination. No toxicologically
significant changes were noted in any of the remaining parental parameters investigated
in this study (i.e. clinical appearance, food consumption, clinical laboratory investigations,
macroscopic examination and organ weights).
Based on these results, the animals in the OECD 422 study (NOTOX Project 498331, BASF
Project 85R0489/11X203) received dose levels of 15, 50 and 150 mg/kg bw/d. At 150 mg/kg
bw/d, dark red foci and irregular surface of the stomach were seen at the macroscopic examination.
In histopathology, corroborative findings in the stomach, consisting of haemorrhage,
and degeneration of the glandular mucosa, inflammation, hyperplasia and hyperkeratosis were
seen. All toxicological effects were limited to the stomach, and could be attributable to the
strong alkaline corrosivity of the test substance. No further toxicologically significant changes
in any parental, reproductive or developmental parameter were observed. The No Observed
Adverse Effect Level (NOAEL) for parental toxicity was 50 mg/kg bw/d.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations checked in table [No.?] were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily
- Mortality: A check was made twice a day on working days or once a day on Saturdays, Sundays or on
public holidays (GD 0-20).
- Clinical symptoms: A cage-side examination was conducted at least once daily before and after treatment period
(GD 0-5 and 20). During treatment period (GD 6-19) all rats were checked daily for any signs
of morbidity, pertinent behavioral changes and/or signs of overt toxicity before administration
as well as within 2 hours and within 5 hours after administration.

Food consumption
The consumption of food was recorded for the intervals GD 0-1, 1-3, 3-6, 6-8, 8-10, 10-13, 13-
15, 15-17, 17-19 and 19-20.

BODY WEIGHT: No
- Time schedule for examinations: All animals were weighed on GD 0, 1, 3, 6, 8, 10, 13, 15, 17, 19 and 20. The body weight
change of the animals was calculated based on the obtained results.
- Furthermore, the corrected body weight gain was calculated after terminal sacrifice (terminal
body weight on GD 20 minus weight of the unopened uterus minus body weight on GD 6).


POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: Necropsy
On GD 20, the dams were sacrificed under isoflurane anesthesia by decapitation, in
randomized order.
After the dams had been sacrificed, they were necropsied and assessed for gross pathology,
in randomized order. The uteri and the ovaries were removed and the following data were
recorded:
- Weight of the unopened uterus
- Number of corpora lutea
- Number and distribution of implantation sites classified as:
• Live fetuses
• Dead implantations:
a) Early resorptions (only decidual or placental tissues visible or according to SALEWSKI
from uteri from apparently non-pregnant animals and the empty uterus horn in the
case of single horn pregnancy)
b) Late resorptions (embryonic or fetal tissue in addition to placental tissue visible)
c) Dead fetuses (hypoxemic fetuses which did not breathe spontaneously after the
uterus had been opened)
After the weight of the uterus had been determined, all subsequent evaluations of the dams
and the gestational parameters were conducted by technicians unaware of treatment group in
order to minimize bias. For this purpose animal numbers were encoded.

OTHER:
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: Dead fetuses (hypoxemic fetuses which did not breathe spontaneously after the
uterus had been opened)
Fetal examinations:
EXAMINATIONS OF THE FETUSES
All fetal analyses were conducted by technicians unaware of the treatment group, in order to
minimize bias.

Examinations of the fetuses after dissection from the uterus
At necropsy each fetus was weighed, sexed, and external tissues and all orifices were examined
macroscopically. The sex was determined by observing the distance between the anus
and the base of the genitalia.
Furthermore, the viability of the fetuses and the condition of placentae, umbilical cords, fetal
membranes, and fluids were examined. The placentas were weighed and their individual
weights were recorded.
Thereafter, the fetuses were sacrificed by a subcutaneous injection of pentobarbital
(Narcoren®; dose: 0.1 mL/fetus).
After these examinations, approximately one half of the fetuses per dam were eviscerated,
skinned and fixed in ethanol; the other half were placed in Harrison’s fluid for fixation.

Soft tissue examination of the fetuses
The fetuses fixed in Harrison’s fluid were examined for any visceral findings according to the
method of BARROW and TAYLOR. After this examination these fetuses were discarded.

Skeletal examination of the fetuses
The skeletons of the fetuses fixed in ethanol were stained according to a modified method of
KIMMEL and TRAMMELL. Thereafter, the skeletons of these fetuses were examined under a
stereomicroscope. After this examination the stained fetal skeletons were archived individually.
Statistics:
1. Simultaneous comparison of all dose groups with the control group using the DUNNETT-test (two-sided) for the hypothesis of equal means (Food consumptiona), body weight, body weight change, corrected body weight gain (net maternal body weight change), carcass weight, weight of unopened uterus, number of corpora lutea, number of implantations, number of resorptions, number of live fetuses, proportions of preimplantation loss, proportions of postimplantation loss, proportions of resorptions, proportion of live fetuses in each litter, litter mean fetal body weight, litter mean placental weight)
2. Pairwise comparison of each dose group with the control group using FISHER'S EXACT test (onesided) for the hypothesis of equal proportions (Female mortality, females pregnant at terminal sacrifice, number of litters with fetal findings)
3. Pairwise comparison of each dose group with the control group using the WILCOXON-test (onesided) for the hypothesis of equal medians (Proportions of fetuses with malformations, variations and/or unclassified observations in each litter)
Historical control data:
yes

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Description (incidence and severity):
No clinical signs or changes of general behavior, which may be attributed to the test substance, were detected in any female at dose levels of 15, 50 or 150 mg/kg bw/d during the entire study period.
Mortality:
no mortality observed
Description (incidence):
There were no substance-related or spontaneous mortalities in any females of all test groups.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
The mean body weights of the dams in test groups 1, 2 and 3 (15, 50 and 150 mg/kg bw/d) were in general comparable to the controls throughout the entire study period. The statistically significantly increased body weight gain value in test group 2 on GD 19-20 is considered as an incidental event.
The corrected body weight gain of test groups 1-3 (15, 50 and 150 mg/kg bw/d) revealed no difference of any biological relevance to the concurrent control group. Moreover, mean carcass weights remained also unaffected by the treatment (for details cf. "Attached background material").
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
The mean food consumption of the dams in test groups 1, 2 and 3 (15, 50 or 150 mg/kg bw/d) was generally comparable to the concurrent control throughout the entire study period. The statistically significantly decreased food consumption value during the pre-treatment period (GD 1-3) in test group 3 as well as the statistically significantly increased value on GD 19-20 (same test group) are considered as incidental events.
Food efficiency:
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:
not examined
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Description (incidence and severity):
No necropsy findings which could be attributed to the test substance were seen in any dam.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed

Maternal developmental toxicity

Number of abortions:
no effects observed
Description (incidence and severity):
For details cf. "Attached background material"
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
For details cf. "Attached background material"
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
For details cf. "Attached background material"
Early or late resorptions:
no effects observed
Description (incidence and severity):
For details cf. "Attached background material"
Dead fetuses:
no effects observed
Description (incidence and severity):
For details cf. "Attached background material"
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
For details cf. "Attached background material"
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.DescriptionIncidenceAndSeverityEffectsOnPregnancyDuration): For details cf. "Attached background material"
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
For details cf. "Attached background material"
Other effects:
no effects observed
Details on maternal toxic effects:
Uterus weight
The mean gravid uterus weights of the animals of test groups 1-3 (15, 50 and 150 mg/kg bw/d)
were not influenced by the test substance. The differences between these groups and the
concurrent control groups revealed no dose-dependency and were assessed to be without
biological relevance.

Reproduction data
The conception rate reached 96% in the control group and 100% in the low-, mid- and highdose
groups (15, 50 and 150 mg/kg bw/d). With these rates, a sufficient number of pregnant
females were available for the purpose of the study.
There were no test substance-related and/or biologically relevant differences between the test
groups 0-3 in conception rate, in the mean number of corpora lutea and implantation sites or
in the values calculated for the pre- and the postimplantation losses, the number of resorptions
and viable fetuses. All observed differences are considered to reflect the normal range of fluctuations
for animals of this strain and age, for details see also historical control data (cf. "Attached background material").

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
The mean fetal weights of test groups 1, 2 and 3 were not influenced by the test substance
and did not show any biologically relevant differences in comparison to the concurrent control
group. For details cf. "Attached background material".
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): For details cf. "Attached background material".
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
For details cf. "Attached background material".
Changes in sex ratio:
no effects observed
Description (incidence and severity):
The sex distribution of the fetuses in test groups 1-3 (15, 50 and 150 mg/kg bw/d) was
comparable to the concurrent control fetuses. For details cf. "Attached background material".
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
For details cf. "Attached background material".
Changes in postnatal survival:
not examined
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
One fetus, each, in test groups 1 and 2 (15 and 50 mg/kg bw/d) had external malformations , both fetuses had associated skeletal malformations. The malformations were not related to dose since the high-dose group showed no malformations. The total incidence of external malformations in treated animals did not differ significantly from the concurrent control group and was covered by the historical control data. For details cf. "Attached background material".
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Several skeletal malformations were detected in fetuses of test groups 0-3 (0, 15, 50 and 150 mg/kg bw/d) affecting the skull, sternum and forelimb. Each, one fetus of test groups 1 and 2 had associated external findings. All other findings were single cases and not related to dose. Except of one finding (misshapen scapula), all findings can be found in the historical control data. Misshapen scapula was only observed in one single fetus in test group 1, not dose-related and, therefore, considered as incidental. All findings were not considered to be treatment-related. For details cf. "Attached background material".
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Two soft tissue variations, i.e. dilated renal pelvis and dilated ureter, were detected in all test groups including the control (0, 15, 50 or 150 mg/kg bw/d). The total incidences in treated animals did not differ significantly from the control group and was comparable to the historical control data. For details cf. "Attached background material".
Other effects:
no effects observed
Details on embryotoxic / teratogenic effects:
Finally, fetal examinations revealed that there is no effect of the compound on the respective morphological structures up to the highest tested dose (150 mg/kg bw/d). For details cf. "Attached background material".

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: tertogenicity / embryotoxicity

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
no
Treatment related:
no

Applicant's summary and conclusion

Conclusions:
Under the conditions of this prenatal developmental toxicity study, the oral administration of 1,8-Diazabicyclo[5.4.0]undec-7-ene to pregnant Wistar rats from implantation to one day prior to the expected day of parturition (GD 6-19) at doses as high as 150 mg/kg bw/d caused neither evidence of maternal nor developmental toxicity. In conclusion, the no observed adverse effect level (NOAEL) for maternal and prenatal developmental toxicity is the highest tested dose of 150 mg/kg bw/d.
Executive summary:

In a prenatal developmental toxicity study the test substance 1,8-Diazabicyclo[5.4.0]undec- 7-ene was administered to pregnant Wistar rats daily by gavage from implantation to one day prior to the expected day of parturition (GD 6-19) to evaluate its potential maternal and prenatal developmental toxicity. Analyses confirmed the correctness of the prepared concentrations and the stability of the test substance in the vehicle. Generally, clinical observations including food consumption and body weight/body weight gain revealed no toxicologically relevant differences between the animals receiving 15, 50 and 150 mg/kg bw/d 1,8-Diazabicyclo[5.4.0]undec-7-ene and controls. No differences of toxicological relevance between the control and the treated groups (15, 50 or 150 mg/kg bw/d) were determined for any reproductive parameters, such as conception rate, mean number of corpora lutea, mean number of implantations, as well as pre- and postimplantation loss. Similarly, no influence of the test substance on fetal weight and sex distribution of the fetuses was noted at any dose. Overall, there was no evidence for toxicologically relevant adverse effects of the test substance on fetal morphology at any dose.