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Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Subject to confirmation of Testing Proposal by ECHA
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 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): 1,8-Diazabicyclo-5,4,0-undecene-7
- Analytical purity:>=98%
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 species / strain selection:
The rat is a frequently used laboratory animal, and there is
comprehensive experience with this animal species. Moreover,
the rat has been proposed as a suitable animal species by the
OECD and the EPA for this type of study.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services
GmbH, Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: [yes]
- Age at study initiation: 35 ± 1 days
- Weight at study initiation: Males: 156.4 - 158.2 / Females: 127.2 - 129.1
- 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: 7 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
29 Mar 2016 Study initiation date: signature of study director -8
30 Mar 2016 Experimental starting date: arrival of the animals
and start of the acclimatization period -7
04 Apr 2016 Randomization of the animals -2
05 Apr 2016 Ophthalmoscopy -1
06 Apr 2016 Start of the administration period 0
27 Jun 2016 Urinalysis 82
30 Jun 2016 FOB1; MA1 a)
04 Jul 2016FOB2; MA2a)
01 Jul 2016FOB3; MA3 b)
05 Jul 2016FOB4; MA4 b)

06 Jul 2016 Ophthalmoscopy 91
06 Jul 2016 Last weighing 91
07 - 08 Jul 2016 Blood sampling and necropsyc) 92
Jul 2016 - Feb 2017 Organ fixation, processing and evaluation
15 Sep 2017 Experimental completion date; draft report to
QAU

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Remarks:
highly deionized water
Details on oral exposure:
1,8-Diazabicyclo[5.4.0]undec-7-ene was applied as a solution. To prepare this solution, the
appropriate amount of test substance was weighed out depending on the desired
concentration. Then, highly deionized water was filled up to the desired volume, subsequently
mixed with a magnetic stirrer. The test-substance preparations were produced daily. The
administration volume was 10 mL/kg body weight.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The various analyses confirmed
• the stability of the test-substance preparations for a period of 1.5 hours at room temperature,
• the correctness of concentrations and given homogeneity in test-substance preparations.
Duration of treatment / exposure:
On day of arrival, the animals were subjected to an acclimatization period during which they
received ground diet and drinking water ad libitum. Prior to the first detailed clinical observation,
the animals were distributed according to weight among the individual test groups, separated
by sex. The weight variation of the animals used did not exceed 20 percent of the mean weight
of each sex. The list of randomization instructions was compiled with a computer.
The test substance was administered daily by gavage for 3 months. Control animals received
only the vehicle. All remaining animals were sacrificed after a fasting period (withdrawal of
food) of at least 16 hours.
Frequency of treatment:
The test substance was administered daily by gavage for 3 months
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.:
40 mg/kg bw/day (actual dose received)
Remarks:
mid dose
Dose / conc.:
120 mg/kg bw/day (actual dose received)
Remarks:
high dose
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based on the results of an OECD 422 study (85R0489/11X203, findings in the stomach at 150 mg/kg bw/d: hemorrhage, degeneration of glandular mucosa, inflammation, hyperplasia, and hyperkeratosis, which could be attributed to the strong corrosivity of the test substance).
Positive control:
No

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations were included. A check for moribund and dead animals was made twice daily on working days and once daily
on Saturdays, Sundays and public holidays. If animals were in a moribund state, they were
sacrificed and necropsied.

DETAILED CLINICAL OBSERVATIONS: Yes / No / Not specified
- Time schedule: daily
Detailed clinical observations (DCO) were performed in all animals prior to the administration
period and thereafter at weekly intervals. The findings were ranked according to the degree of
severity, if applicable. The animals were transferred to a standard arena (50 × 37.5 cm with
sides of 25 cm height). The following parameters were examined:
1. Abnormal behavior when handled
2. Fur
3. Skin
4. Posture
5. Salivation
6. Respiration
7. Activity/ arousal level
8. Tremors
9. Convulsions
10.Abnormal movements
11.Gait abnormalities
12.Lacrimation
13.Palpebral closure
14.Exophthalmos
15.Assessment of the feces discharged during the examination (appearance/ consistency)

BODY WEIGHT: Yes
- Time schedule for examinations: Body weight was determined before the start of the administration period in order to randomize
the animals. During the administration period the body weight was determined on study day 0
(start of the administration period) and thereafter at weekly intervals. The difference between
the body weight on the respective day of weighing and the body weight on study day 0 was
calculated as body weight change.

Food consumption
Food consumption was determined weekly (as representative value over 7 days) for each
cage. The average food consumption/cage was used to estimate the mean food consumption
in grams per animal and day.
Drinking water consumption
Drinking water consumption was observed by daily visual inspection of the water bottles for
any overt changes in volume.


OPHTHALMOSCOPIC EXAMINATION: Yes
The eyes of all animals were examined prior to the start of the administration period. At the
end of the administration period, i.e. study day 91, the eyes of animals in test groups 0 (control)
and 3 (1000 mg/kg bw/d) were examined for any changes using an ophthalmoscope (HEINE
OPTOTECHNIK, Herrsching, Germany) after application of a mydriatic agent (Mydrum,
Chauvin ankerpharm GmbH, Rudolstadt, Germany).

HAEMATOLOGY: Yes


CLINICAL CHEMISTRY: Yes /
- Time schedule for collection of blood:
- Animals fasted: Yes / No / Not specified
- How many animals:
- Parameters checked in table [No.?] were examined.

URINALYSIS: Yes
- Time schedule for collection of urine:
- Metabolism cages used for collection of urine: Yes / No / Not specified
- Animals fasted: Yes / No / Not specified
- Parameters checked in table [No.?] were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations:
- Dose groups that were examined:
- Battery of functions tested: sensory activity / grip strength / motor activity / other:

IMMUNOLOGY: No


OTHER:
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table)
3.10. PATHOLOGY
3.10.1. Necropsy
The animals were sacrificed by decapitation under isoflurane anesthesia. The exsanguinated
animals were necropsied and assessed by gross pathology.
3.10.2. Organ weights
The following weights were determined in all animals sacrificed on schedule:
1. Anesthetized animals
2. Adrenal glands
3. Brain
4. Epididymides
5. Heart
6. Kidneys
7. Liver
8. Ovaries
9. Spleen
10. Testes
11. Thymus
12. Thyroid glands
13. Uterus with cervix
3.10.3. Organ/tissue fixation
The following organs or tissues were fixed in 4% neutral-buffered formaldehyde solution or in
modified Davidson’s solution:
1. All gross lesions
2. Adrenal glands
3. Aorta
4. Bone marrow (femur)
5. Brain
6. Cecum
7. Cervix
8. Coagulating glands
9. Colon
10. Duodenum
11. Epididymides
12. Esophagus
13. Extraorbital lacrimal glands
14. Eyes with optic nerve (modified Davidson’s solution)
15. Femur with knee joint
16. Harderian glands
17. Heart
18. Ileum
19. Jejunum (with Peyer’s patches)
20. Kidneys
21. Larynx
22. Liver
23. Lungs
24. Lymph nodes (mesenteric and axillary lymph nodes)
25. Mammary gland (male and female)
26. Nose (nasal cavity)
27. Ovaries
28. Oviducts
29. Pancreas
30. Parathyroid glands
31. Pharynx
32. Pituitary gland
33. Prostate
34. Rectum
35. Salivary glands (mandibular and sublingual glands)
36. Sciatic nerve
37. Seminal vesicles
38. Skeletal muscle
39. Skin
40. Spinal cord (cervical, thoracic and lumbar cord)
41. Spleen
42. Sternum with marrow
43. Stomach (forestomach and glandular stomach)
44. Testes
45. Thymus
46. Thyroid glands
47. Trachea
48. Urinary bladder
49. Uterus
50. Vagina

HISTOPATHOLOGY: Yes (see table)

3.10.4. Histopathology
The histotechnical processing (trimming, paraplast embedding, cutting, hematoxylin and eosin
(H&E) staining) of all organs (except eyes with optic nerve) of all animals (according to the
study plan, the amendments to the study plan and the BASF raw data) were accomplished by
the test site TPL Path Labs (TPL), Sasbacher Straße 10, 79111 Freiburg, Germany, under the
responsibility of the Principal investigater (PI) Ms Sabrina Zschernitz. Raw data of the study
phase, as well as remaining wet tissue, paraplast blocks and HE-stained slides were sent to
the test facility for archiving for at least the period of time specified in the GLP principles.
Histopathological evaluation of the HE-stained slides was performed by the study pathologist.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
In male animal no. 12 of test group 1 (15 mg/kg bw/d) showed a protruding eyeball from study
day 0 onwards to day 36. In female animal no. 62 of test group 2 (40 mg/kg bw/d) a skin lesion
in the region of the left shoulder was observed from study day 25 to 36.
No clinical findings were observed for all other male and female animals in test groups 1 and
2 (15 and 40 mg/kg bw/d).
Slight salivation within two hours after treatment was observed in all male and female animals
of test group 3 (120 mg/kg bw/d), moderate salivation was observed in 6 males and 5 females
of these and severe salivation was seen in 4 males and one female in this group from study
day 40 onwards on several days. From the temporary, short appearance immediately after
dosing it was concluded that salivation was induced by a bad taste of the test substance or
local affection of the upper digestive tract.
Mortality:
no mortality observed
Description (incidence):
No animal died or was sacrificed moribund during the study.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
No test substance-related changes of mean body weights and mean body weight change
values in both sexes were observed from study day 0 to 91.
In female animals of test group 2 (40 mg/kg bw/d) significantly increased body weight change
was observed from day 0 to 35 (15.8%). Furthermore, non-statistically significant increased
body weight changes occurred from day 0 to 7 (27.6%), 0 to 14 (19.1%), 0 to 21 (18.6%), 0 to
28 (12.2%), 0 to 49 (11.4%), 0 to 56 (11.4%), 0 to 77 (12.7%) and 0 to 84 (10.4%) in females
of test group 2 (40 mg/kg bw/d) and from day 0 to 28 (10.7%) in females of
test group 3 (120 mg/kg bw/d). Because of a missing statistical significance and dosedependency
of these findings, the authors assessed them to be incidental and not related to
treatment.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption was not increased during single intervals in female animals of test groups
1 and 2 (15 and 40 mg/kg bw/d). Concerning test group 2 food consumption was slightly
increased from day 0 to 7, 17.4% deviation versus control, and from study day 70 to 91 with a
maximum of 19.1%. Concerning test group 1 increased food consumption from study day 70
to 91 with a maximum deviation versus control of 26.8% was measured. Because of the
incidental increase in single intervals and missing dose dependency of the finding, it is not
considered to be an treatment-related. In male animals no alterations were observed.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
No overt changes with respect to water consumption were observed visually for animals treated with the test substance.
Ophthalmological findings:
no effects observed
Description (incidence and severity):
No treatment-related findings were observed.
Haematological findings:
no effects observed
Description (incidence and severity):
No treatment-related changes among hematological parameters were observed. At the end of the three-months administration period in males of test groups 2 and 3 (40 and 120 mg/kg bw/d) absolute basophil cell counts were significantly decreased, but the values were within the historical control range (males absolute basophils 0.01-0.02 Giga/L). Therefore, this change was regarded as incidental and not treatment-related.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
No treatment-related changes among clinical chemistry parameters were observed.
Urinalysis findings:
no effects observed
Description (incidence and severity):
No treatment-related changes among urinalysis parameters were observed.
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Deviations from "zero values" were obtained in several animals. However, as most findings were equally distributed between test-substance treated groups and controls, were without a dose-response relationship or occurred in single animals only, these observations were considered to have been incidental.

Immunological findings:
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
The significant increase in male’s relative kidney weight of test group 1 (15 mg/kg bw/d; 8 % compared with control) and decrease of relative ovary weight of test group 2 (45 mg/kg bw/d; 12 % compared with control) and 3 (120 mg/kg bw/d; 14 % compared with control) were regarded to be incidental as the weights were within historical control data (BASF SE 2017; for details see "Attached background material") and no histopathologic finding in test group 3 (120 mg/kg bw/d) animals could explain the weight changes. All other mean relative weight parameters did not show significant differences when compared to the control group 0.

Gross pathological findings:
no effects observed
Description (incidence and severity):
All findings occurred either individually or were biologically equally distributed over control and
treatment groups. They were considered to be incidental or spontaneous in origin and without
any relation to treatment.
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
Motor activity measurement

Comparing the single intervals with the control group, interval 8 in females of test group 1
(15 mg/kg bw/d) and test group 2 (40 mg/kg bw/d) was significantly increased. However, since
these findings were single events and no further significant changes were observed in the
respective sum of all intervals, these findings were regarded incidental and spontaneous in
nature.
Details on results:
All findings occurred either individually or were biologically equally distributed over control and
treatment groups. They were considered to be incidental or spontaneous in origin and without
any relation to treatment.
One female of test group 3 (120 mg/kg bw/d) revealed a malignant lymphoma, that was found
in many organs investigated. It was regarded to have developed spontaneously and as no
other tumors or tumor precursors were observed in the remaining animals of this test group it
was regarded to be not related to treatment. Furthermore, in literature it is reported that Wistar
rats can develop this kind of tumor spontaneously even though they were very young (Son et
al., 2010; Hayashi et al., 1989).

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
120 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed

Target system / organ toxicity

Key result
Critical effects observed:
no
Lowest effective dose / conc.:
120 mg/kg bw/day (actual dose received)
System:
other: No systemic and/or local toxicity observed
Organ:
other: all organs

Any other information on results incl. tables

See "Attached background material".

Applicant's summary and conclusion

Conclusions:
The oral administration of 1,8-Diazabicyclo[5.4.0]undec-7-ene by gavage to male and female Wistar rats for 3 months caused no test substance-related, adverse signs of systemic toxicity up to a dose level of 120 mg/kg bw/d. Under the conditions of the present study the no observed adverse effect level (NOAEL) was 120 mg/kg bw/d for male and female Wistar rats.
Executive summary:

1,8-Diazabicyclo[5.4.0]undec-7-ene was administered by gavage to groups of 10 male and 10 female Wistar rats at dose levels of 0 (test group 0), 15 (test group 1), 40 (test group 2) and 120 mg/kg bw/d (test group 3) over a period of 3 months.

With regard to clinical examinations, signs of general systemic toxicity were not observed up to the highest dose tested 120 mg/kg bw/d.

Concerning clinical pathology no treatment-related, adverse effects were observed up to a dose of the test substance of 120 mg/kg bw/d.

Regarding pathology, all findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.