Registration Dossier

Administrative data

Description of key information

A guideline-compliant screening study (OECD 422) and supporting range-finder are available; studies used oral (gavage) administration.

The key study is a recently conducted 90 day repeated dose toxicity sudy (OECD 408); study uses oral (gavage) administration in drinking water.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

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
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
GLP compliance:
yes (incl. certificate)
Limit test:
no
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
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
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
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
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.
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).
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
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

See "Attached background material".

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.

Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
key study
Study period:
21st December 2011 to 20th August 2012.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Modern study conducted according to GLP and relevant testing guidelines.
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)
Deviations:
not specified
Qualifier:
according to
Guideline:
other: EPA OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test
Deviations:
not specified
Qualifier:
equivalent or similar to
Guideline:
other: EPA OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test
Deviations:
not specified
Qualifier:
equivalent or similar to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:
not specified
Principles of method if other than guideline:
Study was also conducted equivalent to OECD Guideline 407 abd EPA OPPTS 870.3050.
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Crl:WI(Han)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Age at study initiation: Approximately 11 weeks.
- Housing: Pre-mating animals were housed in groups of 5 animals/sex/cage in Macrolon plastic cages (MIV type, height 18 cm). During mating, females were caged together with males on a one-to-one-basis in Macrolon plastic cages (MIII type, height 18 cm). Post-mating, males were housed in their home cage (Macrolon plastic cages, MIV type, height 18 cm) with a maximum of 5 animals/cage. Females were individually housed in Macrolon plastic cages (MIII type, height 18 cm). During lactation, pups were kept with the dam until termination in Macrolon plastic cages (MIII type, height 18 cm). During locomotor activity monitoring of the dams the pups were kept warm in their home cage using bottles filled with warm water. In order to avoid hypothermia of pups, pups were not left without their dam or a bottle filled with warm water for longer than 30-40 minutes. During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cage (48.3 x 26.7 x 20.3 cm).
- Diet (e.g. ad libitum): Free access to pelleted rodent diet
- Water (e.g. ad libitum): Free access to tap-water.
- Acclimation period: At least 5 days prior to start of treatment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24°C
- Humidity (%): 40 to 70%,
- Air changes (per hr): approximately 15 room air changes/hour
- Photoperiod (hrs dark / hrs light): 12-hour light/12-hour dark cycle

IN-LIFE DATES: From: To:
Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily within 30 minutes prior to dosing and were homogenized to a visually acceptable level. Adjustment was made for the density of the test substance.
On the first day of dosing, the pH value of Groups 2-4 formulations were determined. Based on information that the test substance may react with glass, the formulations were prepared in plastic vials.
Formulations were placed on a magnetic stirrer during dosing.

VEHICLE
Water
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were conducted on a single occasion during the treatment phase. Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in vehicle over 30 minutes at room temperature was also determined (highest and lowest concentration). The accuracy of preparation was considered acceptable if the mean measured concentrations were 90-110% of the target concentration.
Homogeneity was demonstrated if the coefficient of variation was ≤ 10%. Formulations were considered stable if the relative difference before and after storage was maximally 10%.
Duration of treatment / exposure:
Males were exposed for 29 days.
Females were exposed for 43 to 57 days
Frequency of treatment:
Once daily, seven days per week.
Remarks:
Doses / Concentrations:
0, 15, 50, 150 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
5 animals per sex per group.
Control animals:
not specified
Details on study design:
- Dose selection rationale:
The doses selected for this study are based on results from a 14-day dose range finding study where the doses were administered were 0, 15, 40, 100 and 200 mg/kg bw/day. At 200 mg/kg bw/day, slight body weight loss or reduced gains were seen along with changes in clinical pathology parameters. Reddish foci on the stomach glandular mucosa were seen for all animals at this dose level during the macroscopic examination. Therefore, 200 mg/kg was considered to be in excess of the MTD for the main study. No adverse effects were noted at 100 mg/kg bw/day. Based on these data, 15, 50 and 150 mg/kg bw/day were chosen for the main study.
Positive control:
No data
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Mortality/viability were examined at least twice daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once daily from start of treatment onwards, detailed clinical observations were conducted for all animals. Once prior to start of treatment and at weekly intervals during the treatment period this was also performed outside the home cage in a standard arena.

BODY WEIGHT: Yes
- Time schedule for examinations: Males and females were weighed on the first day of exposure and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on Days 1 and 4.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
Weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 postcoitum and on Days 1 and 4 of lactation.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study):
Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no treatment related effect was suspected.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Prior to scheduled post-mortem examination.
- Anaesthetic used for blood collection: Yes, isoflurane.
- Animals fasted: Yes, for a maximum of 20 hours.
- How many animals: 5 animals per sex per group.
- Parameters checked: White blood cells (WBC) and Differential leucocyte count, including Neutrophils, Lymphocytes, Monocytes, Eosinophils, Basophils, Red blood cells, Reticulocytes, Red blood cell distribution width (RDW), Haemoglobin, Haematocrit, Mean corpuscular volume (MCV), Mean corpuscular haemoglobin (MCH), Mean corpuscular haemoglobin concentration (MCHC) and Platelets.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Prior to scheduled post-mortem examination.
- Animals fasted: Yes, for a maximum of 20 hours.
- How many animals: 5 animals per sex per group.
- Parameters checked: Alanine aminotransferase (ALAT), Aspartate aminotransferase (ASAT), Alkaline Phosphatase (ALP), Total protein, Albumin, Total Bilirubin, Bile acids, Urea, Creatinine, Glucose, Cholesterol, Sodium, Potassium, Chloride, Calcium and Inorganic Phosphate (Inorg. Phos).

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: The selected males were tested during Week 4 of treatment and the selected females were tested towards the end of the scheduled lactation period (all before blood sampling).
- Battery of functions tested: hearing ability, pupillary reflex, static righting reflex and grip strength.
Sacrifice and pathology:
All males and the selected 5 females/group were deprived of food overnight (with a maximum of 20 hours) prior to planned necropsy, but water was provided. Non-selected females were not deprived of food.
Animals surviving to scheduled necropsy were deeply anaesthetized using isoflurane and subsequently exsanguinated.

All animals were subjected to macroscopic examination of the cranial, thoracic and abdominal tissues and organs, with special attention being paid to the reproductive organs.

Histopathological examinations were conducted on various tissue samples and all histopathology data was reviewed by a second pathologist. All organ and tissue samples were processed, embedded and cut at a thickness of 2-4 micrometers and stained with haematoxylin and eosin. The testes was processed, sectioned at 3-4 micrometers, and stained with PAS/haematoxylin.
Other examinations:
Organ weights were recorded from the 5 selected animals per group, including Adrenal glands, Spleen, Brain, Testes, Epididymides, Thymus, Heart, Uterus, (including cervix), Kidneys, Prostate, Liver, Seminal vesicles including coagulating glands, Ovaries, and Thyroid including parathyroid.
In all remaining males, the epididymides and testes were examined.
Statistics:
If the variables could be assumed to follow a normal distribution, the Dunnett-test (many to- one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
The Fisher Exact-test was applied to frequency data.
The following additional methods of statistical analysis were used:
Motor activity data was subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences followed by the Wilcoxon test to compare the treated groups to the control group.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance.
Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
No mortality occurred during the study period. No toxicologically significant clinical signs were noted up to 150 mg/kg bw/day. At 150 mg/kg bw/day, salivation was noted for six males and four females over a few days towards the end of the treatment period. This may be related to the taste of the test substance, and was considered to be a physiological response rather than a sign of systemic toxicity. Hunched posture and rales were also noted for a few animals on a few occasions. As inflammatory changes were observed in the stomach for animals at this dose level, it is possible these symptoms were indicative of discomfort due to local stomach effects of the test substance. Hunched posture and/or labored respiration were noted for individual animals at 15 and 50 mg/kg bw/day over 1-3 days. Due to the transient nature and limited occurrence, these signs were not considered to be toxicologically relevant.
Broken tail apex was an incidental finding noted for single males in the control and 50 mg/kg bw/day groups which had no relationship to treatment.

BODY WEIGHT AND WEIGHT GAIN
No toxicologically relevant changes in body weights and body weight gain were noted up to 150 mg/kg bw/day.
Individual animals at 150 mg/kg bw/day lost weight over a single time point during the mating and post coitum periods, respectively. Since this occurred for individual animals and was transient for the female, this was not considered to be indicative of systemic toxicity.
Body weight gains were significantly lower for males at 50 mg/kg bw/day on Day 8 of the pre-mating period. As the difference from controls was only slight and occurred in the absence of a dose response, it was not considered to be toxicologically relevant.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
No toxicologically relevant changes in food consumption before or after allowance for body weight were noted.
Females at 150 mg/kg bw/day had significantly lower relative food consumption on Days 11-14 of the post coitum period only. As the effect was transient and was mainly attributable to a very low value obtained for one female, it was not considered to be toxicologically relevant. Furthermore, one male also had lower food consumption corresponding to the days where lower body weights were recorded. Taken together, these data were not considered to reflect systemic toxicity for the group as a whole.

HAEMATOLOGY
There were no toxicologically relevant effects on haematology parameters up to 150 mg/kg bw/day. Activated partial prothrombin time (APTT) was increased for females at 150 mg/kg bw/day. However, this was attributable to relatively high values obtained for 2 females, and in the absence of corroborating effects seen in any other parameters, this was not considered to be toxicologically relevant.
Minor statistically significant differences seen between controls and animals receiving 15 and 50 mg/kg bw/day occurred in the absence of a dose response and were not considered to be biologically relevant.

CLINICAL CHEMISTRY
There were no toxicologically relevant effects on clinical biochemistry parameters up to 150 mg/kg bw/day.
At 150 mg/kg bw/day, cholesterol was higher for males, though in the absence of any relevant effects on the liver seen for any other parameter, this was ultimately not considered to be toxicologically relevant. There was a slight trend toward lower glucose for animals of all treatment groups, though the difference from controls was not statistically significant and was not considered toxicologically relevant.
Calcium was significantly lower for females at 50 mg/kg bw/day, but was not considered to be toxicologically relevant since the difference from controls was only slight and occurred in the absence of a treatment related distribution.

NEUROBEHAVIOUR
Hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all animals.
The variation in motor activity did not indicate a relation with treatment. Males at 15 mg/kg bw/day had significantly higher total movement and ambulatory counts. However, in the absence of a dose response and any relevant clinical signs noted like hyperactivity, the effects were not considered to be treatment related.
All groups showed a similar habituation profile with very high activity in the first interval that decreased over the duration of the test period.

ORGAN WEIGHTS
Absolute and relative kidney weights were dose dependently higher for females of treated groups than controls, but only reached statistical significance at 150 mg/kg bw/day. The same relationship was not observed for males. However, in the absence of any effects on relevant clinical pathology parameters or treatment-related findings noted in the kidneys during the microscopic examination, the increased weights were not considered to be biologically relevant.
There were no other effects on organ weights or organ to body weight ratios at any dose level tested.

GROSS PATHOLOGY
Dark red foci on the stomach glandular mucosa were noted for 5 males at 150 mg/kg bw/day. In addition, irregular surface and dark red discoloration of the glandular mucosa were noted for a single female and dark red discoloration of the mesenteric lymph node was noted for a single male at this dose level.
An isolated, reddish focus on the stomach glandular mucosa was also seen for a single control male, however, due to the increased incidence, the foci seen for males at 150 mg/kg bw/day were considered to be treatment related.
Incidental findings noted for control and/or treated animals included a bent tail, reddish or dark red foci on the thymus, pelvic dilation of the kidneys, dark red foci on the lungs, a tan focus or greenish discoloration of the clitoral gland, and alopecia on the foreleg. These findings did not show a dose related trend and were not considered to be toxicologically relevant. One female at 50 mg/kg bw/day was found with two mummified fetuses in the cervix at the macroscopic examination. As there were no developmental or reproductive findings noted at any dose level, this was not considered to be treatment related.

HISTOPATHOLOGY: NON-NEOPLASTIC
Findings attributed to the test article were limited to the stomach and spleen, mostly for animals at 150 mg/kg bw/day.
Stomach:
Most stomach observations were confined to animals given 150 mg/kg bw/day.
-Minimal to moderate hemorrhage was observed 0/6, 1/5, 1/5, and 7/8 males in the respective 0, 15, 50 and 150 mg/kg bw/day dose groups. Minimal or slight hemorrhage was present in the glandular mucosa of 1/5 15 mg/kg bw/day and 2/5 150 mg/kg bw/day females.
-Moderate degeneration of the glandular mucosa was present in 1/8 150 mg/kg bw/day males. Minimal degeneration was present in the glandular mucosa of 1/5 150 mg/kg bw/day females.
-Minimal or slight inflammation was present in 7/8 males and 1/5 females at 150 mg/kg bw/day.
-Minimal or slight hyperplasia of non-glandular epithelium was present in 4/8 males and 2/5 females at 150 mg/kg bw/day.
-Minimal hyperkeratosis was observed in 3/8 males and 2/5 females at 150 mg/kg bw/day.

Spleen:
The average severity grades of splenic hematopoiesis were 2.0, 2.4, 1.8 and 1.2 in the respective 0, 15, 50 and 150 mg/kg bw/day dose groups for males. Females at 150 mg/kg bw/day were comparable to controls. In consideration that female 150 mg/kg bw/day animals were comparable to controls and that any significant blood loss from the stomachs of 150 mg/kg bw/day males should have resulted in increased levels of normally occurring hematopoiesis, the decrease in average severity may be spurious.

No test article related abnormalities were seen in the reproductive organs.

There were 3/10 150 mg/kg bw/day males with seminiferous tubular degeneration, however, all were unilateral. One was centrally located slight and focal. The other two were focal, minimal (barely noticeable), and consisted of a single centrally located cross sectional tubule (comprised of sertoliform cells as are often seen in association with the rete). The testes from these two animals were essentially normal. PAS stains demonstrated the presence of all sperm development stages in all control and 150 mg/kg bw/day males. Degeneration was not attributed to the test article.

OTHER FINDINGS
No toxicologically relevant effects on reproductive parameters were noted. No toxicologically relevant effects on gestation index and duration, parturition, maternal care and early postnatal pup development (mortality, clinical signs, body weight and macroscopic examination) were observed.
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: At 150mg/kg bw/day, effects on the stomach were observed at macroscopic and microscopic examination.
Critical effects observed:
not specified
Conclusions:
Effects in this study were limited to gastric pathology at the highest dose level of 150 mg/kg bw//d: a NOAEL of 50 mg/kg bw/d is therefore derived.
Executive summary:

In this OECD 422 screening study, rats were exposed to DBU by oral gavage at dose levels of 0, 15, 50 and 150 mg/kg bw/d. Rats of the control group received the vehicle, water, alone. Males were exposed for 29 days (2 weeks prior to mating, during mating, and up to termination). Females were exposed for 43 - 57 days, (during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation).

The following observations and examinations were evaluated: mortality / viability, clinical signs (daily), functional observations and locomotor activity (males: Week 4, females: end of lactation), body weight and food consumption (at least at weekly intervals), clinical pathology (males: Week 4, females: end of lactation), macroscopy at termination, organ weights and histopathology on a selection of tissues, and reproduction/developmental parameters, consisting of mating, fertility and conception indices, precoital time, number of corpora lutea and implantation sites, gestation index and duration, parturition, maternal care, sex ratio and early postnatal pup development (mortality, clinical signs, body weights and macroscopy). Formulations were analyzed during the study to assess accuracy, homogeneity and stability.

At 150 mg/kg bw/d, parental effects on the stomach were seen at the macroscopic examination including dark red foci and irregular surface of the stomach. At the microscopic examination corroborative findings, consisting of haemorrhage, and degeneration of the glandular mucosa, inflammation, hyperplasia and hyperkeratosis were seen. All effects were local and limited to the stomach, and could be attributable to the strong alkaline corrosivity of the test substance. No toxicologically significant changes were noted in any of the remaining parental parameters investigated in this study (i.e. clinical appearance, functional observations, body weight, food consumption, clinical laboratory investigations, macroscopic examination, organ weights, and microscopic examination). No adverse effects were noted at 15 and 50 mg/kg bw/d. Based on these results, the NOAEL for parental toxicity is considered to be 50 mg/kg bw/d.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
120 mg/kg bw/day
Study duration:
subchronic
Species:
rat

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

The repeated dose toxicity of the submission substance is investigated in a combined repeated dose/developmental/reproductive toxicity range-finding study (OECD 422) and its associated 14 -day range-finding study.

A 90 days repeated dose toxicity sudy (OECD 408); study uses oral (gavage) administration in drinking water is available as key-study.

In the key study (OECD 408; BASF SE, 2017) 1,8-Diazabicyclo[5.4.0]undec-7-ene (DBU, CAS No. 6674-22-2) 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 equall 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. Thus, the NOAEL was determined to be 120 mg/kg bw.

In an OECD 422 screening study, rats were exposed to DBU by oral gavage at dose levels of 0, 15, 50 and 150 mg/kg bw/d. At the highest dose level of 150 mg/kg bw/d, effects on the stomach were seen at the macroscopic examination including dark red foci and irregular surface of the stomach. At the microscopic examination corroborative findings, consisting of haemorrhage, and degeneration of the glandular mucosa, inflammation, hyperplasia and hyperkeratosis were seen. All effects were local and limited to the stomach, and could be attributable to the strong alkaline corrosivity of the test substance. No toxicologically significant changes were noted in any of the remaining parental parameters investigated in this study (i.e. clinical appearance, functional observations, body weight, food consumption, clinical laboratory investigations, macroscopic examination, organ weights, and microscopic examination). No adverse effects were noted at 15 and 50 mg/kg bw/d. Based on these results, the NOAEL for parental toxicity is considered to be 50 mg/kg bw/d.

In the dose range-finding study, four groups of three male and three female Wistar Han rats were exposed to DBU by oral gavage at dose levels of 0, 15, 40, 100 and 200 mg/kg bw/d for 14 days. At the highest dose level of 200 mg/kg bw/d, slight body weight loss or reduced gains and reddish foci on the stomach glandular mucosa was seen for all animals at this dose level during the macroscopic examination. No toxicologically significant changes were noted in any of the remaining parameters investigated in this study (i.e. clinical appearance, food consumption, clinical laboratory investigations, macroscopic examination and organ weights). The NOAEL for this study is therefore 100 mg/kg bw/d.

The findings of repeated dose oral toxicity studies therefore indicate local effects as critical, with no clear evidence for systemic toxicity.

Justification for classification or non-classification

The results of the data available do not indicate significant target organ toxicity at relevant dose levels; no classification (STOT-RE) is therefore proposed according to Regulation (EC) No. 1272/2008.