2-dibutylaminoethanol

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Nov 2018 - Dec 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

25 Jun 2018

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

2-dibutylaminoethanol

EC Number:

203-057-1

EC Name:

2-dibutylaminoethanol

Cas Number:

102-81-8

Molecular formula:

C10H23NO

IUPAC Name:

2-(dibutylamino)ethan-1-ol

Test material form:

other: liquid

Details on test material:

- Name of test material (as cited in study report): N,N-Dibutylethanolamine
- Physical state: liquid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: A028-2018
- Expiration date of the lot/batch: 09 Apr 2020
- Purity: 99.8 corr. area-%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: ambient
- Stability under storage 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.
- Solubility and stability of the test substance in the vehicle The stability of Dibutylethanolamine
in corn oil at room temperature for a period of 7 days was proven before the start of the
administration period

FORM AS APPLIED IN THE TEST
- The test substance was applied as an emulsion

OTHER SPECIFICS
- Physical state/ appearance: Liquid/ yellowish, clear
- 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 or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services GmbH, Sulzfeld, Germany
- Age at study initiation: 42 ± 1 days
- Weight at study initiation: males 165.8 - 167.2 / females 131.0 - 134.3
- Housing: The animals were housed together (5 animals per cage and sex) in H-Temp polysulfonate
cages type 2000P supplied by TECNIPLAST, Hohenpeißenberg, Germany
- Diet (e.g. ad libitum): yes
- Water (e.g. ad libitum): yes
- Acclimation period: yes


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 45-65%
- Air changes (per hr): 15 air changes per hour.
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES:
Males Females Phase of study/ Examination /Study day
19 Nov 2018 Study initiation date: signature of study director/-7
20 Nov 2018 Experimental starting date: arrival of the animals and start of the
acclimatization period/-6
23 Nov 2018 Randomization of the animals /3
26 Nov 2018 Ophthalmoscopy/0
26 Nov 2018 Start of the administration period/0
From 28 Jan 2019 onwards Estrous cycle determination From /63
19 Feb 2019 FOB1; MA1 a) /85
20 Feb 2019 FOB2; MA2 b) /86
21 Feb 2019 FOB3; MA3 a) /87
22 Feb 2019 FOB4; MA4 b) /88
female 20 Feb 2019 Urinalysis/86
male 22 Feb 2019 Urinalysis/88
25 Feb 2019 Ophthalmoscopy 91
25 Feb 2019 Last weighing 91
26 Feb 2019 /27 Feb 2019 Blood sampling and necropsy c) /92/93
20 Dec 2019 Experimental completion date; draft report to QAU

a) = First 5 animals of every test group
b) = Second 5 animals of every test group
c) = Fasting period (withdrawal of food) of about 16 to 20 hours
FOB = Functional observational battery
MA = Measurement of motor activity
1..4 = Identification number of the specific examination

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

Dibutylethanolamine was applied as an emulsion. To prepare this emulsion, the appropriate
amount of test substance was weighed out depending on the desired concentration. Then,
corn oil was filled up to the desired volume and subsequently mixed with a magnetic stirrer.
During administration, the test substance preparations were kept homogeneous by stirring
with a magnetic stirrer. The test substance preparations were prepared in such a manner that
the stability was guaranteed. The administration volume was 4 mL/kg body weight.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability of the test substance in corn oil was demonstrated over a period of 7 days at
room temperature. As the mixtures were stored no longer than this time period, the stability
was guaranteed.
Considering the low relative standard deviation in the homogeneity analysis, it can be
concluded that Dibutylethanolamine was distributed homogeneously in corn oil.
The concentration control analyses of test groups 1 to 3 revealed that the values were in the
expected range of the target concentrations, i.e. were always in a range of about 90-110% of
the nominal concentrations at any time point.
Taken together, the results demonstrate the correctness of the concentrations of the test item
in the vehicle for test groups 1 to 3.

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 for 13 weeks. Control animals received only the
vehicle. All remaining animals were sacrificed after a fasting period (withdrawal of food) of at
least 16-20 hours.

Frequency of treatment:

The test substance was administered daily by gavage for 3 months

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Remarks:

control

Doses / concentrations 2

Dose / conc.:

15 mg/kg bw/day (actual dose received)

Remarks:

low dose

Doses / concentrations 3

Dose / conc.:

50 mg/kg bw/day (actual dose received)

Remarks:

mid dose

Doses / concentrations 4

Dose / conc.:

150 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:

Rational for dose setting:
Dibutylethanolamine was tested in a 28-day oral toxicity study (OECD 407; including a 2-weeks
recovery period) using Sprague-Dawley rats (Hatano Research Institute, Food and
Drug Safety Center, commissioned by the Ministry of Health, Labor and Welfare, Japan).
Groups of 5 male and 5 female animals were treated at dose levels of 0, 25, 100 and 400
mg/kg bw/d (corn oil served as vehicle) for 4 weeks; another 5 animals per sex were added
to the control and the high-dose groups in view of the recovery test.
In male and female animals treated at a dose level of 400 mg/kg bw/d, severe clinical signs
of toxicity occurred from study day 4 onwards, i.e. clonic or tonic convulsion, spasm, tremor,
abnormal phonation and pale skin, and subsequently gasping, slow respiration, deep
respiration and prone position. Three male and 5 female animals died during the course of
the treatment period, i.e. one female on study day 11, 2 males and 1 female on study day 15,
1 male and 2 females on study day 18, and 1 female on study day 25. During the pathological
examinations, findings occurred in kidneys, liver and adrenal glands (Hatano Research
Institute, 2004).
No relevant changes were observed for male and female animals treated at 25 and 100 mg/kg
bw/d. The same was true for the animals of the recovery groups, i.e. control and high-dose
group.
At BASF SE, another 28-day study to be used as range-finding study in Wistar rats was
performed (BASF project No. 10C0286/05S039). Dibutylethanolamine was administered by
gavage to groups of 5 male and 5 female Wistar rats at dose levels of 0, 100 and 300 mg/kg
bw/d (corn oil served as vehicle). Due to severe clinical findings in male and female animals
treated at 300 mg/kg bw/d, e.g. tremors, tonic/clonic convulsions and the premature deaths
of 1 male and 2 female animals, the dose level was reduced to 200 mg/kg bw/d from study
day 18 onwards. After dose level reduction, findings occurred only for a few more days but
not towards the end of the administration period. No relevant findings were observed for male
and female animals treated at 100 mg/kg bw/d.
Taking the severe findings in the high-dose group during the administration period into
account and, in addition, considering the fact that the animals expected a three-times longer
administration period, the following dose levels were selected for the present study:

150 mg/kg bw/d a) as high dose
50 mg/kg bw/d a) as intermediate dose
15 mg/kg bw/d a) as low dose

a) mg/kg body weight/day

Positive control:

No

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observation: 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
- 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 high). The following parameters were examined:
Abnormal behavior when handled, fur, skin, posture, salivation, respiration, activity/arousal level,
tremors, convulsions, abnormal movements, impairment of gait, lacrimation, palpebral closure,
exophthalmos, feces (appearance/ consistency), urine, pupil size

BODY WEIGHT: Yes
- 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
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 day 0
was calculated as body weight change.

FOOD CONSUMPTION:
- Food consumption was determined weekly and calculated as mean food consumption in
grams per animal and day.

WATER CONSUMPTION:
- Drinking water consumption was monitored by daily visual inspection of the water bottles for
any changes in volume.

OPHTHALMOSCOPIC EXAMINATION: Yes / No / Not specified
- Prior to the start of the administration period on day 0 the eyes of all animals and on study
day 91 the eyes of the control and high-dose animals were examined for any changes using
an ophthalmoscope after administration of a mydriatic agent


HAEMATOLOGY: Yes
- The following parameters were determined in blood with EDTA-K3 as anticoagulant using a
particle counter:
leukocyte count (WBC), erythrocyte count (RBC), hemoglobin (HCT), hematocrit (HCT),
mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular
hemoglobin concentration (MCHC), platelet count (PLT), differential blood count, reticulocytes (RET),
prothrombin time (Hepato Quick´s test) (HQT)
Furthermore, blood smears were prepared and stained according to WRIGHT without being
evaluated, because of non-ambiguous results of the differential blood cell counts measured
by the automated instrument.


CLINICAL CHEMISTRY: Yes
alanine aminotransferase (ALT), aspartate aminotransferase (AST), Alkaline phosphate (ALP),
γ-Glutamyltransverase (GGT), sodium (Na), potassium (K), chloride (Cl), inorganic phosphate (INP),
calcium (Ca), urea (UREA),creatine (CREA), glucose (GLUC), total bilirubin (TBIL),
total protein (TPROT), albumin (ALB), globulins (GLOB), triglycerides (TRIG), cholesterol (CHOL),
bite acids (TBA), HDL-cholesterol (HDL-CHOL), LDL-cholesterol (LDL-CHOL)

THYROID HORMONES:
- total triiodothyronine (T3), total thyroxine (T4), thyroid stimulating hormone (TSH)

URINALYSIS: Yes
pH, protein (PRO), glucose (GLU), ketones (KET, urobilinogen (UBG), bilirubin (BIL), blood,
specific gravity (SP,GR), sediment, color, turbidity (COL, TURB), volume (VOL)

NEUROBEHAVIOURAL EXAMINATION: Yes
- A functional observational battery (FOB) was performed in all animals at the end of the
administration period starting at about 10:00 h. At least one hour before the start of the FOB
the rats were transferred to single-animal polycarbonate cages type III (floor area of about
800 cm²). Drinking water was provided ad libitum, but no food was offered during the
measurements. The FOB started with passive observations without disturbing the rats,
followed by removal from the home cage, open field observations in a standard arena and
sensory motor tests as well as reflex tests.

Home cage observations:
The animals were observed in their closed home cages; during this period any disturbing
activities (touching the cage or rack, noise) were avoided during these examinations in order
not to influence the behavior of the rats. Attention was paid to:
posture, tremors, convulsions, abnormal movements, gait, other findings.


Open field observations:
The animals were transferred to a standard arena (50 × 50 cm with sides of 25 cm height)
and observed for at least 2 minutes. The following parameters were examined:
behavior on removal from the cage, fur, skin, salivation, nasal discharge, lacrimation,
eyes/pupil size, posture, palpebral closure, respiration, tremors, convulsions, abnormal
movements/stereotypes, gait abnormalitties, activity/arousal level, feces excreted within
2 minutes (appearance/ consistency), urine excreted within 2 minutes (amount/color),
rearing within 2 minutes, other findings

Sensory motor tests/ reflexes:
The animals were then removed from the open field and subjected to following sensory motor
or reflex tests:
reaction to an object being moved towards the face (approach response),
touch sensitivity (touch response), vision (visual placing response), pupillary reflex, pinna reflex,
audition (auditory startle response), coordination of movements (righting response), behavior during
handling, vocalization, pain perception (tail pinch), grip strength of forelimbs, grip strength of
hindlimbs, landing foot-splay test, other findings.

Motor activity assessment
Motor activity (MA) was also measured from 14:00 h onwards on the same day as the FOB
was performed. The examinations were performed using the TSE Labmaster System
supplied by TSE Systems GmbH (Bad Homburg, Germany). For this purpose, the rats were
placed in new clean polycarbonate cages with a small amount of bedding for the duration of
the measurement. Eighteen beams were allocated per cage. The number of beam interrupts
was counted over 12 intervals for 5 minutes per interval. The sequence in which the rats were
placed in the cages was selected at random. On account of the time needed to place the rats
in the cages, the starting time was "staggered" for each animal. The measurement period
began when the 1st beam was interrupted and finished exactly 1 hour later. No food or water
was offered to the rats during these measurements and the measurement room was
darkened after the transfer of the last rat.

IMMUNOLOGY: No

OTHER:
Estrous cycle determination
Vaginal smears for cycle determination were prepared in the morning and evaluated
according to the timetable for at least 3 weeks until the day of sacrifice.

Sperm parameters
Immediately after necropsy and organ weight determination, the right testis and cauda
epididymis were taken from all male animals.
Sperm motility examinations and the preparation of the specimens for sperm morphology
were carried out in a randomized sequence.
The right testis and right cauda epididymis were deep frozen at -20°C until evaluation of the
sperm head count. Initially, sperm morphology and sperm head count (cauda epididymis and
testis) were evaluated for the control (test group 0) and test group 3, only.
Sperm motility, sperm morphology, sperm head count (cauda epididymis),
sperm head count (testis)

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Necropsy
The animals were sacrificed by decapitation under isoflurane anesthesia. The exsanguinated
animals were necropsied and assessed by gross pathology.

Organ weights
The following weights were determined in all animals sacrificed on schedule:
Anesthetized animals (final body weight), Adrenal glands (fixed), Brain, Epididymides, Heart, Kidneys,
LIver, Ovaries(fixed), Pituitary gland (fixed), Prostate (ventral and dorsolateral part together, fixed),
Spleen, Seminal vesicles with coagulating glands (fixed), Testes, Thymus, Thyroid glands (fixed),
Uterus (with cervix).

Organ/tissue fixation
The following organs or tissues were fixed in in 4% neutral-buffered formaldehyde solution or in
modified Davidson`s solution:
All gross lesions, Adrenal glands, Aorta, Bone marrow (femur), Brain, Cecum, Cervix, Coagulating
glands, Colon, Duodenum, Epididymis, left (modified Davidson`s solution), Esophagus, Extraorbital
lacrimal glands,Eyes with optic nerve (modified Davidson`s solution), Femur with knee joint,
Harderian glands, Heart, Ileum, Jejunum (with Peyer`s patches), Kidneys, Larynx, Liver, Lungs,
Lymph nodes (axillary and mesenteric), Mammary gland (male and female), Nose (nasal cavity),
Ovaries, Oviducts, Pancreas, Parathyroid glands, Pharynx, Pituitary gland, Prostate, Rectum,
Salivary glands (mandibular and sublingual), Sciatic nerve, Seminal vesicles, Skeletal muscle, Skin,
Spinal cord (cervical, thoracic and lumbar cord), Spleen, Sternum with marrow, Stomach
(forestomach and glandular stomach), Testes left (modified Davidson`s solution), Thymus,
Thyroid glands, Trachea, Urinary bladder, Uterus, Vagina.


HISTOPATHOLOGY: Yes
Histopathology
Fixation was followed by histotechnical processing, examination by light microscopy and assessment
of findings. All gross lesion, Adrenal glands, Aorta, Bone marrow (frmur), Brain, Cecum, Cervix,
Coagulating glands, Colon,Duodenum, Epididymidis left, Eyes with optic nerve, Femur with knee
joint, Heart, Ileum, Jejunum, Kidneys, Liver, Lungs, Lymph nodes (axillary and mesenteric),
Mammary gland, ´Ovaries, Pancreas, Parathyroid gland, Peyer`s patches, Pituitary glands, Prostate,
Rectum, Salivary glands (mandibular and sublingual glands), Sciatic nerve, Seminal vesicles,
Skeletal muscle, Skin, Spinal cord (cervical, thoracic, lumbar), Spleen, Stomach (forestomach
and glandular stomach),Testes (left), Thymus, Thyroid glands, Trachea, Urinary bladder, Uterus, Vagina.

Other examinations:

Special stains (Oil-red-O and Sudan Black) were performed exemplarily on kidney sections
of 2 animals
Special attention was given for the synchrony of the morphology of the estrous cycle in
ovaries, uterus, cervix, and vagina.
Special attention was given for the male reproductive organs, especially the stage of
seminiferous tubules.
A correlation between gross lesions and histopathological findings was attempted.

Statistics:

Statistics of clinical examinations
Body weight,body weight change: DUNNETT's test (two-sided)
Rearing, grip strength forelimbs, grip strength hindlimbs, footsplaytest, motor activity:
Non-parametric one-wayanalysis using KRUSKALWALLIS test (two-sided).

Statistics of clinical pathology:
Blood parameters: For parameters with bidirectional changes:
Non-parametric one-way analysis using KRUSKAL-WALLIS test.
If the resulting p-value was equal or less than 0.05, a pairwise
comparison of each dose group with the control group was performed using
WILCOXON-test (two-sided) for the hypothesis of equal medians.
For parameters with unidirectional changes:
Pairwise comparison of each dose group with the control group using the
WILCOXON-test (one-sided) for the hypothesis of equal medians.
Urinalysis parameters: WILCOXON-test (one-sided)
Urine pH, volume and specific gravity: Non-parametric one-way analysis using
KRUSKAL-WALLIS test.
If the resulting p-value was equal or less than 0.05, a pairwise
comparison of each dose group with the control group was performed using
WILCOXON-test (two-sided) for the hypothesis of equal medians.
Sperm analysis parameters:Pairwise comparison of each dose group
with the control group using theWILCOXON-test (one-sided) with
Bonferroni-Holm adjustment for thehypothesis of equal medians; If only control
and one dose group are measured, WILCOXON-test (one-sided) without
adjustment were used.For the percentage of abnormal sperms
(ABNORMAL5_C) values < 5% were set to5 % (cut off 5%)

Statistics of pathology:
Weight parameters;Non-parametric one-way analysis using KRUSKALWALLIS H test
(two-sided). If the resulting p-value was equal or less than 0.05, a pairwise comparison of each
test group with the control group was performed using WILCOXON-test (two-sided)
for the equal medians.

Results and discussion

Results of examinations

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

see attached document: summary clinical observation
All male and all female animals of test group 3 (150 mg/kg bw/d) showed slight salivation
directly after treatment on several days of the application period. Salivation was also
observed in 5 male and 3 female animals of test group 2 (50 mg/kg bw/d) and in 6 male
animals in test group 1 (15 mg/kg bw/d).
From the temporary, short appearance immediately after dosing it was concluded the findings
were induced by a bad taste of the test substance or local affection of the upper digestive
tract. The effect was related to the test substance but assessed as being non-adverse as no
lesions in the upper digestive tract were observed in male and female animals during
pathological examinations.
A protruding right eyeball was observed one in female animal of test group 1 (15 mg/kg bw/d)
between study days 91-93. This finding was considered to be incidental and not related
to treatment.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

see attached document; summary bw_bwc
No test substance-related, adverse effects on body weight development were obtained in any
test group (15, 50 and 150 mg/kg bw/d).
Mean body weights and body weight change values of male and female animals did not show
any significant deviations to the control.

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

no effects observed

Ophthalmological findings:

effects observed, non-treatment-related

Description (incidence and severity):

see attached document: summary ophthalmological findings
No treatment-related findings were observed.
All other apparent findings were assessed as being incidental in nature since they occurred
in control as well as in treated animals and did not show a dose-response relationship
see attached document: ophthalmological findings

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

see attached document: summary haematological and chlinical chemistry
At the end of the administration period, in females of test groups 1 and 2 (15 and 50 mg/kg
bw/d) absolute and relative lymphocyte counts were significantly decreased whereas relative
neutrophil counts were significantly increased. Additionally, in females of test group 2 (50
mg/kg bw/d) relative, large unstained cell (LUC) counts were significantly decreased.
However, all mentioned alterations were not dose-dependent and, therefore, they were
regarded as incidental and not treatment-related.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

see attached document: summary haematological and chlinical chemistry
At the end of the administration period, in males of test group 3 (150 mg/kg bw/d) potassium
levels were significantly increased whereas chloride levels were significantly decreased.
Chloride values were within, potassium values slightly above the historical control range
(males, chloride 96.4-106.1 mmol/L, potassium 4.52-5.04 mmol/L). The unique potassium
increase among these individuals was regarded to be potentially treatment-related but nonadverse
(ECETOC Technical Report No. 85, 2002), whereas the chloride decrease was
regarded as incidental and not treatment-related.

Thyroid hormones
No treatment-related alterations of T3, T4 and TSH levels were observed.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

At the study end, in males of test group 2 (50 mg/kg bw/d) higher incidences of triple
phosphate crystals were observed in the urine sediment. This change was not dosedependent
and, therefore, it was regarded as incidental and not treatment-related.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

see attached document: FOB
Deviations from "zero values" were obtained in several rats. However, as most findings were
equally distributed between test-substance treated groups and controls, were without a doseresponse
relationship or occurred in single rats only, these observations were considered to
have been incidental.
Open field observations: No test substance-related effects were observed.
Sensorimotor tests/reflexes: No test substance-related effects were observed.
Quantitative parameters No test substance-related effects were observed.
In male animals of test group 2 (50 mg/kg bw/d), grip strength of hindlimbs was significantly
increased (+17%), but a dose-response relationship did not occur. The deviation was
assessed as being spontaneous in nature.

Immunological findings:

not examined

Description (incidence and severity):

see attached document: summary organ and body weight
All mean absolute and relative weight parameters did not show significant differences when
compared to the control group 0.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

see attached document: summary gross lesions and microscopic findings
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.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Vacuolation of epithelial cells of the collecting ducts was characterized by macrovesicular
clear vacuoles in the collecting duct epithelium of the inner medulla. The outer part of the
inner medulla was most prominently affected with a gradual decrease towards the tip of
papilla. The vacuoles did not stain with HE, Sudan Black or Oil-Red-O. Vacuolation affected
8 out of 10 males and 1 out of 10 females of test group 3 (150 mg/kg bw/d), with a severity
ranging from minimal to severe in males, the one female animal was only minimally affected.
No signs of necrosis, cellular degeneration, inflammation or edema were present.
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.

Description (incidence and severity):

see attached document: summary motor activiy
Regarding the overall motor activity as well as single intervals, no test substance-related and
relevant deviations to the control animals were noted for male and female animals of test
groups 1-3 (15, 50, and 150 mg/kg bw/d).
In female animals of test group 2 (50 mg/kg bw/d) and test group 3 (150 mg/kg bw/d), the
overall motor activity was significantly higher when compared to the controls. However, a
clear dose-response relationship did not occur, and the changes were assessed to be
incidental and not related to treatment.
Comparing the single intervals with the control groups, significantly higher values were
measured for male animals of test group 1 (15 mg/kg bw/d) at interval No. 8 and for male
animals of test group 3 (150 mg/kg bw/d) at interval Nos. 11 and 12.
Significantly higher values were also obtained for female animals of test groups 2 (50 mg/kg
bw/d) and 3 (150 mg/kg bw/d) at interval No. 5 as well as for female animals of test group 2
at interval No. 6.
All changes were regarded to be incidental and not related to treatment as single intervals
were not changed in a dose-dependent manner.

see attached document: summary estrous cycle
Estrous cycle
No test substance-related, adverse effects on estrous cycles were obtained in any test group
(15, 50 and 150 mg/kg bw/d).

see attached document: summary spermanalysis
Sperm analysis
Concerning motility of the sperms and the incidence of abnormal sperms in the cauda
epididymidis as well as sperm head counts in the testis and in the cauda epididymidis
no treatment-related effects were observed

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

50 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Target system / organ toxicity

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

150 mg/kg bw/day (actual dose received)

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Any other information on results incl. tables

   

Kidneys	Male animals				Female animals
Test group (mg/kg bw/d)	0 (0)	1 (15)	2 (50)	3 (150)	0 (0)	1 (15)	2 (50)	3 (150)
No. of animals	10	10	10	10	10	10	10	10
Vacuolation,collecting ducts	0	0	0	8	0	0	0	1
·      Grade1				4				1
·      Grade2				1
·      Grade3				2
·      Grade4				1

 

Applicant's summary and conclusion

Conclusions:

The administration of Dibutylethanolamine by gavage for 3 months to male and female Wistar
rats caused test substance-related findings at a dose level of 150 mg/kg bw/d taking the
vacuolation of the collecting duct epithelium in kidneys into account. Therefore, under the conditions of the present study the NOAEL was 50 mg/kg bw/d for male
and female Wistar rats.

Executive summary:

Dibutylethanolamine was administered orally by gavage to groups of 10 male and 10 female

Wistar rats at dose levels of 0 mg/kg b/day (mg/kg bw/d; test group 0), 15 mg/kg

bw/d (test group 1), 50 mg/kg bw/d (test group 2) and 150 mg/kg bw/d (test group 3) over a

period of 3 months. Corn oil served as vehicle, control animals were dosed daily with the

vehicle only.

With regard to clinical examinations, signs of general systemic toxicity were not observed

even at a dose level of 150 mg/kg bw/d of Dibutylethanolamine. In addition, no test

substance-related effects on estrous cycle length and the number of cycles were obtained

Concerning clinical pathology, no treatment-related, adverse effects were observed up to a

dose of the compound of 150 mg/kg bw/d.

The unique potassium increase observed for male animals of test group 3 (150 mg/kg bw/d)

was considered to be potentially treatment-related but regarded to be non-adverse (ECETOC

Technical Report No. 85, 2002). A relation to the kidney-related findings observed in these

male animals of the test group 3 was excluded.

Regarding pathology, neither treatment-related weight changes nor gross lesions were

observed.

The target organ were the kidneys. In 8 out of 10 males and 1 out of 10 females of test group

3, macrovesicular vacuolation of the epithelial cells of the collecting ducts was observed. No

signs of necrosis, degeneration or inflammation were present.

Therefore, under the conditions of the present study the NOAEL was 50 mg/kg bw/d for male

and female Wistar rats.