2-dibutylaminoethanol

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP Guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

(22 Mar 1996; Inhalation exposure is conducted according to OECD 413 (7 September 2009))

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

BASF SE, Experimental Toxicology and Ecology, Ludwigshafen, Germany

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sulzfeld (According to a written statement from the breeder, male and female animals were derived from different litters. This was necessary to rule out the possibility of sibling mating.)
- Age at study initiation: about 10 - 11 weeks
- Housing: individually in Makrolon type M III cages (exceptions: during mating (1 male/1 female per cage), during rearing up to PND 4 (1 dam with her litter); for motor activity (MA) measurements the animals were housed individually in polycarbonate cages
- Diet: Ground Kliba maintenance diet mouse / rat “GLP” (Provimi Kliba SA, Kaiseraugst, Switzerland); ad libitum, but the animals did not have access to feed during the exposure.
- Water: ad libitum, but the animals did not have access to water during the exposure
- Acclimation period: at least 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 30 - 70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 / 12

Route of administration:

inhalation: aerosol

Type of inhalation exposure (if applicable):

nose/head only

Vehicle:

air

Details on exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Head / nose inhalation system (V ca. 155 L and 90 L, respectively); the snouts of the rats projected into the inhalation chamber to inhale the atmosphere.
- Method of holding animals in test chamber: The rats are restrained in glass exposure tubes.
- Source and rate of air: conditioned supply air: test group 0: 5.7 – 6.3 m³/h, test groups 1 - 3: 4.2 - 4.8 m³/h; compressed air (filtered air pressurized to about 6 bar): 5.1 - 5.7 m³/h
- Method of conditioning air: activated charcoal filtered air conditioned to about 50% ± 20% relative humidity and 22°C ± 2°C.
- System of generating particulates/aerosols: For each concentration, a respective constant amount of the test substance will be supplied to a two component atomizer by means of a metering pump. The aerosol was generated with compressed air into the inhalation system.
- Temperature, humidity, pressure: 21.7 - 24.1°C, 39.3 - 63.8%, positive pressure
- Air change rate: about 67 times / hour

TEST ATMOSPHERE
- Brief description of analytical method used: online by propane-calibrated total hydrocarbon analyzer (FID).
- Samples taken from breathing zone: yes

Details on mating procedure:

- M/F ratio per cage: 1: 1
- Length of cohabitation: overnight for a maximum of 2 weeks
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentrations of the inhalation atmospheres were analyzed online by propane-calibrated total hydrocarbon analyzer (FID). By means of response factor provided by the manufacture, the measured concentration of propane in each test group less the background concentration, was converted to concentration of the test substance. To verify the correctness of the FID measurement, absorption samples were drawn from the atmospheres. The absorption samples were analyzed by gas chromatography in all test groups. Daily means were calculated based on three measured samples per concentration and exposure. From the daily mean values of each concentration, mean concentrations and standard deviations for the entire study were derived. In these groups, the constancy of concentrations in the inhalation systems in the chambers were continuously monitored using total hydrocarbon analyzers. The control group was analyzed on three days during the exposure period. The analyses were carried out at the Inhalation and Analytical Chemistry Laboratory of the Experimental Toxicology and Ecology of BASF SE.

Duration of treatment / exposure:

6 h /day

Frequency of treatment:

- males: daily (7 days per week); male animals were sacrificed on study day 29 (28 days exposure) after the beginning of the administration, and examined
- females: daily (7 days per week); females were allowed to litter and rear their pups until day 4 after parturition, after sacrifice and necropsy of the pups on PND 4 the parental female animals were exposed to the respective concentrations of test substance for 6 hours a day total 4 exposure days on 4 consecutive days before scheduled killing(study day 51);
(exception: no exposure on the day of FOB / MA)

Details on study schedule:

screening test

Remarks:

Doses / Concentrations:
25, 75, 225 mg/m³
Basis:
other: target concentration

Remarks:

Doses / Concentrations:
20.6, 72.1, 236.3 mg/m³
Basis:
analytical conc.

Remarks:

Doses / Concentrations:
21.5, 86.0, 301.0 mg/m³
Basis:
nominal conc.

No. of animals per sex per dose:

10

Control animals:

other: control group was exposed to conditioned air

Details on study design:

- Dose selection rationale: The concentrations were based on available data.

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: The clinical condition of the test animals was recorded once during the pre-exposure period and on non-exposure days and at least 3 times (before, during and after exposure) on exposure days. During exposure only a group wise examination was possible. Abnormalities and changes were documented daily for each animal.
A check for moribund or dead animals was made twice daily on working days or once daily (Saturday, Sunday or on public holidays). If animals were in a moribund state, they were sacrificed and necropsied.


DETAILED CLINICAL OBSERVATIONS: Yes
All animals were subjected to detailed clinical observations outside their cages once before the beginning of the administration period (day 0) and subsequently once a week (in the morning). For observation, the animals were therefore be removed from their cages and placed in a standard arena (50 x 37.5 x 25 cm). The scope of examinations and the scoring of the findings that are observed was based on the current index of findings in PDS ToxData and includes but is not limited to the following parameters listed: abnormal behavior in “handling”, fur, skin, posture, salivation, respiration, activity/arousal level, tremors, convulsions, abnormal movements, gait abnormalities, lacrimation, palpebral closure, exophthalmos, assessment of the feces discharged during the examination (appearance/consistency), assessment of the urine discharged during the examination, pupil size.


BODY WEIGHT: Yes
In general, the body weight of the male and female parental animals was determined once a week at the same time of the day (in the morning) until sacrifice. The body weight change of the animals was calculated from these results. The following exceptions are notable for the female animals: During the mating period the parental females were weighed on the day of positive evidence of sperm (GD 0) and on GD 7, 14 and 20. Females with litter were weighed on the day of parturition (PND 1) and on PND 4. After the pups were sacrificed the females were exposed for 4 consecutive days. The F0 females were weight once before the exposure period and once on the last exposure day.
Females without positive evidence of sperm, without litter or waiting for necropsy, were weighed weekly. These body weight data were solely used for the calculations of the dose volume.


FOOD CONSUMPTION:
Food consumption was determined once a week for male and female parental animals, with the following exceptions:
Food consumption was not determined after the 2nd premating week (male parental animals) and during the mating period (male and female F0 animals). Food consumption of the F0 females with evidence of sperm was determined on gestation days (GD) 0 - 7, 7 - 14, and 14 - 20. Food consumption of F0 females, which gave birth to a litter was determined on PND 1 - 4. Food consumption of the females during the 4 exposure days after necropsy of the pups.
Food consumption was not determined in females without positive evidence of sperm during gestation periods and in females without litter during lactation period.


WATER CONSUMPTION: No

OTHER: hematology, clinical chemistry, neurobehavioural examination

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no

PARAMETERS EXAMINED
The following parameters were examined in [F1 ] offspring: number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain (pups were weighed on the day after birth (PND 1) and on PND 4), clinical observations

GROSS EXAMINATION OF DEAD PUPS: yes, for external and internal abnormalities

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals (male animals were sacrificed on study day 29 (28 days exposure) after the beginning of the administration)
- Maternal animals: All surviving animals (females were allowed to litter and rear their pups until day 4 after parturition, after sacrifice and necropsy of the pups on PND 4 the parental female animals were exposed to the respective concentrations of test substance for 6 hours a day total 4 exposure days on 4 consecutive days before scheduled sacrifice (study day 51)).

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations.

HISTOPATHOLOGY / ORGAN WEIGHTS:

The following weights were determined in all animals sacrificed on schedule: anesthetized animals, epididymides, testes;
The following weights were determined in 5 animals per sex/test group sacrificed on schedule (females with litters only, same animals as used for clinical pathological examinations): adrenal glands, brain, heart, kidneys, liver, lung, spleen, thymus.

Histopathological assessment: all gross lesions, adrenal glands, aorta, bone marrow (femur), brain, cecum, cervix, coagulating glands, colon, duodenum, eyes with optic nerve, esophagus, extraorbital lacrimal glands, epididymides, femur with knee joint, heart, ileum, jejunum (with Peyer’s patches), kidneys, larynx, liver, lungs, lymph nodes (tracheobronchial, mediastinal and mesenteric), mammary gland (male and female), nose (nasal cavity), ovaries, oviducts, pancreas, parathyroid glands, pharynx, pituitary gland, prostate gland, rectum, salivary glands (mandibular and sublingual), sciatic nerve, seminal vesicles, skeletal muscle, spinal cord (cervical, thoracic and lumbar cord), spleen, sternum with marrow, stomach (forestomach and glandular stomach), testes, thymus, thyroid glands, trachea, urinary bladder, uterus, vagina; special attention is given on stages of spermatogenesis in the testes.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring were sacrificed at PND 4

GROSS NECROPSY
All pups were examined externally and eviscerated; their organs were assessed macroscopically.
All stillborn pups and all pups that died before PND 4 were examined externally, eviscerated and their organs were assessed macroscopically.
All pups without notable findings or abnormalities were discarded after their macroscopic evaluation. Animals with notable findings or abnormalities were evaluated on a case-by-case basis, depending on the type of finding.

Statistics:

Body weight and body weight change (parental animals and pups; for the pup weights, the litter means were used), gestation days, implantation sites, pups delivered, life pups day x: DUNNETT test (two-sided); Male and female mating indices, male and female fertility indices, gestation index, females delivering, females with liveborn pups, females with stillborn pups, females with all stillborn pups: FISHER'S EXACT test (one-sided); Mating days until day 0 pc, viability index: WILCOXON test (one-sided) with BONFERRONI-HOLM adjustment; weight parameters (determined in pathology): KRUSKAL-WALLIS and WILCOXON test (two-sided)

Reproductive indices:

For the males, mating and fertility indices were calculated for F1 litters according to the following formulas:

Male mating index (%) = (number of males with confirmed mating* / number of males placed with females) x 100
* defined by a female with vaginal sperm or with implants in utero

Male fertility index (%) = (number of males proving their fertility* / number of males placed with females) x 100
* defined by a female with implants in utero

For the females, mating, fertility and gestation indices were calculated for F1 litters according to the following formulas:

number of females mated*
Female mating index (%) = (number of females mated* / number of females placed with males) x 100
* defined as the number of females with vaginal sperm or with implants in utero


Female fertility index (%) = (number of females pregnant* / number of females mated**) x 100
* defined as the number of females with implants in utero
** defined as the number of females with vaginal sperm or with implants in utero

Gestation index (%) = (number of females with live pups on the day of birth / number of females pregnant*) x 100
* defined as the number of females with implants in utero

Offspring viability indices:

The total number of pups delivered and the number of liveborn and stillborn pups were noted, and the live birth index was calculated for F1 litters according to the following formula:

Live birth index (%) = (number of liveborn pups at birth / total number of pups born) x 100

The implantations were counted and the postimplantation loss (in %) was calculated according the following formula:

Postimplantation loss (%) = [(number of implantations – number of pups delivered) / number of implantations] x 100

 

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

see below

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Test group 3 (target concentration 225 mg/m³)

• Lower mean body weights F0 females on gestation days (GD) 7
(- 5.7 %, p < 0.05) and 14 (5.2 %, p < 0.05)
• Reduced body weight gain of the F0 male animals during premating period (-12.9 g
versus -1.8 g in the control, p < 0.05)
• Lower mean body weight gain of F0 female animals from gestation day 0 to 7 (-37 %,
p < 0.01)

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Test group 3 (target concentration 225 mg/m³)
• Decreased food consumption during pre-mating (p < 0.05)
o in male animals between study days 0-7 (-9 %) and 7-14 (-11%)
o in female animals between study days 0-7 (-7%) and 7-12 (-5%).
• Decreased food consumption during gestation of parental female animals
o between gestation day 0-7 (-11%, p < 0.01)
o between gestation day 7-14 (-11%, p < 0.01)
o between gestation day 14-21 (-11%, p < 0.01)

Test group 2 (target concentration 75 mg/m³)
• Decreased food consumption during gestation
o in female animals between study days 0 - 7 (-9%, p < 0.05).

Test group 1 (target concentration 25 mg/m³)
• No adverse effect observed

Food efficiency:

not specified

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Immunological findings:

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test group 3 (target concentration 225 mg/m³)
Nasal cavity, level I
• Degeneration /regeneration transitional epithelium in 2/10 male (graded minimal) and
8/10 female animals (graded minimal in 7/10 and slight in 1/10 animals)
• Metaplasia, squamous, transitional epithelium graded minimal in 1/10 male and 3/10
female animals.
• Degeneration /regeneration respiratory epithelium in 2/10 male animals graded
minimal.
Nasal cavity, level II
• Degeneration /regeneration olfactory epithelium graded minimal in one female animal.

Test group 2 (target concentration 75 mg/m³)
Nasal cavity, level I
• Degeneration /regeneration transitional epithelium in one female animal (graded
minimal)

Test group 1 (target concentration 25 mg/m³)
• No adverse effect observed

Larynx
Treatment – related findings in the larynx were epithelial alteration characterized by minmal
to slight modification of epithelial cells (i.e., three to four cell layers, focally flattened and
stratified) indicating beginning metaplastic transformation and minimal to slight squamous
metaplasia (characterized by three to four cell layers of flattened, stratified epithelium with no
signs of keratinization and only affecting the epiglottis when graded minimal and up to five
cell layers of flattened, stratified epithelium, occasionally minimal focal keratinization,
with/without focal desquamation of superficial cells when graded slight) (Kaufmann et al.,
2009).

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

Clinical observations
In the high concentration (225 mg/m³) group food consumption during pre-mating was
significantly decreased in male animals between study days 0 to 7 (-9 %) and 7 to 14 (-11%)
as well as in female animals between study days 0 and 7 (-7%) and 7 and 12 (-5%). These
findings were considered to be substance-related.
During gestation the food consumption in the high concentration F0 females (225 mg/m³) was
significantly decreased during whole gestation period. It was from gestation day (GD) 0 to 7
(-11%), from GD 7 to 14 (-11%) and from GD 14 to 20 (-11%). It was significantly decreased
in the mid concentration group (75 mg/m³) between GD 0 to 7 (-9%).
No other findings were observed for male and female animals in test group 1 and 2 (25 and
75 mg/m³).

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

not specified

Reproductive performance:

no effects observed

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS):
There were no test substance-related or spontaneous mortalities in any of the groups. During the pre-exposure period, the pre-mating and the mating period the animals showed no clinical signs and findings different from normal. During the post-mating day the male animals showed no clinical signs and findings different from normal. During the gestation period six female animals of the control group, eight female animals of the low concentration (25 mg/m³), eight female animals of the mid concentration (75 mg/m³) and seven female animals of the high concentration (225 mg/m³) showed vaginal discharge. As this finding was distributed evenly in the controls and the groups exposed to the test substance, it was considered to be treatment-related (head-nose exposure), but not substance-related. During the lactation period the female animals showed no clinical signs and findings different from normal. During the 4-day exposure period of the females after the pups were sacrificed the animals showed no clinical signs and findings different from normal.
The detailed clinical observations did not reveal any abnormalities in male and female animals of all test groups.


BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS):
Premating period: The mean body weights of the test substance exposed male and female animals were not statistically significantly different from the control group 0.
Mating period: The mean body weights of the test substance exposed male animals were not statistically significantly different from the control group 0, although the mean body weights of the males test group 3 seems to be slightly lower than those of other groups.
Gestation:
The mean body weights of group 3 females were slightly lower than the control on gestation days (GD) 7 and 14 (p < 0.05).
Lactation:
The mean body weights of F0 female animals were not statistically significantly different from the control group 0 during lactation period.
After PND 4:
The mean body weights of F0 female animals after PND 4 were not statistically different to the controls.

Body weight change / during the exposure period:
The body weight change of the F0 male animals of the high concentration group (225 mg/m³) was statistically significantly lower than the controls during premating period (-12.9 g, p < 0.05) at the beginning of the mating period. This effect was diminished in course of the exposure and was not observed any further in the second week of the pre-mating period, as well as during mating period.
During pre-mating period, the mean body weight changes of the female animals were not statistically different when compared with the control.
From gestation day 0 to 7, the mean body weight change of F0 female animals was significantly lower (-37 %, p < 0.01) than the controls. This effect was not observed any further at later time points. The body weight changes were in other test groups not significantly different to the control.

In high concentration (225 mg/m³) food consumption during pre-mating was significantly decreased in male animals between study days 0 - 7 (-9 %) and 7 - 14 (-11%) as well as in female animals between study days 0 - 7 (-7%) and 7 - 14 (-5%). These findings were considered to be substance-related. During the gestation the food consumption in the high concentration (225 mg/m³) was significantly decreased in female animals during the whole period (-11%) and in the mid concentration (75 mg/m³) was significantly decreased in female animals between study days 0 - 7 (-9%). No other findings were observed for male and female animals in test group 1 and 2 (25 and 75 mg/m³).

The effects on food consumption and body weight / body weight change might be an indirectly consequence of the local irritation in the nasal cavity. They are therefore considered to be treatment-related.


REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS):
Male reproduction data:
For all F0 parental males, which were placed with females to generate F1 pups, copulation was confirmed. Thus, the male mating index was 100% in all groups including the controls. Fertility was proven for most of the F0 parental males within the scheduled mating interval for F1 litter. One low-concentration male (25 mg/m³ - No. 17) did not generate implants in the mated female (No. 117). The animal No. 17 showed some changes in testes and epididymides, which might have impaired fertility, even if animals with similar findings in the same group did produce offspring. Even though, the male fertility index ranged between 90% and 100% without showing any relation to exposure concentration. This reflects the normal range of biological variatio inherent in the strain of rats used for this study.

Female reproduction and delivery data:
The female mating index calculated after the mating period for F1 litter was 100% in all test groups. The mean duration until sperm was detected (GD 0) varied between 2.1 and 3.1 days without any relation to exposure concentrations. All sperm positive rats delivered pups or had implants in utero with the following exception: Low-dose female No. 117 (mated with male No. 17) did not become pregnant. The fertility index varied between 90% in test group 1, 100 % test groups 2, 3 and in control. These values reflect the normal range of biological variation inherent in the strain of rats used for this study. The non-pregnant female did not have any relevant gross lesions. The mean duration of gestation was similar in all test groups (i.e. between 21.8 and 22.1 days). The gestation index were 100% in all test groups as well as control.

Implantation was not affected by the treatment since the mean number of implantation sites was comparable between all test substance-treated groups and the controls, taking normal biological variation into account (10.4 / 11.9 / 11.9 and 10.5 implants/dam in test groups 0 - 3 (0, 25, 75 and 225 mg/m³). There were no statistically significant differences in post-implantation loss between the groups (6.5% / 7.7% / 7.2% / 9.7%), and the mean number of F1 pups delivered per dam remained unaffected (9.7 / 11.0 / 11.0 and 9.4 pups/dam at 0, 25, 75 and 225 mg/m³).
The rate of liveborn pups was also not affected by the test substance, as indicated by live birth indices of 100% (test group 1, 2 and control), 97.9% (test group 3). Two stillborn pups (2.1 %) were only in test group 3 animals. However, this is within the normal range of biological variation inherent in the strain of rats used for this study.

The sex distribution and sex ratios of live F1 pups on the day of birth and PND 4 did not show substantial differences between the control and the test substance-treated groups; slight differences were regarded to be spontaneous in nature.


ORGAN WEIGHTS (PARENTAL ANIMALS):
When compared with control group 0 (=100%), the following mean relative weights were significantly increased or decreased in one or more test groups: males: epididymides (group 1: 88%, group 3: 90%), spleen (group 1: 137%), testes (group 2: 93%, group 3: 92%); females: liver (group 3: 92%)
When compared with control group 0 (=100%), the following mean absolute weights were significantly increased or decreased in one or more test groups: males: epididymides (group 1: 88%); females: thymus (group 1: 129%)
Weight changes in thymus, spleen and liver were regarded as incidental as no dose response relationship and/or histopathological correlates were detected. The decrease in epididymides and testes weights was considered to be treatment-related even in the absence of a clear dose response relationship or, in the case of the testes, no changes in relative weights as there was a histopathological correlate (see histopathology).
All other mean absolute or relative weight parameters did not show significant differences when compared to the control group 0.


GROSS PATHOLOGY (PARENTAL ANIMALS):
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.


HISTOPATHOLOGY (PARENTAL ANIMALS):
Treatment-related findings were observed in level I of larynx, and level I and II of nasal cavity, in males and females as well as in epididymides and testes in male animals:

Larynx: Treatment – related findings in the larynx were epithelial alteration characterized by slight modification of epithelial cells (i.e., three to four cell layers, focally flattened and stratified) indicating beginning metaplastic transformation and minimal to slight squamous metaplasia (characterized by three to four cell layers of flattened, stratified epithelium with no signs of keratinization and only affecting the epiglottis when graded minimal and up to five cell layers of flattened, stratified epithelium, occasionally minimal focal keratinization, with / without focal desquamation of superficial cells when graded slight) (Kaufmann et al., 2009).
Laryngeal epithelial alteration were regarded as non-adverse according to the literature (Kaufmann W. et al., Exp Toxicol Pathol, 2009 Nov, 61(6): 591-603).

Nasal cavity, level I: Degeneration / regeneration of transitional and respiratory epithelium was characterized by variable vacuolation, presence of few apoptotic bodies, minimal infiltrates of inflammatory cells, increased size and basophilia of nuclei and minimal disorganization of cells. Metaplasia, squamous was characterized by flattened epithelial cells with variably present minimal keratinization.
Nasal cavity, level II: One male test group 3 (225 mg/m³) animal (No 134) showed minimal degeneration / regeneration of the olfactory epithelium, in level I it showed degeneration/regeneration of the transitional epithelium).
Degeneration / regeneration of nasal epithelia was regarded as adverse.

Testes: Tubular degeneration was observed in a higher incidence in treated test groups. This finding was characterized by randomly affected (not stage specific) tubules with sloughed spermatogenic cells, vacuolation of the spermatogenic epithelium or missing germ cell layers.

Epididymides: Debris in the epididymides was characterized by sloughed spermatogenic cells and noted with increased incidence in treated animals.

This effect in the testes is likely due to the technical exposure scenario (heat [e.g. Brock WJ et al., 1996] and possibly evading movements by the animals leading to pressing backwards in tubes), rather than being a direct effect of the test substance as this finding is also found in control animals. These findings did generally not affect fertility with one possible exception in one group 1 male animal (No. 17). All findings in testes and epididymides were assessed as treatment - related and adverse but not substance-related.

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.


OTHER FINDINGS (PARENTAL ANIMALS): see also "Repeated dose toxicity"

Key result

Dose descriptor:

NOAEC

Remarks:

(reproductive and developmental parameters)

Effect level:

236.3 mg/m³ air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: highest dose tested, no adverse effects observed

Remarks on result:

other: Generation: P/F1

Key result

Dose descriptor:

NOAEC

Remarks:

(local)

Effect level:

20.6 mg/m³ air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: histopathology lesions of nasal epithelia

Dose descriptor:

NOAEC

Remarks:

(systemic)

Effect level:

236.3 mg/m³ air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Clinical signs:

no effects observed

Mortality / viability:

mortality observed, non-treatment-related

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

Ophthalmological findings:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Urinalysis findings:

no effects observed

Sexual maturation:

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

Behaviour (functional findings):

no effects observed

Developmental immunotoxicity:

no effects observed

VIABILITY (OFFSPRING):
The mean number of delivered F1 pups per dam and the rates of liveborn were evenly distributed about the groups. The respective values reflect the normal range of biological variation inherent in the strain used in this study. The low rate of stillborn pups in test group 3 was within the biological variation of this stain and was considered to be not related to the test substance.
The viability index indicating pup mortality during lactation (PND 0 - 4) varied between 98.5 % (test group 2), 99.1 % (test group 1) and 100% (test group 3 and control) without showing any association to the treatment.
The sex distribution and sex ratios of live F1 pups on the day of birth and PND 4 did not show substantial differences between the control and the test substance-treated groups; slight differences were regarded to be spontaneous in nature.

CLINICAL SIGNS (OFFSPRING):
There were no test substance related adverse clinical signs observed in any of surviving F1 generation pups of the different test groups.

BODY WEIGHT (OFFSPRING):
No test compound-related influence on F1 pup body weights and pup body weight change were noted in all test groups.

Two female runts were seen in the control and two female runts in test group 3 (225 mg/m³). As there were as many runts in the control as in test group 3, this finding was considered to be incidental and not substance-related.

GROSS PATHOLOGY (OFFSPRING):
No findings were observed at gross necropsy in any male or female pups of all test groups. One male and one female pup of test group 2 (75 mg/m³) could not be assessed because they had been cannibalized. Because no pup was cannibalized in high concentration groups, this finding is considered as incidental.

Key result

Dose descriptor:

NOAEC

Generation:

F1

Effect level:

>= 236.3 mg/m³ air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Developmental toxicity

Reproductive effects observed:

not specified

Conclusions:

Under the conditions of this OECD 422 combined repeated dose toxicity study with the
reproduction/developmental toxicity screening test in Wistar rats the following NOAEC (no
observed adverse effect concentration) were determined for dibutylethanolamine after
inhalation exposure:
The NOAEC for local toxicity at the respiratory tract was 20.6 mg/m³ for the F0 females
and males based on histological findings in nasal cavity.
The NOAEC for general, systemic toxicity was 72.1 mg/m³ for the F0 females and males.
However, it has to be noted, that the only finding for systemic toxicity was the lower body
weight gain, which was considered to be secondary to local toxicity. No other systemic
toxicity was evident by examination of hematology, clinical chemistry and histopathology of
other organs.
The NOAEC for reproductive performance and fertility was 236.3 mg/m³ for the F0
parental rats.
The NOAEC for developmental toxicity in the F1 offspring was 236.3 mg/m³.

Executive summary:

DISCUSSION

During the exposure period, the target concentrations were met well and were maintained as

constant and stable as could be provided with the presented generation techniques in the

concentration range tested.

No relevant clinical signs of toxicity were observed in exposed parental animals during the

premating and mating phases. However, food consumption was reduced slightly in high

concentration males during premating, in high concentration females during premating and

gestation period. In females of mid concentration group, food consumption was reduced

during gestation days 0 and 7. Consistently to the reduced food consumption, slightly

reduced body weight gain was observed. During gestation stage, mean body weights of

group 3 females were significantly reduced on gestation day 7 and 14. These unspecific

findings might be an indirect consequence of the local irritation in the nasal cavity. They are

considered to be treatment-related and adverse.

Generally, no toxicologically relevant reproductive including fertility or developmental

difference was observed between animals exposed to measured concentrations of 20.6, 72.1

and 236.3 mg/m³ and controls. These concentrations of dibutylethanolamine did not

adversely impact the reproduction of these rats, nor did treatment impact delivery and pup

viability. Furthermore, none of the F1 generation pups showed any evidence of

developmental toxicity in response to dibutylethanolamine.

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

dose of the compound of 225 mg/m3.

Regarding pathology, target organs were the level I of the larynx, and level I and II of the

nasal cavity, in males and females as well as epididymis and testis in male animals.

Laryngeal epithelial alteration in many male and female animals of all treated test groups (25,

75, 225 mg/m³) and minimal to slight squamous metaplasia without inflammation in male and

female animals of test groups 2 and 3 (75 and 225 mg/m³) were regarded as non-adverse

according to the literature (Kaufmann et al., 2009).

Degeneration / regeneration of nasal epithelia in level I in 2/10 males (transitional and/or

olfactory) and 8/10 females (transitional) as well as squamous metaplasia of transitional

epithelium in one male and 3/10 female animals of test group 3 (225 mg/m³) and in one

female of test group 2 (transitional) (75 mg/m³) as well as degeneration / regeneration of the

olfactory epithelium in level II noted in one test group 3 (225 mg/m³) female was regarded as

adverse.

Tubular degeneration of the testis and resulting debris in the epididymides were observed in

control and treated animals of all test groups. These findings did generally not affect fertility

with one possible exception in one group 1 male animal (No. 17). The incidence of these

findings was, however, higher in treated animals correlating with decreased absolute and

relative epididymis weights and absolute testis weights. (Tubular degeneration: control [4/10]

test group 1 [6/10] test group 2 [7/10] test group 3 [8/10]; Debris in the epididymides: control

[2/10] test group 1 [4/10] test group 2 [4/10] test group 3 [7/10]). The findings in testis and

epididymides have been observed frequently in head-nose exposed control animals in the

past. The historical control data are presented in part III. In one case the incidence was as

high as 100%. The grading of this finding ranged from minimal (grade 1) to extreme (grade

5). As the control animals were exposed only to clean conditioned air, this finding was

considered to be attributed to the exposure condition, but not to any substances. In the

literature, this effect in the testis was discussed as an effect associated with technical

exposure scenario (heat [e.g. Brock WJ et al., 1996] and possibly evading movements by the

animals leading to pressing backwards in tubes), rather than being a direct effect of the test

substance.

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.

Effect on fertility: via oral route

Endpoint conclusion:

no study available

Effect on fertility: via inhalation route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

236.3 mg/m³

Study duration:

subacute

Species:

rat

Quality of whole database:

Available data is reliable and sufficient for assessment.

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

A GLP-compliant investigation of the toxicological effects resulting from repeated inhalation exposure of 2-dibutylaminoethanol to Wistar rats was performed following OECD guideline 422 (BASF SE, 2013). Groups of ten male and ten female rats (F0 animals) per test group were exposed nose-only to a dynamic atmosphere of 2-dibutylaminoethanol aerosol for 6 hours per day on each day. The duration of treatment covered a 2-week pre-mating and 2-week mating period in both sexes, 1 day post-mating in males, and the entire gestation period of the females. After the lactation period and after necropsy of the pups total all parental females were exposed to the test substance on 4 consecutive days. The target concentrations were 25, 75 and 225 mg/m³ (measured concentrations: 20.6, 72.1, 236.3 mg/m³). A concurrent control group was exposed to conditioned air. The test did not cause any adverse effects with regard to reproductive and developmental parameters, but only transiently reduced food consumption, body weight and body weight gain. In histopathology lesions of nasal epithelia was observed in animals exposed to 72.1 and 236.3 mg/m³ test substance. Thus, concerning systemic toxicity, reproductive and developmental parameters the NOAEC for 2-dibutylaminoethanol was determined to be 236.3 mg/m³ for male and female animals. Considering histopathological changes the NOAEC for local effects was 20.6 mg/m³.

Effects on developmental toxicity

Description of key information

- oral, (OECD 414), rat: NOAEL (maternal and prenatal toxicity) = 60 mg/kg bw/day

- inhalative, screening (OECD422), rat: NOAEC(reproductive and developmental parameters; males and females) = 236.3 mg/m³, NOAEC(systemic; males and females) = 236.3 mg/m³ (highest dose tested), NOAEC(local, males and females) = 20.6 mg/m³

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

05Mar2019 to 27Nov2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

25 Jun 2018

GLP compliance:

yes

Limit test:

no

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: room temperature
- 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 oily preparation

OTHER SPECIFICS
- Physical state/ appearance: Liquid/ yellowish, clear
- Homogeneity: Given

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Age at study initiation: 10-12 weeks
- Weight at study initiation: 163.5 - 218.2
- Housing: During the study period, the rats were housed individually in Polycarbonate cages type III
supplied by TECNIPLAST, Hohenpeißenberg, Germany and Becker & Co., Castrop-Rauxel,
Germany (floor area about 800 cm²). Individual housing is preferred over group housing as the
close individual monitoring of food and water consumption as well as of clinical signs of toxicity
in pregnant animals is crucial during this period of increased sensitivity. In addition, the control
for signs of abortion or fetal loss can only be done in a reliable fashion with single-housed
animals
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: GD 0 - GD 6

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

-IN-LIFE DATES; From: To:
Study initiation date: 04 Mar 2019
Experimental starting date:(arrival of 1st cohort of test animals) 05 Mar 2019
Supply of animals/ Beginning of acclimatization (GD 0) Beginning oftreatment/
End of acclimatization (GD 6)
End of treatment (GD 19)
Blood sampling / Sacrifice of the animals (GD 20)
1st cohort 05 Mar 2019 11 Mar 2019 24 Mar 2019 25 Mar 2019
2nd cohort 06 Mar 2019 12 Mar 2019 25 Mar 2019 26 Mar 2019
3rd cohort 07 Mar 2019 13 Mar 2019 26 Mar 2019 27 Mar 2019
Experimental completion date: (draft to QAU*) 27 Nov 2019
* QAU = Quality Assurance Unit

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test substance preparations were prepared at the beginning of the administration period
and thereafter at intervals, which took into account the period of established stability. The
preparations were kept at room temperature.
For the test substance preparations, the specific amount of test substance was weighed,
topped up with corn oil in a graduated flask and intensely mixed with a magnetic stirrer until it
was completely dissolved.

TEST GROUPS AND DOSES
Test group Dose [mg/kg bw/d] Concentration [g/100 mL] Volume [mL/kg bw/d] .
0: 0 [mg/kg bw/d] 0 [g/100 mL] 4a)
1: 20 [mg/kg bw/d] 0.50[g/100 mL] 4b)
2: 60 [mg/kg bw/d] 1.50 [g/100 mL] 4b)
3: 200[mg/kg bw/d] 5.00 [g/100 mL] 4b)
a) corn oil
b) Test substance preparations in corn oil

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analytical verifications of the stability of the test substance in corn oil over a period of 7 days
at room temperature had been verified prior to the start of the study. Samples of the test
substance preparations were sent twice (at the beginning of administration and after the end
of study to the analytical laboratory for verification of the concentrations.

Details on mating procedure:

Impregnation procedure: purchased timed pregnant

The animals were paired by the breeder (“time-mated”); the day of evidence of mating
(= detection of vaginal plug/sperm) was referred to as GD 0. The animals arrived on the same
day (GD 0) at the experimental laboratory. The following day was designated as “GD 1”. The
animals were acclimated to the laboratory conditions between start of the study (beginning of
the experimental phase) and first administration (GD 6).

Duration of treatment / exposure:

The test substance preparations were administered to the animals once a day orally by gavage,
from implantation to one day prior to the expected day of parturition (GD 6 to GD 19), always
at approximately the same time in the morning. The animals of the control group were treated
with the vehicle (corn oil) in the same way. The volume administered each day was 4 mL/kg
body weight. The calculation of the administration volume was based on the most recent
individual body weight.

Frequency of treatment:

The test substance preparations were administered to the animals once a day orally by gavage,
from implantation to one day prior to the expected day of parturition (GD 6 to GD 19), always
at approximately the same time in the morning.

Dose / conc.:

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

Remarks:

control

Dose / conc.:

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

Remarks:

low dose

Dose / conc.:

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

Remarks:

mid dose

Dose / conc.:

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

Remarks:

high dose

No. of animals per sex per dose:

25

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
In a range-finding study (BASF project No. 10C0286/05S039), Dibutylethanolamine was
administered by gavage to groups of five non-pregnant female Wistar rats for 28-days at dose
levels of 0, 100 and 300 mg/kg body weight/day (mg/kg bw/d). Corn oil served as vehicle. A
dose of 300 mg/kg bw/d induced neurobehavioral changes in females from study day 10
onwards. Clinical findings consisted of tremors in all females, twitching in three and lateral
position in two out of five females. Furthermore, mortalities were seen in each one female on
study days 14 and 17. From study day 18 onwards, this dose level was reduced to 200 mg/kg
bw/d. In females, clonic convulsions occurred still in one animal on study day 18. Twitching
was observed in up to two females on study days 19 and 20. All mentioned findings occurred
between 15 min and 2 hours after administration but not thereafter. All other parameters were
not affected. At the lowest dose of 100 mg/kg bw/d, no relevant findings were observed.

In a rat maternal toxicity study (BASF project No. 10R0286/05R038), Dibutylethanolamine
was administered orally by repeated gavage to up to seven pregnant Wistar rats per test group
from gestation day (GD) 6 through GD 19. Doses of 200 and 70 mg/kg bw/d were selected
based on the findings observed in the above-mentioned range-finding study. At 200 mg/kg
bw/d, signs of systemic toxicity were observed. They consisted of a statistically significant
reduction of water (GD 6-8: 25% below control) and food (GD 6-13: 31%; GD 6-19: 12% below
control) consumption, a statistically significant decrease in body weight change (including a
body weight loss during GD 6-8: -1.4 g vs. 6.9 g in control) and a decrease in net weight change
(27% below control). At 70 mg/kg bw/d, no relevant, adverse findings were observed.
Based on the findings presented in the two studies above, 200 mg/kg bw/d was selected as
high-dose in the following prenatal development toxicity study to show some maternal toxicity
as stated in the respective OECD test guideline No. 414.
The following dose levels were chosen for the present prenatal
developmental toxicity study in Wistar rats:
20 mg/kg body weight/day: as low-dose level
60 mg/kg body weight/day: as mid-dose level
200 mg/kg body weight/day: as high-dose level #
The oral route was selected since this has proven to be suitable for the detection of a toxicological hazard.

# Because of severe clinical findings, such as twitching, convulsions, tremor and/or abdominal
position after treatment, in almost all animals, the high-dose group (200 mg/kg bw/d) was
sacrificed prematurely on GD 13/12/11 of the study. The animals were discarded without further
examinations. No organs or tissues were fixed, but the pregnancy status was evaluated.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

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

BODY WEIGHT: Yes
All animals were weighed on GD 0, 1, 3, 6, 8, 10, 13, 15, 17, 19 and 20. The body weight
change of the animals was calculated based on the obtained results.
Corrected (net) body weight gain
Furthermore, the corrected body weight gain was calculated after terminal sacrifice (terminal
body weight on GD 20 minus weight of the unopened uterus minus body weight on GD 6).

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
The consumption of food was recorded for the intervals GD 0-1, 1-3, 3-6, 6-8, 8-10, 10-13,
13-15, 15-17, 17-19 and 19-20.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
-The consumption of water was recorded for the intervals GD 0-1, 1-3, 3-6, 6-8, 8-10, 10-13,
13-15, 15-17, 17-19 and 19-20.


POST-MORTEM EXAMINATIONS: Yes / No / No data
- Sacrifice on gestation day 20
- Organs examined: Necropsy

OTHER:
After the dams had been sacrificed, they were necropsied and assessed for gross pathology.
Thyroid glands (including parathyroid glands) were sampled.

Thyroid hormones
The concentrations of TSH were determined:
Total triiodothyronine (T3), Total thyroxine (T4), Thyroid stimulating hormone (TSH)
The following weights were determined in all dams sacrificed on schedule:
Thyroid glands (with parathyroid glands) (fixed)
Thyroid glands were weighed together (left and right).

Ovaries and uterine content:

After the dams had been sacrificed, they were necropsied and assessed for gross pathology.
Thyroid glands (including parathyroid glands) were sampled.
The uteri and the ovaries were removed and the following data were recorded:
Weight of the unopened uterus, Number of corpora lutea,
Number and distribution of implantation sites classified as: Live fetuses,
Dead implantations:
Early resorptions (only decidual or placental tissues visible or according to SALEWSKI
from uteri from apparently non-pregnant animals and the empty uterus horn in the
case of single horn pregnancy)
Late resorptions (embryonic or fetal tissue in addition to placental tissue visible)
Dead fetuses (hypoxemic fetuses which did not breathe spontaneously after the
uterus had been opened)
After the weight of the uterus had been determined, all subsequent evaluations of the dams
and the gestational parameters (except of gross pathology including organ sampling and
weights) were conducted.

Fetal examinations:

At necropsy each fetus was weighed, sexed, and external tissues and all orifices were
examined macroscopically. The sex was determined by observing the distance between the
anus and the base of the genitalia.
Furthermore, the viability of the fetuses and the condition of placentas, umbilical cords, fetal
membranes, and fluids were examined. The placentas were weighed and their individual
weights were recorded.
Thereafter, the fetuses were sacrificed by a subcutaneous injection of pentobarbital
(Narcoren®; dose: 0.1 mL/fetus).
The anogenital distance (defined as the distance from the center of the anal opening to the
base of the genital tubercle) measurements were conducted, using a measuring ocular, on all
liveborn fetuses.
After these examinations, approximately one half of the fetuses per dam were eviscerated,
skinned and fixed in ethanol; the other half was placed in Harrison’s fluid for fixation.

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

Skeletal examination of the fetuses
The skeletons of the fetuses fixed in ethanol were stained according to a modified method of
KIMMEL and TRAMMELL. Thereafter, the skeletons of these fetuses were examined under a
stereomicroscope. After this examination the stained fetal skeletons were retained individually.

Statistics:

Simultaneous comparison of all dose groups with the control group using theDUNNETT-test
(two-sided) for the hypothesis of equal means.:
Water consumptiona), food consumptiona), body weight, body weight change, corrected body
weight gain (net maternal body weight change), carcass weight, weight of unopened uterus,
number of corpora lutea, number of implantations, number of resorptions, number of live fetuses,
proportions of preimplantation loss, proportions of postimplantation loss, proportions of resorptions,
proportion of live fetuses in each litter, litter mean fetal body weight, litter mean placental weight,
anogenital distance, anogenital index
Pairwise comparison of eachdose group with the control group using FISHER'S EXACT test
(one-sided) for the hypothesis of equal proportions: Female mortality, females pregnantat terminal
sacrifice, number of litters with fetal findings.
Pairwise comparison of each dose group with the control group using the WILCOXONtest
(one-sided) for the hypothesis of equal medians.

Indices:

Conception rate, pre-impantation loss, post-implantation loss

Historical control data:

yes

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

For almost all females of test group 3 (200 mg/kg bw/d) severe clinical findings were recorded
during the treatment period (within the 2-hour examination interval after treatment):
twitching after treatment in 23 females from GD 7 onwards,
convulsion after treatment in 6 females from GD 10 onwards,
tremor after treatment in 3 females on GD 9 and 11,
abdominal position after treatment in 17 females from GD 8 onwards.
As mentioned above, all females of test group 3 were sacrificed prematurely on GD 13/12/11.
Furthermore, most females (23 out of 25) of the high-dose group and 6 females of the middose
group showed transient salivation during the treatment period. Salivation occurred in the
respective animals within the 2-hour examination interval after treatment (i.e. 0-2h) and was
initially observed on GD 7 (200 mg/kg bw/d) or GD 18 (60 mg/kg bw/d). Salivation occurred
most probably due to the bad taste of the test substance, was assessed to be a local effect
and, therefore, considered as not adverse.
No clinical signs or changes of general behavior, which may be attributed to the test substance,
were detected in any female of test group 1 (20 mg/kg bw/d).
see attached document: summary of maternal clinical observation

Description (incidence):

Due to severe clinical findings in almost all animals of test group 3, the high-dose group of 200 mg/kg bw/d was sacrificed prematurely due to animal
welfare reasons on GD 13/12/11 ) of the study.
There were no further test substance-related or spontaneous mortalities in any females of test
groups 0-2 (0, 20 or 60 mg/kg bw/d).

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

see attached document: summary bw_bwc
From GD 8 onwards until premature sacrifice on GD 11-13 the mean body weights of the highdose
dams (200 mg/kg bw/d) were lower in comparison to the concurrent control group,
attaining statistical significance on GD 10 (-4%). The average body weight gain of the highdose
dams (200 mg/kg bw/d) was distinctly and statistically significantly reduced during GD 6-
10 showing a body weight loss during GD 6-8. These effects were assessed as
treatmentrelated and adverse.
In test group 2 (60 mg/kg bw/d) the mean body weights were generally comparable to the
concurrent control group. The average body weight gain was statistically significantly reduced
on GD 6-8 but recovered afterwards and was comparable to the control values again
throughout the remaining study period. If calculated for the entire treatment period (GD 6-19),
the mean body weight gain of the mid-dose dams was comparable to the concurrent control
group.
The mean body weights and the average body weight gain of the low-dose dams (20 mg/kg
bw/d) were generally comparable to the concurrent control group throughout the entire study
period.
Corrected (net) body weight gain
The corrected body weight gain of test groups 1 and 2 (20 and 60 mg/kg bw/d) revealed no
difference of any biological relevance to the corresponding control group. Moreover, mean
carcass weights remained unaffected by the treatment.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

see attached document: summary food consumption
The mean food consumption of the high-dose dams (200 mg/kg bw/d) was statistically
significantly reduced from GD 6 onwards until premature sacrifice on GD 11-13 (up to 31%
below control). This was assessed as treatment-related and adverse.
The mean food consumption of the mid- and low-dose dams (60 and 20 mg/kg bw/d) was
generally comparable to the concurrent control group throughout the entire study period.

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

effects observed, treatment-related

Description (incidence and severity):

see attached document; summary maternal water consumption
The mean water consumption of the dams in test group 3 (200 mg/kg bw/d) was statistically
significantly reduced from GD 6 onwards until premature sacrifice during GD 11-13 (up to 21%
in comparison to the concurrent control). This was assessed as treatment-related and adverse.
In test group 2 (60 mg/kg bw/d) the mean water consumption was statistically significantly
reduced during GD 6-10 (up to 12% below control) but recovered afterwards and was
comparable to the control values again until scheduled sacrifice on GD 20.
The mean water consumption of the dams in test group 1 (20 mg/kg bw/d) was comparable to
the concurrent control group throughout the entire study period.

Ophthalmological findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

see attached document: summary gravid uterine weitghts and bwc
The mean gravid uterus weights of the low- and mid-dose animals (20 and 60 mg/kg bw/d)
were not influenced by the test substance. The differences between these groups and the
control group revealed no dose-dependency and were assessed to be without biological
relevance.
Absolute and relative organ weights
All mean absolute and relative weight parameters did not show significant differences when
compared to the control group 0.

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

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.

Other effects:

no effects observed

Description (incidence and severity):

See attached document: Incidence of microscopic findings
Thyroid hormones
At gestation day 20, in dams of test groups 1 and 2 (20 and 60 mg/kg bw/d) no treatment related
alterations of T3, T4 and TSH were observed.
Gross lesions
No macroscopic findings of thyroid glands were noted in test groups 0 (control), 1 (20 mg/kg
bw/d) and 2 (60 mg/kg bw/d).

Number of abortions:

no effects observed

Pre- and post-implantation loss:

effects observed, non-treatment-related

Description (incidence and severity):

A 100% post-implantation loss was observed in one female each in test groups 1 and 2. In one lowdose
dam (20 mg/kg bw/d) one early resorption was recorded after staining the empty
uterus at C-section (i.e. pregnant by stain), whereas for one mid-dose dam (60 mg/kg bw/d)
9 early and 3 late resorptions were recorded (no viable fetuses). In the other dams of test
groups 1 and 2 post-implantation losses and number of resorptions were comparable to the
control. Since only one dam per test group was affected without relation to dose, these isolated
findings were regarded as incidental and not treatment-related.

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

see attached document: summary of reproduction data
There were no test substance-related and/or biologically relevant differences between the test
groups 0-2 in conception rate, in the mean number of corpora lutea and implantation sites or
in the values calculated for the pre- and post-implantation losses, the number of resorptions
and viable fetuses. All observed differences are considered to reflect the normal range of
fluctuations for animals of this strain and age.

Early or late resorptions:

effects observed, non-treatment-related

Description (incidence and severity):

In one lowdose dam (20 mg/kg bw/d) one early resorption was recorded after staining the empty
uterus at C-section (i.e. pregnant by stain), whereas for one dam in the mid-dose (60 mg/kg bw/d)
9 early and 3 late resorptions were recorded (no viable fetuses).

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Details on maternal toxic effects:

There were no test substance-related and/or biologically relevant differences between the test
groups 0-2 in conception rate, in the mean number of corpora lutea and implantation sites or
in the values calculated for the pre- and post-implantation losses, the number of resorptions
and viable fetuses. All observed differences are considered to reflect the normal range of
fluctuations for animals of this strain and age.
Regarding pathology, no treatment-related findings were noted. 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.

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

body weight and weight gain

clinical signs

food consumption and compound intake

water consumption and compound intake

Fetal body weight changes:

no effects observed

Description (incidence and severity):

see attached document: summary placental and fetal bw
The mean fetal weights of test groups 1 and 2 (20 and 60 mg/kg bw/d) were not influenced by
the test substance and did not show any biologically relevant differences in comparison to the
control group.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The sex distribution of the fetuses in test groups 1 and 2 (20 and 60 mg/kg bw/d) was
comparable to the control fetuses.

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

not examined

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

One low-dose male fetus an umbilical hernia was recordrd. Since the finding was not related to dose,
it was not assessed as treatment-related.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

For only one low-dose fetus a skeletal malformation was recorded: malpositioned and bipartite
sternebra (unchanged cartilage). Since the finding was not related to dose, it was not assessed
as treatment-related.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Two soft tissue variations were detected, i.e. short innominate and dilated renal pelvis, both in
test groups 0 and 1. The incidences of these variations were neither statistically significantly
nor dose-dependently increased in the treated groups. Therefore, they were not assessed as
treatment-related.

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Two unclassified external observations were recorded. A skin tag was seen in one low-dose
fetus (20 mg/kg bw/d), furthermore, placentae fused were seen in one control litter. These
findings were not considered biologically relevant, since they were single events without
relation to dose.
For all test groups, skeletal variations of different bone structures were observed, with or
without effects on corresponding cartilages. The observed skeletal variations were related to
several parts of fetal skeletons and appeared without a relation to dose The overall affected
fetuses/litter incidences of skeletal variations were comparable to the historical control data.

Details on embryotoxic / teratogenic effects:

Soft tissue malformations did not occur in any fetus in this study. There were noted external
and skeletal malformations in two fetuses of the low-dose group.. These observations in single
fetuses were unrelated to dose and both can be found in the historical control data, thus they
are considered as incidental events.
External variations did not occur in any fetus in this study. Two soft tissue variations and a range of
skeletal variations were noted in all test groups including the controls. Two out of three incidences
showed no relation to dosing. The skeletal variations are equally distributed about the different
test groups, if normal biological variation is taken into account, and can be found in the historical
control data at a comparable frequency.
Unclassified soft tissue observations did not occur in any of the fetuses in this study. A
spontaneous origin is assumed for the unclassified external and skeletal cartilage observations
which were observed in several fetuses of all test groups The distribution and type of these findings
do not suggest any relation to treatment.

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: teratogenicity / embryotoxicity

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Treatment related:

no

Total soft tissue variations

 

		Test group 0 0 mg/kg bw/d	Test group 1 20 mg/kg bw/d	Test group 2 60 mg/kg bw/d
Litter Fetuses	N N	25 133	24 121	24 121
Fetal incidence	N (%)	3 (2.3)	3 (2.5)	0.0
Litter incidence	N (%)	3 (12)	2 (8.3)	0.0
Affected fetuses/litter	Mean %	2.0	2.5	0.0

 

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

Total fetal skeletal variations

 

		Test group 0 0 mg/kg bw/d	Test group 1 20 mg/kg bw/d	Test group 2 60 mg/kg bw/d
Litter Fetuses	N N	25 144	24 129	24 132
Fetal incidence	N (%)	138 (96)	127 (98)	129 (98)
Litter incidence	N (%)	25 (100)	24 (100)	24 (100)
Affected fetuses/litter	Mean %	95.9	98.6	97.9

 

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

 

 

 

Occurrence of statistically significantly increased fetal skeletal         

variations (expressed as mean percentage of affected fetuses/litter)

 

Finding	Test group 0 0 mg/kg bw/d	Test group 1 20 mg/kg bw/d	Test group 2 60 mg/kg bw/d	HCD Mean % (range)
Incomplete ossification of basisphenoid	10.4	32.8**	18.4	22.4 (5.3 - 43.2)
Incomplete ossification of supraoccipital; unchanged cartilage	11.8	17.2	23.6*	22.4 (3.0 - 51.2)
Incomplete ossification of skull; unchanged cartilage	3.5	9.2*	5.0	7.5 (2.1 - 13.4)

 

mg/kg bw/d = milligram per kilogram body weight per day; HCD = Historical control data; % = per cent

* = p≤0.05 (Wilcoxon-test [one-sided]) ** = p≤0.01 (Wilcoxon-test [one-sided])

 

Conclusions:

Under the conditions of this prenatal developmental toxicity study, the oral administration of
Dibutylethanolamine to pregnant Wistar rats from implantation to one day prior to the
expected day of parturition (GD 6-19) caused evidence of distinct maternal toxicity, such as
severe clinical findings (twitching, convulsions, tremor, abdominal position), a reduction of
water and food consumption and a decrease in body weight/body weight gain. Due to the
above-mentioned findings, treatment of the high-dose was stopped and the dams were
euthanized without further examinations on GD 13/12/11 of the study.
In conclusion, the no observed adverse effect level (NOAEL) for maternal toxicity is the
mid-dose of 60 mg/kg bw/d.
Examination of the low- and mid-dose fetuses was performed and showed no adverse findings.
In conclusion, the no observed adverse effect level (NOAEL) for prenatal developmental
toxicity is 60 mg/kg bw/d.
Under the conditions of this study the test item is not teratogenic.

Executive summary:

In a prenatal developmental toxicity study, the test substance Dibutylethanolamine was

administered to pregnant Wistar rats daily by gavage from implantation to one day prior to the

expected day of parturition (GD 6-19) to evaluate its potential maternal and prenatal

developmental toxicity.

Analyses confirmed the correctness of the prepared concentrations and the stability of the test

substance in the vehicle.

Concerning clinical examination, strong signs of systemic maternal toxicity was observed at

the highest test group of 200 mg/kg bw/d Dibutylethanolamine. Since severe clinical findings

(twitching, convulsions, tremor, abdominal position) were observed in almost all animals, the

high-dose group was sacrificed prematurely due to animal welfare reasons. Treatment of that

test group was stopped and the dams were euthanized without further examinations on GD

13/12/11 of the study. The next lower dose of 60 mg/kg bw/d showed no adverse clinical

findings.

Water and food consumptions were statistically significantly reduced in high-dose dams during

GD 6-13 (up to 21% and 31% below control, respectively). Furthermore, high-dose dams

showed a statistically significant decrease in body weight (change) including a body weight

loss during GD 6-8. The above-mentioned findings were assessed as treatment-related and

adverse.

Mid-dose dams showed a reduction in water consumption in the beginning of treatment (GD

6-10: up to 12% below control) but recovered afterwards to control levels. Food consumption

was not affected in mid-dose dams. Body weight change was statistically significantly reduced

on GD 6-8 but recovered afterwards and body weight in general was comparable to the control

values. These two marginal findings in the mid-dose group were only observed in the beginning

of treatment but not thereafter. They were considered to be not sufficient to proof adversity and

therefore, they were not assessed as treatment-related and adverse.

In the low dose group (20 mg/kg bw/d), no adverse findings were observed.

Concerning thyroid hormone levels, no treatment-related, adverse effect was observed up to a

dose of the compound of 60 mg/kg bw/d.

Regarding pathology, no treatment-related findings were noted. 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.

No differences of toxicological relevance between the control and the treated groups (20 or 60

mg/kg bw/d) were determined for any reproductive parameters, such as conception rate, mean

number of corpora lutea, mean number of implantations, as well as pre- and post-implantation

loss. Similarly, no toxicologically relevant influence of the test substance on sex distribution

and anogenital distance/index of the fetuses was noted at any dose.

Overall, there was no evidence for toxicologically relevant adverse effects of the test substance

on fetal morphology at any dose.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

60 mg/kg bw/day

Study duration:

subchronic

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

236.3 mg/m³

Study duration:

subchronic

Species:

rat

Quality of whole database:

Available data is reliable and sufficient for assessment.

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

In a prenatal developmental toxicity study, Dibutylethanolamine was administered at doses of 20, 60 and 200 mg/kg bw/d to pregnant Wistar rats daily by gavage from implantation to one day prior to the expected day of parturition (GD 6-19) to evaluate its potential maternal and prenatal developmental toxicity. Pronounced maternal toxicity was observed at the high dose of 200 mg/kg bw/d. Since severe clinical findings (twitching, convulsions, tremor, abdominal position) were observed in almost all animals of this dose, the high-dose group was sacrificed prematurely due to animal welfare reasons. Treatment of that test group was stopped and the dams were euthanized without further examinations on GD 13/12/11 of the study. The next lower dose of 60 mg/kg bw/d showed no adverse clinical findings.

Water and food consumptions were statistically significantly reduced in high-dose dams during GD 6-13 (up to 21% and 31% below control, respectively). Furthermore, high-dose dams showed a statistically significant decrease in body weight (change) including a body weight loss during GD 6-8.

Mid-dose (60 mg/kg bw/d) dams showed a reduction in water consumption in the beginning of treatment (GD 6-10: up to 12% below control) but recovered afterwards to control levels. Body weight change was statistically significantly reduced on GD 6-8 but recovered afterwards.

In the low dose group (20 mg/kg bw/d), no adverse findings were observed.

No differences of toxicological relevance between the control and the treated groups (20 or 60 mg/kg bw/d) were determined for any reproductive parameters, such as conception rate, mean number of corpora lutea, mean number of implantations, as well as pre- and post-implantation loss. Similarly, no toxicologically relevant influence of the test substance on sex distribution and anogenital distance/index of the fetuses was noted at any dose. There was no evidence for toxicologically relevant adverse effects on fetal morphology at any dose.

In conclusion, the no observed adverse effect level (NOAEL) for maternal and prenatal developmental toxicity was 60 mg/kg bw/d.

A GLP-compliant investigation of the toxicological effects resulting from repeated inhalation exposure of 2-dibutylaminoethanol to Wistar rats was performed following OECD guideline 422 (BASF SE, 2013). Groups of ten male and ten female rats (F0 animals) per test group were exposed nose-only to a dynamic atmosphere of 2-dibutylaminoethanol aerosol for 6 hours per day on each day. The duration of treatment covered a 2-week pre-mating and 2-week mating period in both sexes, 1 day post-mating in males, and the entire gestation period of the females. After the lactation period and after necropsy of the pups total all parental females were exposed to the test substance on 4 consecutive days. The target concentrations were 25, 75 and 225 mg/m³ (measured concentrations: 20.6, 72.1, 236.3 mg/m³). A concurrent control group was exposed to conditioned air. The test did not cause any adverse effect with regard to reproductive and developmental parameters, but only transiently reduced food consumption, body weight and body weight gain. In histopathology lesions of nasal epithelia was observed in animals exposed to 72.1 and 236.3 mg/m³ test substance. Thus, concerning systemic toxicity, reproductive and developmental parameters the NOAEC for 2-dibutylaminoethanol was determined to be 236.3 mg/m³ for male and female animals. Considering histopathological changes the NOAEC for local effects was 20.6 mg/m³.

Justification for classification or non-classification

Dangerous Substance Directive (67/548/EEC)

The test substance has not to be classified under Directive 67/548/EEC.

 

Classification, Labeling, and Packaging Regulation (EC) No. 1272/2008

The test substance has not to be classified under Regulation (EC) No.1272/2008.