2-dibutylaminoethanol

Toxicity to reproduction

Administrative data

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

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

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

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

Details on test material:

- Name of test material (as cited in study report): Dibutylethanolamine
- Physical state: Liquid / clear, colorless
- Analytical purity: > 99%
- Lot/batch No.: B 5661 vom 17.05.2011
- Test substance No.: 05/0286-3
- Storage condition of test material: Room temperature (The stability under storage conditions over the exposure period was guaranteed.)
- Homogeneity: Homogeneous

Test animals

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

Administration / exposure

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

Doses / concentrations

Doses / concentrations 1

Remarks:

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

Doses / concentrations 2

Remarks:

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

Doses / concentrations 3

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.

Examinations

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

 

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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 / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

not specified

Reproductive performance:

no effects observed

Details on results (P0)

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"

Effect levels (P0)

1

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

2

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

3

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'

Results: F1 generation

General toxicity (F1)

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

Developmental neurotoxicity (F1)

Behaviour (functional findings):

no effects observed

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

no effects observed

Details on results (F1)

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.

Effect levels (F1)

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

Overall reproductive toxicity

Reproductive effects observed:

not specified

Applicant's summary and conclusion

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.