2-dibutylaminoethanol

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

short-term repeated dose toxicity: inhalation

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: vapour

Type of inhalation exposure:

nose/head only

Vehicle:

air

Remarks on MMAD:

MMAD / GSD: The test substance in the concentration to be tested is an aerosol. Therefore no cascade impactor measurement had been performed.

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Head / nose inhalation system (Volume 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

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, were 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)

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: The control group was exposed to conditioned air

Details on study design:

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

Examinations

Observations and examinations performed and frequency:

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


OPHTHALMOSCOPIC EXAMINATION: No


HAEMATOLOGY: Yes
- Time schedule for collection of blood: on study days 13 (females) and 29 (males)
- Anaesthetic used for blood collection: Yes (Isoflurane anesthesia)
- Animals fasted: Yes
- How many animals: the first 5 surviving male animals per group and the first 5 surviving female animals on study days 13 (females) and 29 (males)
- Parameters checked: leukocytes, erythrocytes, hemoglobin, hematocrit, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelets, differential blood count, reticulocytes; clotting tests were carried out using a ball coagulometer (AMAX destiny plus model; Trinity biotech, Lemgo, Germany); parameter of clotting test: prothrombin time; furthermore, blood smears were prepared and stained according to WRIGHT without being evaluated, because of non-ambiguous results of the differential blood cell counts measured by the automated instrument;


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on study days 13 (females) and 29 (males)
- Animals fasted: Yes
- How many animals: the first 5 surviving male animals per group and the first 5 surviving female animals on study days 13 (females) and 29 (males)
- Parameters checked: alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, gamma-glutamyl transferase, sodium, potassium, chloride, inorg. phosphate, calcium, urea, creatinine, glucose, total bilirubin, total protein, albumin, globulins, triglycerides, cholesterol, bile acids


URINALYSIS: No


NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: males: at the end of the administration period; females: at the end of the premating period (no inhalation exposure will take place on the day of FOB examination)
- Dose groups that were examined: 5 parental male and female animals per group
- Battery of functions tested: The FOB started with passive observations without disturbing the animals, followed by removal from the home cage, open field observations in a standard arena and sensorimotor tests as well as reflex tests. Homecage observation (posture, tremors, convulsions, abnormal movements, impairment of gait, other findings); open field observation (behavior when removed from cage, fur, skin, salivation, nose discharge, lacrimation, eyes / pupil size, posture, palpebral closure, respiration, tremors, convulsions, abnormal movements/stereotypy, impairment of gait, activity / arousal level, feces excreted within 2 minutes (number of scybala discharged/appearance/ consistency), urine excreted within 2 minutes (amount/color), number of rearings within 2 minutes); sensory-motoric test / reflexes (approach response, touch response, vision (“Visual placing response”), pupillary reflex, pinna reflex, audition (“startle response”), coordination of movements (“righting response”), behavior during handling, vocalization, pain perception (“tail pinch”), other findings, grip strength of forelimbs and hindlimbs, landing foot splay test), motor activity measurement (Multi-Varimex system supplied by Columbus Instruments Int. Corp., Ohio, USA)

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
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.

HISTOPATHOLOGY: Yes (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)

Statistics:

Food consumption (parental animals), 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; feces, rearing, grip strength forelimbs, grip strength hind-limbs, landing foot-splay test, motor activity: KRUSKAL-WALLIS and WILCOXON test (one- or two-sided); blood parameters: KRUSKAL-WALLIS and WILCOXON test (two-sided); weight parameters (determined in pathology): KRUSKAL-WALLIS and WILCOXON test (two-sided)

Results and discussion

Results of examinations

Details on results:

CLINICAL SIGNS AND MORTALITY:
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 WEIGHT GAIN:
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.


FOOD CONSUMPTION:
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.


HAEMATOLOGY:
No treatment-related changes among hematological parameters were observed.


CLINICAL CHEMISTRY:
No treatment-related changes among clinical chemistry parameters were observed. In male rats of test group 3 (225 mg/m3), globulin values were lower compared to controls. However, this parameter was not dose-dependently changed. Additionally, this was the only changed parameter in clinical pathology. Therefore, this alteration was regarded as incidental and not treatment-related.


NEUROBEHAVIOUR:
Home cage observations: No test substance-related or spontaneous findings were observed in male and female animals of all test groups during the home cage observation.
Open field observations: The open field observations did not reveal any test substance-related findings in male and female animals of all test groups.
Sensorimotor tests/reflexes: There were no test substance-related findings in male and female animals of all test groups.
Quantitative Parameters: In general no test substance-related impaired parameters were observed in male and female animals of all test groups. In F0 females of test group 1, slightly though significantly higher grip strength of forelimbs was determined. This parameter was not increased in other groups, it was considered to be incidental due to the lack of concentration-response relationship.
Motor activity measurement (MA):
Overall motor activity (summation of all intervals): No statistically significant changes on motor activity data (summation of all intervals) was observed in the male and female animals of all dose groups in comparison to the concurrent control group. Single intervals: No abnormalities were detected.


ORGAN WEIGHTS:
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:
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:
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: see also "Repeated dose toxicity"

Effect levels

1

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: histopathological lesions of nasal epithelia

2

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

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'

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Table 1: Relative increase / decrease of absolute weights in males
Absolute weights	Males
Group (mg/m³)	1 (25)	2 (75)	3 (225)
Epididymides	88%**	93%	90%**
Spleen	137%**	96%	90%
Testes	98%	93%*	92%*
*: p <= 0.05, **: p <= 0.01
Table 2: Relative increase / decrease of absolute weights in females
Absolute weights	Females
Group (mg/m³)	1 (25)	2 (75)	3 (225)
Liver	102%	103%	89%*
*: p <= 0.05
Table 3: Relative increase / decrease of absolute weights in males
Absolute weights	Males
Group (mg/m³)	1 (25)	2 (75)	3 (225)
Epididymides	88%**	94%	94%
**: p <= 0.01
Table 4: Relative increase / decrease of absolute weights in females
Absolute weights	Females
Group (mg/m³)	1 (25)	2 (75)	3 (225)
Thymus	129%*	96%	107%
*: p <= 0.05