Registration Dossier

Toxicological information

Repeated dose toxicity: inhalation

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Administrative data

Endpoint:
repeated dose toxicity: inhalation
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
22 May 2012 (Experimental starting date (Arrival of test animals)) to 18 Apr 2013 (Experimental completion date (check the raw data for its completeness)
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: U.S. EPA OPPTS Guidelines 870.3650
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht, Mainz, Germany
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
other: liquid
Details on test material:
- Name of test material (as cited in study report): Dibutylethanolamine
- Molecular formula (if other than submission substance): C10H23NO
- Molecular weight (if other than submission substance): 173.3
- Smiles notation (if other than submission substance): CCCCN(CCO)CCCC
- InChl (if other than submission substance): 1S/C10H23NO/c1-3-5-7-11(9-10-12)8-6-4-2/h12H,3-10H2,1-2H3
- Structural formula attached as image file (if other than submission substance): see Fig.
- Substance type: organic (Alkanolamine)
- Physical state: Liquid / clear, colorless
- Analytical purity: 99.5 g/100g (H-NMR); 99.08 area-% on a RTX-5-Amine column; 99.19 area-% on a DB-1701 column (GC)
- Stability under test conditions: homogeneous
- Storage condition of test material: room temperature
- Other: The Sponsor is responsible for compliance for all test substance information and their storage, except for those test substance investigations commissioned by the test facility.

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS: Wistar Crl:WI(Han)
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH; Sandhofer Weg 7, 97633 Sulzfeld
- Age at study initiation: about 10 - 11 weeks; male/female (no siblings)
- Weight at study initiation: not reported
- Fasting period before study: no
- Housing: individually in Makrolon type M III cages (floor area of about 800 cm²) except mating period during which one male and one female were housed together. Pregnant animals and their litters were housed together until PND 4 (end of lactation). For motor activity (MA) measurements the animals were housed individually in polycarbonate cages supplied by TECNIPLAST, Hohenpeißenberg, Germany with wire covers from Ehret, Emmendingen, Germany (floor area of about 800 cm²) and small amounts of bedding material (the present supplier is documented in the raw data).

Pregnant females were provided with nesting material (cellulose wadding) toward the end of gestation.

Dust-free wooden bedding was used in this study (the present supplier is documented in the raw data). Wooden gnawing blocks (Type NGM E-022), supplied by Abedd® Lab. and Vet. Service GmbH, Vienna, Austria, were added for environmental enrichment.

The cages with the test animals were arranged on the racks in such a way that uniform experimental conditions (ventilation and light) were ensured.

- Diet (e.g. ad libitum): ad libitum (Kliba maintenance diet mouse-rat “GLP” meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland) except the exposure period. Besides, the animals were sacrificed after a fasting period (withdrawal of food) for at least 16-20 hours.
- Water (e.g. ad libitum): ad libitum (from water bottles) except the exposure period.
- Acclimation period: 8 days.

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

IN-LIFE DATES: From: To:

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure:
nose only
Vehicle:
air
Remarks on MMAD:
MMAD / GSD: No details are given
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Inhalation apparatus type INA
The inhalation atmosphere was maintained inside aerodynamic exposure systems (INA 120, volume V ≈ 155 L, BASF SE until study day 7 (control group) or INA 60, volume V ≈ 90 L, BASF SE from study day 8 through to the end of the study (control and test groups)) consisting of a cylindrical inhalation chamber made of stainless steel sheeting and cone shaped outlets and inlets.
Generation procedure:

The test substance was used unchanged.

For each concentration the test substance was supplied to a two-component atomizer at a constant rate by means of a metering pump. The aerosol was generated with compressed air into the inhalation system. Due to the high vapor pressure of the test substance, the liquid droplets evaporate spontaneously and form vapor inhalation atmospheres. The control group was exposed to conditioned air.

- Method of holding animals in test chamber: The rats were restrained in glass exposure tubes. Their snouts projected into the inhalation chamber and thus they inhaled the aerosol
- Source and rate of air: conditioned supply air and humidified air (without further details),
- Method of conditioning air: filtered through activated charcoal

- System of generating particulates/aerosols:
• Continuous infusion pumps PERFUSOR (B. Braun Melsungen AG, Melsungen, Germany)
• Two-component atomizers (stainless steel, Model 970; Düsen-Schlick GmbH, Untersiemau/Coburg, Germany)

- Temperature, humidity, pressure in air chamber:
Conditioned supply air is activated charcoal filtered air conditioned to about 50% ± 20% relative humidity and 22°C ± 2°C. Compressed air is filtered air pressurized to about 6 bar.

- Air flow rate and air change rate: The test pump rates, substance flow and air flows were scheduled (presented in table 2 in "Any othet information on materials and methods").
- Method of particle size determination: The test substance in the concentration to be tested is a vapor. Therefore no cascade impactor measurement had been performed.
- Treatment of exhaust air: A positive pressure was maintained inside the exposure systems by adjusting the air flow of the exhaust air system. This ensured that the aerosol in the breathing zones of the animals was not diluted by laboratory air.

In order to accustom the animals to exposure they were treated with supply air under conditions comparable to exposure on two days before start of exposure (pre-exposure period).

TEST ATMOSPHERE
- Brief description of analytical method used:
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.

- Samples taken from breathing zone: yes

VEHICLE: air
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
MEASUREMENT OF THE EXPOSURE CONDITIONS
The atmospheric concentrations were measured online by propane-calibrated total hydrocarbon analyzer (FID) and confirmed by GC analyses of absorption samples.
Recording of exposure parameters is presented in table 3 in "Any othet information on materials and methods".
No surveillance of the oxygen content in the inhalation system was performed. The air change within the inhalation systems was judged to be sufficient to prevent oxygen depletion by the breathing of the animals and the concentrations of the test substance used could not have a substantial influence on oxygen partial pressure.

Principles of recording with the automated measuring system:

Each parameter was measured at appropriate measuring points using suitable measuring equipment (sensors, orifice plates etc.). The measurements were standardized (0 20 or 4 20 mA) and transferred to instrumentation consoles. There, the measured values were displayed in an analogous way (where this is provided for) and some were used as actual value for regulating the specific parameter.

In addition, the measured values were scanned every 10 seconds, converted from analog to digital, transferred to a personal computer, displayed on its screen, and saved on hard disk. The computer checked the arriving values against preset threshold values, displayed warnings if violations of thresholds occurred and recorded the start and the end of threshold violations for each measured parameter affected. After the end of each exposure all data gathered during this exposure were backed up on optical media.

Daily protocols were prepared from the recorded values using suitable software. The protocols include start and stop times of exposure and possible threshold violations, and daily means of each parameter. The records saved on optical media and the printed daily records are considered as raw data.
Duration of treatment / exposure:
Males (28 exposure days):
a) 14 days premating
b) up to 14 days mating
c) Sacrifice after a minimum of 28 days after the first application (on day 29)

Females (50 exposure days)
a) 14 days premating
b) up to 14 days mating
c) during the pregnancy up to and including GD 19
d) after necropsy of the pups total 4 exposures on 4 consecutive days including the day before scheduled killing (on day 51)
Frequency of treatment:
6h/day; 5 days/week
Doses / concentrations
Remarks:
Doses / Concentrations:
20.6, 72.1, 236.3 mg/m³
Basis:
nominal conc.
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
Based on available data, the following concentrations were selected for the present study:

225 mg/m³ as the high concentration causing adverse effect
75 mg/m³ as the mid concentration causing some effect
25 mg/m³ as the low concentration and the expected no adverse effect concentration
- Other: no exposure on the day of functional observation battery/motor activity measurement (FOB/MA). Observations of FOB/BA were performed on study day 12 in females and on study day 26 in males.
- Rationale for animal assignment (if not random): randomized
Positive control:
None.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
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.
- 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. Individual data of daily observations can be found in the raw data.

The parturition and lactation behavior of the dams was generally evaluated in the mornings in combination with the daily clinical inspection of the dams. Only particular findings (e.g. inability to deliver) were documented on an individual dam basis.

On weekdays (except Saturday, Sunday and public holidays) the parturition behavior of the dams was inspected in the afternoons in addition to the evaluations in the mornings.

The day of parturition was considered the 24-hour period from about 15.00 h of one day until about 15.00 h of the following day.


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once before the first exposure and weekly thereafter.

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 will be based on the current index of findings in PDS ToxData® and includes but is not limited to the following parameters listed:
1. Abnormal behavior in “handling”
2. Fur
3. Skin
4. Posture
5. Salivation
6. Respiration
7. Activity/arousal level
8. Tremors
9. Convulsions
10. Abnormal movements
11. Gait abnormalities
12. Lacrimation
13. Palpebral closure
14. Exophthalmos
15. Assessment of the feces discharged during the examination (appearance/consistency)
16. Assessment of the urine discharged during the examination
17. Pupil size

BODY WEIGHT: Yes
- Time schedule for examinations: once a week at the same time of the day (in the morning) until sacrifice.
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
Generally, 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.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: at the time of the detailed clinical observations, the animals were tested for the presence of exophthalmos.
- Dose groups that were examined: all animals.

HAEMATOLOGY: Yes
- Time schedule for collection of blood:towards the end of the exposure period
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: 5 animals per sex and group
- Parameters checked in table [4] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: towards the end of the exposure period
- Animals fasted: Yes
- How many animals: 5 animals per sex and group
- Parameters checked in table [4] were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: A functional observational battery was performed in 5 parental male and 5 parental female animals per group and for the males the FOB were carried out at the end of the administration period, for the females at the end of the premating period.
- Dose groups that were examined: all dose groups.
- Battery of functions tested: sensory activity, grip strength, motor activity

OTHER:
Sacrifice and pathology:
GROSS PATHOLOGY: Yes. All parental animals were sacrificed under pentobarbitone anesthesia by exsanguination from the abdominal aorta and vena cava. The exsanguinated animals were necropsied and assessed by gross pathology, special attention being given to the reproductive organs.
HISTOPATHOLOGY: Yes (see table 5)
Other examinations:
Organ weights
The following weights were determined in all animals sacrificed on schedule:

1. Anesthetized animals
2. Epididymides
3. 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):

1. Adrenal glands
2. Brain
3. Heart
4. Kidneys
5. Liver
6. Lung
7. Spleen
8. Thymus
Statistics:
Statistical methods are summarised in "Overall remarks, attachments".

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
body weights were slightly reduced in male and female animals during premating and mating period. During gestation stage, mean body weights of group 3 females were significantly reduced on gestation day 7 and 14.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
food consumptions were slightly reduced in male and female animals during premating and mating period and significantly decreased during gestation stage.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
increased: epididymides, testes, spleen, liver and thymus
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Treatment – related findings in the larynx, nasal cavities, testes and epididymides.
Histopathological findings: neoplastic:
no effects observed
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 post-mating period one female animal of the control group (No. 104) showed vaginal discharge.

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.
One sperm positive low concentration female (No. 117) did not deliver F1 pups.
The detailed clinical observations did not reveal any abnormalities in male and female animals of all test groups.

BODY WEIGHT AND WEIGHT GAIN

Body weight in 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.

Body weigh in 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.
Body weight in 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).
Body weight during lactation and on PND 4:
The mean body weights of F0 female animals were not statistically significantly different from the control group 0.
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).

OPHTHALMOSCOPIC EXAMINATION (included in detailed clinical observation)
No effects.

HAEMATOLOGY
Prior to blood sampling, male rats were dosed for four weeks and female rats were dosed for two weeks.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

On the day of the performance of the Functional Observation Battery, the animals were not exposed to the test substances. Observations were performed on study day 12 in females and on study day 26 in males.
- 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): No statistically significant changes on motor activity data (summation of all intervals) as well as on single intervals were observed in the male and female animals of all dose groups in comparison to the concurrent control group.

ORGAN WEIGHTS

When compared with control group 0 (=100%), the mean absolute weights presented in the tables 2-5 were significantly increased or decreased in one or more test groups (statistically significant changes printed in bold).

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.

Fertility:
The mating pair (Nos. 17/117), which did not produce offspring did not show relevant gross lesions.


HISTOPATHOLOGY: NON-NEOPLASTIC

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 epididymis and testis in male animals with incidences and grading shown in the tables 6-9 below.

HISTOPATHOLOGY: NEOPLASTIC (if applicable) No neoplastic lesions are reported.

OTHER FINDINGS
- Male and female reproduction and delivery data were similar to controls (please refer to section 7.8.1. and 7.8.2. for detailed information);
- Pup number, status at delivery, viabilit/mortality, sex ratio, clinical observations, pup body weight and pup necropsy observations were all similar to controls (please refer to section 7.8.1. and 7.8.2. for detailed information).

Effect levels

open allclose all
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: based on histopathological changes in larynx and nasal cavity
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'
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: the highest dose tested, no adverse effects observed

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

The atmospheric concentrations were measured online by FID and confirmed by GC analyses of absorption samples. The concentrations measured by FID are presented in table 1. GC analyses confirmed largely the values of FID measurement. Details see part II of the report.

 

Table 1: Study means and standard deviations of test substance concentrations measured by FID

Test group

Target concentration
(mg/m³)

Measured concentration (mg/m³)

Nominal concentration (mg/m³)

Effectiveness of vapor generation
(%)

Mean

SD

1

25

20.6

6.0

21.5

95.8

2

75

72.1

20.0

86.0

83.8

3

225

236.3

39.5

301.0

78.5

The vapor generation effectiveness was as expected for these high concentrations.

Measurementsconcerning operation conditions

The air flows were constantly maintained in the desired range. An air change of about 67 times per hour can be calculated by dividing the supply air flow through the volume of each inhalation system.

 

Relative humidities in the inhalation systems ranged between 39.3 and 63.8 %. In the test groups the relative humidity was slightly lower than those in the control chamber, because the test substance was sprayed by means of compressed air, which is dry. Nonetheless, all values were within the range suggested by the respective testing guidelines. 

The temperatures in the inhalation systems ranged between 21.7 and 24.1°C.

Food, drinking water and bedding enrichment analyses

On the basis of duration of use and the analytical findings with respect to chemical and microbiological contaminants the food, drinking water and bedding enrichment were found to be suitable.

Absolute and relative organ weights

When compared with control group 0 (=100%), the mean absolute weights presented in the tables 2-5 were significantly increased in one or more test groups (statistically significant changes printed in bold):

Table 2: Relative increase 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 3: Relative increase of absolute weights in females 

Absolute weights

Females

Group

(mg/m³)

1

(25)

2

(75)

3

(225)

Liver

102%

103%

89%*

* : p <= 0.05, **: p <= 0.01

Table 4: Relative increase of relative weights in males 

Relative weights

Males

Group

(mg/m³)

1

(25)

2

(75)

3

(225)

Epididymides

88%**

94%

94%

*: p <= 0.05, **: p <= 0.01

Table 5: Relative increase of relative weights in females

Relative weights

Females

Group

(mg/m³)

1

(25)

2

(75)

3

(225)

Thymus

129%*

96%

107%

*: p <= 0.05, **: p <= 0.01

 

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 epididymis and testis weights was considered to be treatment-related even in the absence of a clear dose response relationship or, in the case of the testis, no changes in relative weights as there was a histopathological correlate.

All other mean absolute or relative weight parameters did not show significant differences when compared to the control group 0.

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 epididymis and testis in male animals with incidences and grading shown in the table below:

 

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). Table 6: Incidence and grading of histological findings in larynx

 

Male animals

Female animals

Test group

(mg/m³)

0

(0)

1

(25)

2

(75)

3

(225)

0

(0)

1

(25)

2

(75)

3

(225)

No. of animals

10

10

10

10

9

10

10

10

Epithelial alteration

1

6

8

5

1

6

8

8

  • Grade 1

1

6

8

4

1

6

4

8

  • Grade 2

 

 

 

1

 

 

4

 

Metaplasia, squamous

 

 

2

5

 

 

1

2

  • Grade 1

 

 

2

4

 

 

1

2

  • Grade 2

 

 

 

1

 

 

 

 

Nasal cavity, level I

 

The following treatment- related findings were noted in the nasal cavity:

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.

 

Table 7: Incidence and grading of histological findings in nasal cavity

 

Male animals

Female animals

Test group

(mg/m³)

0

(0)

1

(25)

2

(75)

3

(225)

0

(0)

1

(25)

2

(75)

3

(225)

No. of animals

10

10

10

10

9

10

10

10

Degeneration /regeneration transitional epithelium

 

 

 

2

 

 

1

8

  • Grade 1

 

 

 

2

 

 

1

7

  • Grade 2

 

 

 

 

 

 

 

1

Metaplasia, squamous, transitional epithelium

 

 

 

1

 

 

 

3

  • Grade 1

 

 

 

1

 

 

 

3

Degeneration /regeneration respiratory epithelium

 

 

 

2

 

 

 

 

  • Grade 1

 

 

 

2

 

 

 

 

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)

 

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.

Table 8: Incidence and grading of histological findings in testes


 

Male animals

Test group

(mg/m³)

0

(0)

1

(25)

2

(75)

3

(225)

No. of animals

10

10

10

10

Degeneration, tubular

4

6

7

8

  • Grade 1

3

2

2

3

  • Grade 2

 

3

3

4

  • Grade 3

1

1

2

1

Sperm plug

 

 

 

1

  • present

 

 

 

1

 

 

Epididymides

 

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

 

Table 9: Incidence and grading of histological findings in epididymides

 

Male animals

Test group

(mg/m³)

0

(0)

1

(25)

2

(75)

3

(225)

No. of animals

10

10

10

10

Debris

2

4

4

7

  • present

2

4

4

7

 

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.

 

Fertility

The male partner of the mating pair (Nos. 17/117), which did not produce offspring showed testicular degeneration and debris in the epididymis which might have impaired fertility even if animals with similar findings in the same group did produce offspring.

Applicant's summary and conclusion

Conclusions:
Inhalation exposure to dibutylethanolamine to maximum of 236.3 mg/m³ during 2 weeks premating, mating, gestation period 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. No adverse parental or pup findings were evident at any concentration. In histopathology lesions of nasal epithelia was observed in animals exposed to 72.1 and 236.3 mg/m³ test substance. Thus, concerning reproductive and developmental parameters the no observed adverse effect concentration (NOAEC) for dibutylethanolamine was determined to be >236.3 mg/m3. Considering histopathological changes the NOAEC for dibutylethanolamine was 20.6 mg/m³.
Executive summary:

To evaluate the toxicity profile of Dibutylethanolamine (DBEA) after inhalation exposure, groups of ten male and ten female Wistar rats (F0 animals) per test group were exposed nose-only to dynamic atmosphere of DBEA 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 (correspond to measured concentrations 20.6, 72.1 and 236.3 mg/m³, respectively). A concurrent control group was exposed to conditioned air. For adaptation to the experimental conditions all animals were kept in glass restraining tubes identical to those used in the study and were exposed nose-only to fresh air on two days before start of the exposure period.

After 2 weeks of premating treatment the F0 animals were mated to produce F1 generation pups. Mating pairs were from the same test group. Mating was discontinued as soon as sperm was detected in the vaginal smear. F0 animals were examined for their reproductive performance including determination of the number of implantation sites and the calculation of post-implantation loss for all F0 females.

 

A detailed clinical observation (DCO) was performed in all animals before initial test substance exposure and, as a rule, thereafter at weekly intervals. Clinical observation was performed at least three times on exposure days and once a day during the other days.

 

Food consumption of the F0 parents was determined once weekly during premating. In dams food consumption was determined for gestation days 0 - 7, 7 - 14, 14 - 20 and lactation days 1 - 4. During the 4 exposure days after necropsy of the pups the food consumption was determined also.

 

Body weights of F0 parents were determined once a week, in males throughout the study and in females during premating. During gestation and lactation period, F0 females were weighed on gestation days (GD) 0, 7, 14 and 20,on the day after parturitionpostnatal day [PND] 1) and on PND 4. After the pups are sacrificed the females that were exposed for 4 consecutive days were weight once before the exposure period and once on the last exposure day.

 

A functional observational battery (FOB) was performed and motor activity was measured in 5 parental males and females per group. The FOB of the female animals was on study day 12. The male animals were performed at the end of the exposure period on study day 26.

 

The pups were sexed and examined for macroscopically evident changes on PND 0. They were weighed on PND 1 and on PND 4. Their viability was recorded. At necropsy on PND 4, all pups were sacrificed with CO2, under isoflurane anesthesia, and examined macroscopically for external and visceral findings.

 

Clinico-chemical and hematological examinations were performed in 5 animals per sex and group towards the end of the exposure period.

 

A complete necropsy including gross pathological evaluation and weighing of selected organs was performed. Organs and tissues were examined histopathologically as required by the corresponding test guidelines.

Results

Test group 3 (225 mg/m³)

 

Significantly decreased food consumption during pre-mating (p < 0.05) in male animals between study days 0-7 (-9 %) and 7-14 (-11%) and in female animals between study days 0-7 (-7%) and 7-14 (-5%);

Significantly decreased food consumption during gestationin female animals during the whole period (-11%,p < 0.05);

Significantly lowered mean body weights F0 females on gestation days (GD) 7 (-5.7 %, p < 0.05) and 14 (5.2 %, p < 0.05);

Significantly 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);

Significantly lowered mean body weight gain of F0 female animals from gestation day 0 to 7 (-37 %, p < 0.01).

 

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 epitheliumgraded 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 male animal.

 

Test group 2 (75 mg/m³)

Significantly decreased food consumption during gestation in female animals between study days 0 - 7 (-9%, p < 0.05).

 

Nasal cavity, level I

 

Degeneration /regeneration transitional epithelium in one female animal (graded minimal)

 

Test group 1 (25 mg/m³)

 

No adverse effect observed

 

Conclusion

Inhalation exposure to dibutylethanolamine to maximum of 236.3 mg/m³ during 2 weeks premating, mating, gestation period 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. No adverse parental or pup findings were evident at any concentration.In histopathology lesions of nasal epithelia was observed in animals exposed to 72.1 and 236.3 mg/m³ test substance. Thus, concerning reproductive and developmental parametersthe no observed adverse effect concentration (NOAEC) for dibutylethanolamine was determined to be >236.3 mg/m3. Considering histopathological changes the NOAEC fordibutylethanolaminewas 20.6 mg/m³.