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REACH

2,2'-butyliminodiethanol

EC number: 203-055-0 | CAS number: 102-79-4

Life Cycle description
Uses advised against

Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:: developmental toxicity
Type of information:: experimental study
Adequacy of study:: key study
Study period:: 05Mar2019 to 27Nov2019
Reliability:: 1 (reliable without restriction)
Rationale for reliability incl. deficiencies:: guideline study

Cross-referenceopen all close all

Cross-reference 1

Reason / purpose for cross-reference:: reference to other study

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

experimental study

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

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)

Deviations:

Qualifier:

according to guideline

Guideline:

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

Deviations:

Qualifier:

according to guideline

Guideline:

other: U.S. EPA OPPTS Guidelines 870.3650

Deviations:

GLP compliance:

yes (incl. QA statement)

Remarks:

Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht, Mainz, Germany

Limit test:

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system 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:

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

Dose / conc.:

20.6 mg/m³ air (nominal)

Dose / conc.:

72.1 mg/m³ air (nominal)

Dose / conc.:

236.3 mg/m³ air (nominal)

No. of animals per sex per dose:

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.

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".

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

Endocrine findings:

not specified

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

not specified

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

Neuropathological findings:

not specified

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

Other effects:

not specified

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, viability/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).

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

Critical effects observed:

not specified

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 (%)
Test group	Target concentration (mg/m³)	Mean	SD	Nominal concentration (mg/m³)	Effectiveness of vapor generation (%)
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)

(25)

(75)

(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)

(25)

(75)

(225)

Epididymides

88 %**

94 %

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

Table 5: Relative increase of relative weights in females

Relative weights

Females

Group

(mg/m³)

(25)

(75)

(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.

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 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.

Cross-reference 2

Reason / purpose for cross-reference:: reference to other study

Reference

Endpoint:: screening for reproductive / developmental toxicity
Type of information:: experimental study
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
Reason / purpose for cross-reference:: reference to same study
Reason / purpose for cross-reference:: reference to other study
Qualifier:: according to guideline
Guideline:: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:: no
Qualifier:: according to guideline
Guideline:: other: Inhalation exposure following OECD - Guideline method 413
Deviations:: no
Qualifier:: according to guideline
Guideline:: other: U.S. EPA OPPTS Guidelines 870.3650
Deviations:: no
GLP compliance:: yes (incl. QA statement)
Remarks:: Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht, Mainz, Germany
Limit test:: no
Species:: rat
Strain:: Wistar
Sex:: male/female
Details on test animals or test system 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) escept 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:
Route of administration:: inhalation: vapour
Type of inhalation exposure (if applicable):: nose only
Vehicle:: air
Details on 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 other 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
Details on mating procedure:: - Impregnation procedure: [cohoused]
- If cohoused: The animals were paired by placing the female in the cage of the male mating partner from about 15:00-16.00 h until 06.00-07.00 h of the following morning.
- M/F ratio per cage: 1/1
- Length of cohabitation: 2 weeks
Throughout the mating period, each female animal was paired with a predetermined male animal from the same test group.
- Proof of pregnancy: [sperm in vaginal smear] referred to as [day 0 ] of pregnancy
- Any other deviations from standard protocol: Deviations from the specified times were possible on weekends and public holidays (specified in the raw data).
- After successful mating each pregnant female was caged (how): individually. Pregnant animals and their litters were housed together until PND 4 (end of lactation).
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
Details on study schedule:: Not applicable
Dose / conc.:: 20.6 mg/m³ air (analytical)
Dose / conc.:: 72.1 mg/m³ air (analytical)
Dose / conc.:: 236.3 mg/m³ air (analytical)
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

- Rationale for animal assignment (if not random): randomized
- 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.
Positive control:: None
Parental animals: Observations and examinations:: 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

OTHER:
- Functional observation battery (FOB): home cage and open field observations as well sensory motor tests/reflexes have been performed;
- Motor activity measurement (MA);
- Opththalmoscopis observations (included in the detailed clinical observation);
- Haematology and clinical chemistry.
Oestrous cyclicity (parental animals):: Not examined
Sperm parameters (parental animals):: Parameters examined in [P] male parental generations:
[testis weight, epididymis weight]
Litter observations:: STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: [no] All pups delivered from the F0 parents (F1 litter) were examined as soon as possible on the day of birth to determine the total number of pups, the sex and the number of liveborn and stillborn pups in each litter. At the same time, the pups were also being examined for macroscopically evident changes. Pups, which died before this initial examination, were defined as stillborn pups.

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, physical or behavioural abnormalities, other:]
In general, a check was made for any dead or moribund pups twice daily on workdays (once in the morning and once in the afternoon) or as a rule, only in the morning on Saturdays, Sundays or public holidays.
The live pups were examined daily for clinical symptoms (including gross-morphological findings) during the clinical inspection of the dams and documented for each pup.
The pups were weighed on the day after birth (PND 1) and on PND 4. Pups' body weight change was calculated from these results. The individual weights were always determined at about the same time of the day (in the morning). “Runts” were defined on the basis of the body weights on PND 1. "Runts" are pups that weigh less than 75 % of the mean weight of the respective control pups.

GROSS EXAMINATION OF DEAD PUPS:
[yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead.]
Postmortem examinations (parental animals):: SACRIFICE
- Male animals: All surviving animals [on day 29]
- Maternal animals: All surviving animals [on PND 9: after the necropsy of the pups 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.]

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table [4] were prepared for microscopic examination and weighed, respectively.

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
Postmortem examinations (offspring):: SACRIFICE
- The F1 offspring were sacrificed at [4] days of age.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:
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.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGTHS
Not reported. 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.

- Soft tissue examinations: Yes: [all per litter], macroscopically
- Skeletal examinations: Yes: [all per litter], macroscopically
- Head examinations: Yes: [all per litter], macroscopically
Statistics:: Statistical methods are summarised in "Overall remarks, attachments".
Reproductive indices:: The pairing partners, the number of mating days until vaginal sperm was detected in the female animals, and the gestational status of the females were recorded for F0 breeding pairs.

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

The pairing partners, the number of mating days until vaginal sperm were detected, and gestational status were recorded for F0 females.

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

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
Offspring viability indices:: The number and percentage of dead pups on the day of birth (PND 0) and of pups dying between PND 1-4 (lactation period) were determined. Pups which died accidentally or were sacrificed due to maternal death were not included in these calculations. The number of live pups/litter was calculated on the day after birth, and on lactation day 4. The viability index was calculated according to the following formula:

Viability index (%) = (number of live pups on day 4 after birth / number of live pups on the day of birth) x 100.

On the day of birth (PND 0) the sex of the pups was determined by observing the distance between the anus and the base of the genital tubercle; normally, the anogenital distance is considerably greater in male than in female pups. The sex of the pups was finally confirmed at necropsy.

The sex ratio was calculated at day 0 and day 4 after birth according to the following formula:

Sex ratio = (number of live male or female pups on day 0/4 / number of live male and female pups on day 0/4) x 100.
Clinical signs:: no effects observed
Description (incidence and severity):: 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.
Dermal irritation (if dermal study):: not examined
Mortality:: no mortality observed
Description (incidence):: There were no test substance-related or spontaneous mortalities in any of the groups.
Body weight and weight changes:: effects observed, treatment-related
Description (incidence and severity):: Food consumption and 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.

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).

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 and compound intake (if feeding study):: effects observed, treatment-related
Description (incidence and severity):: food consumption and 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 efficiency:: not examined
Water consumption and compound intake (if drinking water study):: not examined
Ophthalmological findings:: not examined
Haematological findings:: no effects observed
Clinical biochemistry findings:: no effects observed
Endocrine findings:: not specified
Urinalysis findings:: not examined
Behaviour (functional findings):: not specified
Immunological findings:: not specified
Organ weight findings including organ / body weight ratios:: effects observed, treatment-related
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:: not specified
Other effects:: not examined
Reproductive function: oestrous cycle:: not examined
Reproductive function: sperm measures:: effects observed, treatment-related
Description (incidence and severity):: increased weight of testes and epididymides
Reproductive performance:: no effects observed
Description (incidence and severity):: 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 variation inherent in the strain of rats used for this study.

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.
Dose descriptor:: NOAEC
Effect level:: > 236.3 mg/m³ air (analytical)
Based on:: test mat.
Sex:: male/female
Basis for effect level:: other: No adverse effect with regard to reproductive and developmental parameters.
Clinical signs:: no effects observed
Description (incidence and severity):: There were no test substance related adverse clinical signs observed in any of surviving F1 generation pups of the different test groups.
Dermal irritation (if dermal study):: not examined
Mortality / viability:: no mortality observed
Description (incidence and severity):: 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.
Body weight and weight changes:: no effects observed
Description (incidence and severity):: 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.
Food consumption and compound intake (if feeding study):: not examined
Food efficiency:: not examined
Water consumption and compound intake (if drinking water study):: not examined
Ophthalmological findings:: not examined
Haematological findings:: not examined
Clinical biochemistry findings:: not examined
Urinalysis findings:: not examined
Sexual maturation:: no effects observed
Description (incidence and severity):: 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 weight findings including organ / body weight ratios:: not examined
Gross pathological findings:: no effects observed
Description (incidence and severity):: 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.
Histopathological findings:: not examined
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: No adverse effect with regard to reproductive and developmental parameters.
Reproductive effects observed:: not specified
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 vapours of DBEA at target concentrations of 25, 75 and 225 mg/m³ (correspond to measured concentrations 20.6, 72.1 and 236.3 mg/m³, respectively) for 6 hours per day on each day (BASF SE, 2013, Report No.87R0286/05I017, GLP, OECD 422). 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 total exposure amounts to 28 and 50 day in males and females, respectively. The test substance did not cause any adverse effect with regard to reproductive and developmental parameters. The male mating index was 100 % in all groups including the controls. The male fertility index ranged between 90 % and 100 % without showing any relation to exposure concentration. The female mating and fertility indices and duration of gestation were similar to controls. 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. There were no statistically significant differences in post-implantation loss between the groups and the mean number of F1 pups delivered per dam remained unaffected. The rate of liveborn pups was also not affected by the test substance. 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 viability index and sex ration of pups did not show any association to the treatment. There were no test substance related adverse clinical signs observed in any of surviving F1 generation pups of the different test groups. No test compound-related influence on F1 pup body weights and pup body weight change were noted in all test groups. No findings were observed at gross necropsy in any male or female pups of all test groups. Systemic toxicity effects were only transiently reduced food consumption, body weight and body weight gain in parental animals. In their histopathology, lesions of nasal epithelia were observed in animals exposed to 72.1 and 236.3 mg/m³ test substance. Thus, concerning systemic toxicity, reproductive and developmental parameters the NOAEC for 2-dibutylaminoethanol was determined to be 236.3 mg/m³ for male and female animals. Considering histopathological changes, the NOAEC for local effects was 20.6 mg/m³.

Data source

Reference

Reference Type:: study report
Title:: Unnamed
Year:: 2020
Report date:: 2020

Materials and methods

Test guideline

Qualifier:: according to guideline
Guideline:: OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:: 25 Jun 2018

GLP compliance:: yes
Limit test:: no

Test material

Test material information

Test material form:: other: liquid
Details on test material:: - Name of test material (as cited in study report): N,N-Dibutylethanolamine
- Physical state: liquid

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

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- The stability of the test substance under storage conditions over the test period was guaranteed by the sponsor, and the sponsor holds this responsibility.
- Solubility and stability of the test substance in the vehicle The stability of Dibutylethanolamine (DBEA) in corn oil at room temperature for a period of 7 days was proven before the start of the administration period

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

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

Test animals

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

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

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

Administration / exposure

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

TEST GROUPS AND DOSES
Test group Dose [mg/kg bw/d] Concentration [g/100 mL] Volume [mL/kg bw/d] .
0: 0 [mg/kg bw/d] 0 [g/100 mL] 4a)
1: 20 [mg/kg bw/d] 0.50[g/100 mL] 4b)
2: 60 [mg/kg bw/d] 1.50 [g/100 mL] 4b)
3: 200[mg/kg bw/d] 5.00 [g/100 mL] 4b)
a) corn oil
b) Test substance preparations in corn oil
Analytical verification of doses or concentrations:: yes
Details on analytical verification of doses or concentrations:: Analytical verifications of the stability of the test substance in corn oil over a period of 7 days at room temperature had been verified prior to the start of the study. Samples of the test substance preparations were sent twice (at the beginning of administration and after the end of study to the analytical laboratory for verification of the concentrations.
Details on mating procedure:: Impregnation procedure: purchased timed pregnant

The animals were paired by the breeder (“time-mated”); the day of evidence of mating (= detection of vaginal plug/sperm) was referred to as GD 0. The animals arrived on the same day (GD 0) at the experimental laboratory. The following day was designated as “GD 1”. The animals were acclimated to the laboratory conditions between start of the study (beginning of the experimental phase) and first administration (GD 6).
Duration of treatment / exposure:: The test substance preparations were administered to the animals once a day orally by gavage, from implantation to one day prior to the expected day of parturition (GD 6 to GD 19), always at approximately the same time in the morning. The animals of the control group were treated with the vehicle (corn oil) in the same way. The volume administered each day was 4 mL/kg body weight. The calculation of the administration volume was based on the most recent individual body weight.
Frequency of treatment:: The test substance preparations were administered to the animals once a day orally by gavage, from implantation to one day prior to the expected day of parturition (GD 6 to GD 19), always at approximately the same time in the morning.

Doses / concentrationsopen all close all

Doses / concentrations 1

Dose / conc.:: 0 mg/kg bw/day (actual dose received)
Remarks:: control

Doses / concentrations 2

Dose / conc.:: 20 mg/kg bw/day (actual dose received)
Remarks:: low dose

Doses / concentrations 3

Dose / conc.:: 60 mg/kg bw/day (actual dose received)
Remarks:: mid dose

Doses / concentrations 4

Dose / conc.:: 200 mg/kg bw/day (actual dose received)
Remarks:: high dose

No. of animals per sex per dose:: 25
Control animals:: yes, concurrent vehicle
Details on study design:: - Dose selection rationale:
In a range-finding study (BASF project No. 10C0286/05S039), Dibutylethanolamine (DBEA) was administered by gavage to groups of five non-pregnant female Wistar rats for 28-days at dose levels of 0, 100 and 300 mg/kg body weight/day (mg/kg bw/d). Corn oil served as vehicle. A dose of 300 mg/kg bw/d induced neurobehavioral changes in females from study day 10 onwards. Clinical findings consisted of tremors in all females, twitching in three and lateral position in two out of five females. Furthermore, mortalities were seen in each one female on study days 14 and 17. From study day 18 onwards, this dose level was reduced to 200 mg/kg bw/d. In females, clonic convulsions occurred still in one animal on study day 18. Twitching was observed in up to two females on study days 19 and 20. All mentioned findings occurred between 15 min and 2 hours after administration but not thereafter. All other parameters were not affected. At the lowest dose of 100 mg/kg bw/d, no relevant findings were observed.

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

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

Examinations

Maternal examinations:: CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

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

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

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

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

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

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

Thyroid hormones
The concentrations of TSH were determined:
Total triiodothyronine (T3), Total thyroxine (T4), Thyroid stimulating hormone (TSH)
The following weights were determined in all dams sacrificed on schedule:
Thyroid glands (with parathyroid glands) (fixed)
Thyroid glands were weighed together (left and right).
Ovaries and uterine content:: After the dams had been sacrificed, they were necropsied and assessed for gross pathology.
Thyroid glands (including parathyroid glands) were sampled.
The uteri and the ovaries were removed and the following data were recorded: Weight of the unopened uterus, Number of corpora lutea, Number and distribution of implantation sites classified as: Live fetuses, Dead implantations (Early resorptions (only decidual or placental tissues visible or according to SALEWSKI from uteri from apparently non-pregnant animals and the empty uterus horn in the case of single horn pregnancy); Late resorptions (embryonic or fetal tissue in addition to placental tissue visible), Dead fetuses (hypoxemic fetuses which did not breathe spontaneously after the uterus had been opened))

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

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

Skeletal examination of the fetuses
The skeletons of the fetuses fixed in ethanol were stained according to a modified method of KIMMEL and TRAMMELL. Thereafter, the skeletons of these fetuses were examined under a stereomicroscope. After this examination the stained fetal skeletons were retained individually.
Statistics:: Simultaneous comparison of all dose groups with the control group using theDUNNETT-test (two-sided) for the hypothesis of equal means.:
Water consumption), food consumption), body weight, body weight change, corrected body weight gain (net maternal body weight change), carcass weight, weight of unopened uterus, number of corpora lutea, number of implantations, number of resorptions, number of live fetuses, proportions of preimplantation loss, proportions of postimplantation loss, proportions of resorptions, proportion of live fetuses in each litter, litter mean fetal body weight, litter mean placental weight, anogenital distance, anogenital index
Pairwise comparison of each dose group with the control group using FISHER'S EXACT test (one-sided) for the hypothesis of equal proportions: Female mortality, females pregnant at terminal
sacrifice, number of litters with fetal findings.
Pairwise comparison of each dose group with the control group using the WILCOXONtest (one-sided) for the hypothesis of equal medians.
Indices:: Conception rate, pre-impantation loss, post-implantation loss
Historical control data:: yes

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:: effects observed, treatment-related
Description (incidence and severity):: For almost all females of test group 3 (200 mg/kg bw/d) severe clinical findings were recorded during the treatment period (within the 2-hour examination interval after treatment):
twitching after treatment in 23 females from GD 7 onwards,
convulsion after treatment in 6 females from GD 10 onwards,
tremor after treatment in 3 females on GD 9 and 11,
abdominal position after treatment in 17 females from GD 8 onwards.
As mentioned above, all females of test group 3 were sacrificed prematurely on GD 13/12/11.
Furthermore, most females (23 out of 25) of the high-dose group and 6 females of the middose group showed transient salivation during the treatment period. Salivation occurred in the respective animals within the 2-hour examination interval after treatment (i.e. 0-2h) and was initially observed on GD 7 (200 mg/kg bw/d) or GD 18 (60 mg/kg bw/d). Salivation occurred most probably due to the bad taste of the test substance, was assessed to be a local effect and, therefore, considered as not adverse.
No clinical signs or changes of general behavior, which may be attributed to the test substance, were detected in any female of test group 1 (20 mg/kg bw/d).
see attached document: summary of maternal clinical observation
Description (incidence):: Due to severe clinical findings in almost all animals of test group 3, the high-dose group of 200 mg/kg bw/d was sacrificed prematurely due to animal welfare reasons on GD 13/12/11 ) of the study.
There were no further test substance-related or spontaneous mortalities in any females of test groups 0-2 (0, 20 or 60 mg/kg bw/d).
Body weight and weight changes:: effects observed, treatment-related
Description (incidence and severity):: see attached document: summary bw_bwc
From GD 8 onwards until premature sacrifice on GD 11-13 the mean body weights of the highdose dams (200 mg/kg bw/d) were lower in comparison to the concurrent control group, attaining statistical significance on GD 10 (-4 %). The average body weight gain of the highdose dams (200 mg/kg bw/d) was distinctly and statistically significantly reduced during GD 6-10 showing a body weight loss during GD 6-8. These effects were assessed as treatment related and adverse.
In test group 2 (60 mg/kg bw/d) the mean body weights were generally comparable to the concurrent control group. The average body weight gain was statistically significantly reduced on GD 6-8 but recovered afterwards and was comparable to the control values again throughout the remaining study period. If calculated for the entire treatment period (GD 6-19), the mean body weight gain of the mid-dose dams was comparable to the concurrent control group.
The mean body weights and the average body weight gain of the low-dose dams (20 mg/kg bw/d) were generally comparable to the concurrent control group throughout the entire study period.
Corrected (net) body weight gain
The corrected body weight gain of test groups 1 and 2 (20 and 60 mg/kg bw/d) revealed no difference of any biological relevance to the corresponding control group. Moreover, mean carcass weights remained unaffected by the treatment.
Food consumption and compound intake (if feeding study):: effects observed, treatment-related
Description (incidence and severity):: see attached document: summary food consumption
The mean food consumption of the high-dose dams (200 mg/kg bw/d) was statistically significantly reduced from GD 6 onwards until premature sacrifice on GD 11-13 (up to 31 % below control). This was assessed as treatment-related and adverse.
The mean food consumption of the mid- and low-dose dams (60 and 20 mg/kg bw/d) was generally comparable to the concurrent control group throughout the entire study period.
Water consumption and compound intake (if drinking water study):: effects observed, treatment-related
Description (incidence and severity):: see attached document; summary maternal water consumption
The mean water consumption of the dams in test group 3 (200 mg/kg bw/d) was statistically significantly reduced from GD 6 onwards until premature sacrifice during GD 11-13 (up to 21 % in comparison to the concurrent control). This was assessed as treatment-related and adverse.
In test group 2 (60 mg/kg bw/d) the mean water consumption was statistically significantly reduced during GD 6-10 (up to 12 % below control) but recovered afterwards and was comparable to the control values again until scheduled sacrifice on GD 20.
The mean water consumption of the dams in test group 1 (20 mg/kg bw/d) was comparable to the concurrent control group throughout the entire study period.
Ophthalmological findings:: not examined
Urinalysis findings:: not examined
Behaviour (functional findings):: not examined
Immunological findings:: not examined
Organ weight findings including organ / body weight ratios:: effects observed, non-treatment-related
Description (incidence and severity):: see attached document: summary gravid uterine weights and bwc
The mean gravid uterus weights of the low- and mid-dose animals (20 and 60 mg/kg bw/d) were not influenced by the test substance. The differences between these groups and the control group revealed no dose-dependency and were assessed to be without biological relevance.
Absolute and relative organ weights
All mean absolute and relative weight parameters did not show significant differences when compared to the control group 0.
Histopathological findings: non-neoplastic:: effects observed, non-treatment-related
Description (incidence and severity):: All findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.
Other effects:: no effects observed
Description (incidence and severity):: See attached document: Incidence of microscopic findings
Thyroid hormones
At gestation day 20, in dams of test groups 1 and 2 (20 and 60 mg/kg bw/d) no treatment related alterations of T3, T4 and TSH were observed.
Gross lesions
No macroscopic findings of thyroid glands were noted in test groups 0 (control), 1 (20 mg/kg bw/d) and 2 (60 mg/kg bw/d).

Maternal developmental toxicity

Number of abortions:: no effects observed
Pre- and post-implantation loss:: effects observed, non-treatment-related
Description (incidence and severity):: A 100 % post-implantation loss was observed in one female each in test groups 1 and 2. In one lowdose dam (20 mg/kg bw/d) one early resorption was recorded after staining the empty uterus at C-section (i.e. pregnant by stain), whereas for one mid-dose dam (60 mg/kg bw/d) 9 early and 3 late resorptions were recorded (no viable fetuses). In the other dams of test groups 1 and 2 post-implantation losses and number of resorptions were comparable to the control. Since only one dam per test group was affected without relation to dose, these isolated findings were regarded as incidental and not treatment-related.
Total litter losses by resorption:: no effects observed
Description (incidence and severity):: see attached document: summary of reproduction data
There were no test substance-related and/or biologically relevant differences between the test groups 0-2 in conception rate, in the mean number of corpora lutea and implantation sites or in the values calculated for the pre- and post-implantation losses, the number of resorptions and viable fetuses. All observed differences are considered to reflect the normal range of fluctuations for animals of this strain and age.
Early or late resorptions:: effects observed, non-treatment-related
Description (incidence and severity):: In one lowdose dam (20 mg/kg bw/d) one early resorption was recorded after staining the empty uterus at C-section (i.e. pregnant by stain), whereas for one dam in the mid-dose (60 mg/kg bw/d) 9 early and 3 late resorptions were recorded (no viable fetuses).
Dead fetuses:: no effects observed
Changes in pregnancy duration:: no effects observed
Changes in number of pregnant:: no effects observed
Details on maternal toxic effects:: There were no test substance-related and/or biologically relevant differences between the test groups 0-2 in conception rate, in the mean number of corpora lutea and implantation sites or in the values calculated for the pre- and post-implantation losses, the number of resorptions and viable fetuses. All observed differences are considered to reflect the normal range of fluctuations for animals of this strain and age.
Regarding pathology, no treatment-related findings were noted. All findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Effect levels (maternal animals)

: Key result
Dose descriptor:: NOAEL
Effect level:: 60 mg/kg bw/day (actual dose received)
Based on:: test mat.
Basis for effect level:: body weight and weight gain; clinical signs; food consumption and compound intake; water consumption and compound intake

Results (fetuses)

Fetal body weight changes:: no effects observed
Description (incidence and severity):: see attached document: summary placental and fetal bw
The mean fetal weights of test groups 1 and 2 (20 and 60 mg/kg bw/d) were not influenced by the test substance and did not show any biologically relevant differences in comparison to the control group.
Changes in sex ratio:: no effects observed
Description (incidence and severity):: The sex distribution of the fetuses in test groups 1 and 2 (20 and 60 mg/kg bw/d) was comparable to the control fetuses.
Changes in litter size and weights:: no effects observed
Changes in postnatal survival:: not examined
External malformations:: effects observed, non-treatment-related
Description (incidence and severity):: One low-dose male fetus an umbilical hernia was recorded. Since the finding was not related to dose, it was not assessed as treatment-related.
Skeletal malformations:: effects observed, non-treatment-related
Description (incidence and severity):: For only one low-dose fetus a skeletal malformation was recorded: mal positioned and bipartite
sternebra (unchanged cartilage). Since the finding was not related to dose, it was not assessed as treatment-related.
Visceral malformations:: effects observed, non-treatment-related
Description (incidence and severity):: Two soft tissue variations were detected, i.e. short innominate and dilated renal pelvis, both in test groups 0 and 1. The incidences of these variations were neither statistically significantly nor dose-dependently increased in the treated groups. Therefore, they were not assessed as treatment-related.
Other effects:: effects observed, non-treatment-related
Description (incidence and severity):: Two unclassified external observations were recorded. A skin tag was seen in one low-dose fetus (20 mg/kg bw/d), furthermore, placentae fused were seen in one control litter. These findings were not considered biologically relevant, since they were single events without relation to dose.
For all test groups, skeletal variations of different bone structures were observed, with or without effects on corresponding cartilages. The observed skeletal variations were related to several parts of fetal skeletons and appeared without a relation to dose The overall affected fetuses/litter incidences of skeletal variations were comparable to the historical control data.
Details on embryotoxic / teratogenic effects:: Soft tissue malformations did not occur in any fetus in this study. There were noted external and skeletal malformations in two fetuses of the low-dose group.. These observations in single fetuses were unrelated to dose and both can be found in the historical control data, thus they are considered as incidental events.
External variations did not occur in any fetus in this study. Two soft tissue variations and a range of skeletal variations were noted in all test groups including the controls. Two out of three incidences showed no relation to dosing. The skeletal variations are equally distributed about the different test groups, if normal biological variation is taken into account, and can be found in the historical control data at a comparable frequency.
Unclassified soft tissue observations did not occur in any of the fetuses in this study. A spontaneous origin is assumed for the unclassified external and skeletal cartilage observations which were observed in several fetuses of all test groups. The distribution and type of these findings do not suggest any relation to treatment.

Effect levels (fetuses)

: Key result
Dose descriptor:: NOAEL
Effect level:: 60 mg/kg bw/day (actual dose received)
Based on:: test mat.
Sex:: male/female
Basis for effect level:: other: teratogenicity / embryotoxicity

Fetal abnormalities

: Key result
Abnormalities:: no effects observed

Overall developmental toxicity

: Key result
Developmental effects observed:: no
Treatment related:: no

Any other information on results incl. tables

Total soft tissue variations

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

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

Total fetal skeletal variations

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

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

Occurrence of statistically significantly increased fetal skeletal

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

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

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

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

Applicant's summary and conclusion

Conclusions:: Under the conditions of this prenatal developmental toxicity study, the oral administration of Dibutylethanolamine to pregnant Wistar rats from implantation to one day prior to the expected day of parturition (GD 6-19) caused evidence of distinct maternal toxicity, such as severe clinical findings (twitching, convulsions, tremor, abdominal position), a reduction of water and food consumption and a decrease in body weight/body weight gain. Due to the above-mentioned findings, treatment of the high-dose was stopped and the dams were euthanized without further examinations on GD 13/12/11 of the study. In conclusion, the no observed adverse effect level (NOAEL) for maternal toxicity is the mid-dose of 60 mg/kg bw/d. Examination of the low- and mid-dose fetuses was performed and showed no adverse findings. In conclusion, the no observed adverse effect level (NOAEL) for prenatal developmental toxicity is 60 mg/kg bw/d. Under the conditions of this study the test item is not teratogenic.
Executive summary:: In a prenatal developmental toxicity study, the test substance Dibutylethanolamine (DBEA) was administered to pregnant Wistar rats daily by gavage from implantation to one day prior to the expected day of parturition (GD 6-19) to evaluate its potential maternal and prenatal developmental toxicity. Analyses confirmed the correctness of the prepared concentrations and the stability of the test substance in the vehicle. Concerning clinical examination, strong signs of systemic maternal toxicity was observed at the highest test group of 200 mg/kg bw/d Dibutylethanolamine. Since severe clinical findings (twitching, convulsions, tremor, abdominal position) were observed in almost all animals, the high-dose group was sacrificed prematurely due to animal welfare reasons. Treatment of that test group was stopped and the dams were euthanized without further examinations on GD 13/12/11 of the study. The next lower dose of 60 mg/kg bw/d showed no adverse clinical findings. Water and food consumptions were statistically significantly reduced in high-dose dams during GD 6-13 (up to 21 % and 31 % below control, respectively). Furthermore, high-dose damsshowed a statistically significant decrease in body weight (change) including a body weight loss during GD 6-8. The above-mentioned findings were assessed as treatment-related and adverse. Mid-dose dams showed a reduction in water consumption in the beginning of treatment (GD 6-10: up to 12 % below control) but recovered afterwards to control levels. Food consumption was not affected in mid-dose dams. Body weight change was statistically significantly reduced on GD 6-8 but recovered afterwards and body weight in general was comparable to the control values. These two marginal findings in the mid-dose group were only observed in the beginning of treatment but not thereafter. They were considered to be not sufficient to proof adversity and therefore, they were not assessed as treatment-related and adverse. In the low dose group (20 mg/kg bw/d), no adverse findings were observed. Concerning thyroid hormone levels, no treatment-related, adverse effect was observed up to a dose of the compound of 60 mg/kg bw/d. Regarding pathology, no treatment-related findings were noted. All findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment. No differences of toxicological relevance between the control and the treated groups (20 or 60 mg/kg bw/d) were determined for any reproductive parameters, such as conception rate, mean number of corpora lutea, mean number of implantations, as well as pre- and post-implantation loss. Similarly, no toxicologically relevant influence of the test substance on sex distribution and anogenital distance/index of the fetuses was noted at any dose. Overall, there was no evidence for toxicologically relevant adverse effects of the test substance on fetal morphology at any dose.

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2,2'-butyliminodiethanol

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Food, drinking water and bedding enrichment analyses

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Maternal developmental toxicity

Effect levels (maternal animals)

Results (fetuses)

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Fetal abnormalities

Overall developmental toxicity

Any other information on results incl. tables

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