Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

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

Data source

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

Materials and methods

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

Test material

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

Test animals

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

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

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

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

- Diet (e.g. ad libitum): ad libitum (Kliba maintenance diet mouse-rat “GLP” meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland) 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:

Administration / exposure

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 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
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
Doses / concentrationsopen allclose all
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

Examinations

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 perfomed;
- 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.
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.


Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
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.
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.
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
Other effects:
not examined

Reproductive function / performance (P0)

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

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)

There were no test substance-related or spontaneous mortalities in any of the groups.
During the pre-exposure period, the pre-mating and the mating period the animals showed no clinical signs and findings different from normal.

During the post-mating day the male animals showed no clinical signs and findings different from normal. During the 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 FOOD CONSUMPTION (PARENTAL ANIMALS)

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.


REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)

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.


ORGAN WEIGHTS (PARENTAL ANIMALS)
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 (PARENTAL ANIMALS)
All findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.
The mating pair (Nos. 17/117), which did not produce offspring did not show relevant gross lesions.

HISTOPATHOLOGY (PARENTAL ANIMALS)
Treatment-related findings were observed in level I of larynx, and level I and II of nasal cavity, in males and females as well as in epididymis and testis in male animals with incidences and grading shown in the tables 6-9 below.

OTHER FINDINGS (PARENTAL ANIMALS)
No treatment-related changes among hematological parameters were observed. 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.
- Functional Observational Battery (FOB) nd Motor activity measurement (MA): 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. No test substance-related or spontaneous findings were observed in male and female animals of all test groups (please refer to section 7.5.2. for detailed information).

Effect levels (P0)

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.

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined

Details on results (F1)

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

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

BODY WEIGHT (OFFSPRING)
No test compound-related influence on F1 pup body weights and pup body weight change were noted in all test groups.
Two female runts were seen in the control and two female runts in test group 3 (225 mg/m³). As there were as many runts in the control as in test group 3, this finding was considered to be incidental and not substance-related

SEXUAL MATURATION (OFFSPRING)
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

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

Effect levels (F1)

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.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

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

 

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

Test group

Target concentration
(mg/m³)

Measured concentration (mg/m³)

Nominal concentration (mg/m³)

Effectiveness of vapor generation
(%)

Mean

SD

1

25

20.6

6.0

21.5

95.8

2

75

72.1

20.0

86.0

83.8

3

225

236.3

39.5

301.0

78.5

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

Measurements concerning operation conditions

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

 

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

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

Food, drinking water and bedding enrichment analyses

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

Absolute and relative organ weights

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

Table 2: Relative increase of absolute weights in males

Absolute weights

Males

Group

(mg/m³)

1

(25)

2

(75)

3

(225)

Epididymides

88%**

93%

90%**

Spleen

137%**

96%

90%

Testes

98%

93%*

92%*

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

Table 3: Relative increase of absolute weights in females 

Absolute weights

Females

Group

(mg/m³)

1

(25)

2

(75)

3

(225)

Liver

102%

103%

89%*

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

Table 4: Relative increase of relative weights in males 

Relative weights

Males

Group

(mg/m³)

1

(25)

2

(75)

3

(225)

Epididymides

88%**

94%

94%

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

Table 5: Relative increase of relative weights in females

Relative weights

Females

Group

(mg/m³)

1

(25)

2

(75)

3

(225)

Thymus

129%*

96%

107%

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

 

Weight changes in thymus, spleen and liver were regarded as incidental as no dose response relationship and/or histopathological correlates were detected.

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

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

Histopathology

Treatment-related findings were observed in level I of larynx, and level I and II of nasal cavity, in males and females as well as in epididymis and testis in male animals with incidences and grading shown in the table below:

 

Larynx

Treatment – related findings in the larynx were epithelial alteration characterized by slight modification of epithelial cells (i.e., three to four cell layers, focally flattened and stratified) indicating beginning metaplastic transformation and minimal to slight squamous metaplasia (characterized by three to four cell layers of flattened, stratified epithelium with no signs of keratinization and only affecting the epiglottis when graded minimal and up to five cell layers of flattened, stratified epithelium, occasionally minimal focal keratinization, with/without focal desquamation of superficial cells when graded slight). Table 6: Incidence and grading of histological findings in larynx

 

Male animals

Female animals

Test group

(mg/m³)

0

(0)

1

(25)

2

(75)

3

(225)

0

(0)

1

(25)

2

(75)

3

(225)

No. of animals

10

10

10

10

9

10

10

10

Epithelial alteration

1

6

8

5

1

6

8

8

  • Grade 1

1

6

8

4

1

6

4

8

  • Grade 2

 

 

 

1

 

 

4

 

Metaplasia, squamous

 

 

2

5

 

 

1

2

  • Grade 1

 

 

2

4

 

 

1

2

  • Grade 2

 

 

 

1

 

 

 

 

Nasal cavity, level I

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

Degeneration / regeneration of transitional and respiratory epithelium was characterized by variable vacuolation, presence of few apoptotic bodies, minimal infiltrates of inflammatory cells, increased size and basophilia of nuclei and minimal disorganization of cells.

Metaplasia, squamous was characterized by flattened epithelial cells with variably present minimal keratinization.

 

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

 

Male animals

Female animals

Test group

(mg/m³)

0

(0)

1

(25)

2

(75)

3

(225)

0

(0)

1

(25)

2

(75)

3

(225)

No. of animals

10

10

10

10

9

10

10

10

Degeneration /regeneration transitional epithelium

 

 

 

2

 

 

1

8

  • Grade 1

 

 

 

2

 

 

1

7

  • Grade 2

 

 

 

 

 

 

 

1

Metaplasia, squamous, transitional epithelium

 

 

 

1

 

 

 

3

  • Grade 1

 

 

 

1

 

 

 

3

Degeneration /regeneration respiratory epithelium

 

 

 

2

 

 

 

 

  • Grade 1

 

 

 

2

 

 

 

 

Nasal cavity, level II:

One male test group 3 (225 mg/m³) animal (No 134) showed minimal degeneration / regeneration of the olfactory epithelium, in level I it showed degeneration/regeneration of the transitional epithelium)

 

Testes

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

Table 8: Incidence and grading of histological findings in testes


 

Male animals

Test group

(mg/m³)

0

(0)

1

(25)

2

(75)

3

(225)

No. of animals

10

10

10

10

Degeneration, tubular

4

6

7

8

  • Grade 1

3

2

2

3

  • Grade 2

 

3

3

4

  • Grade 3

1

1

2

1

Sperm plug

 

 

 

1

  • present

 

 

 

1

 

Epididymides

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

 

Table 9: Incidence and grading of histological findings in epididymides

 

Male animals

Test group

(mg/m³)

0

(0)

1

(25)

2

(75)

3

(225)

No. of animals

10

10

10

10

Debris

2

4

4

7

  • present

2

4

4

7

 

All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

 

Fertility

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

Applicant's summary and conclusion

Conclusions:
Inhalation exposure to dibutylethanolamine to maximum of 236.3 mg/m³ during 2 weeks premating, mating, gestation period did not cause any adverse effect with regard to reproductive and developmental parameters, but only transiently reduced food consumption, body weight and body weight gain. No adverse parental or pup findings were evident at any concentration. In histopathology lesions of nasal epithelia was observed in animals exposed to 72.1 and 236.3 mg/m³ test substance. Thus, concerning reproductive and developmental parameters the no observed adverse effect concentration (NOAEC) for dibutylethanolamine was determined to be >236.3 mg/m3. Considering histopathological changes the NOAEC for dibutylethanolamine was 20.6 mg/m³.
Executive summary:

To evaluate the toxicity profile of Dibutylethanolamine (DBEA) after inhalation exposure, groups of ten male and ten female Wistar rats (F0 animals) per test group were exposed nose-only to 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.