2-dibutylaminoethanol

Endpoint summary

Description of key information

Repeated dose toxicity:
- oral, subacute (OECD407), rat: NOAEL(systemic toxicity; males and females) = 100 mg/kg bw, LOAEL(systemic toxicity) = 400 mg/kg bw (Mortality, depressed body weight gain, decreased food consumption, variations in clinico-chemical parameters and histological changes)

- oral,sub-chronic (OECD 408), rat: NOAEL 50 mg/kg bw/day, kidneys: vacuolation of the epithelial cells

- inhalative, screening (OECD422), rat: NOAEC(systemic; males and females) = 236.3 mg/m³ (highest dose tested), NOAEC(reproductive and developmental parameters; males and females) = 236.3 mg/m³, NOAEC(local, males and females) = 20.6 mg/m³

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP Guideline study with acceptable restrictions (original study report in japanese, english translation available; mortalities at the highest dose group).

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Atsugi Breeding Center, Charles River Laboratories Japan, Inc.
- Age at study initiation: 4 weeks old
- Weight at study initiation: Males: 149.4 – 168.4 g (Avg. 158.3 g); Females: 114.7 – 140.6 g (Avg. 125.4 g)
- Housing: Individually in metal cages with wire-mesh floor
- Diet: Free access to pellets (CE-2, Clea Japan, Inc.)
- Water: Free access to tap water (supplied by Hadano City Waterworks Bureau)
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 - 25
- Humidity (%): 40 - 75
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 / 12

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: The test substance was weighed for each concentration and treated by adding the vehicle corn oil by means of a graduated cylinder, so as to prepare test samples of the respective concentrations. The administered volume was 5 mL/kg, and the fluid volume to be administered was calculated for each individual, based on the body weight values which were the latest at the time of each administration.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentrations of the test substance in the test samples for administration were determined by gas chromatography.

Duration of treatment / exposure:

28 days

Frequency of treatment:

daily

Remarks:

Doses / Concentrations:
25, 100, 400 mg/kg bw
Basis:
actual ingested

No. of animals per sex per dose:

Males, 10; females, 10 (0, 400 mg/kg; 5 animals each for 14 day recovery period)
Males, 5; females, 5 (25, 100 mg/kg)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Doses were determined based on the results of a preliminary test.
- Post-exposure recovery period in satellite groups: yes, in control and high dose group for 14 days (5 animals each; however, 3 males and 5 females in the 400 mg/kg group died at the dosing period, and therefore, the recovery test was not performed for females.)

Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: For all cases, general conditions including life or death were observed every day (once or more before and after administration during the dosing period, once a day during the recovery test period). Observation after administration was carried out at least once approximately 30 minutes after administration (approximately 2 hours after administration for the animals in the course of the measurement of locomotor activity), and in addition, another observation was made depending on the condition of expression in the findings.

BODY WEIGHT: Yes
- Time schedule for examinations: On day 1, 2 and 4 of administration at week 1 of administration, and afterward twice a week, i.e. on day 1 and 4 of each week from week 2 of administration to week 2 of recovery test, all cases were measured for body weight before administration. Body weights were measured also on the final day of the dosing period (day 28 of administration) and on the final day of the recovery test period (day 14 of recovery test). Moreover, body weights were measured on the day of autopsy, in order to calculate the values of organ weight to body weight.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption determined and mean daily diet consumption calculated as g food/day : Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: All the cases autopsied as scheduled underwent 18–20 hour fasting before blood collection (before sacrifice/autopsy). After that, blood was collected from the abdominal caudal vena cava.
- Anaesthetic used for blood collection: Yes (pentobarbital sodium)
- Animals fasted: Yes
- How many animals: As far as possible, blood was collected from 1 each of the respective groups (males in the order of the control, low, medium and high dose groups, and females in the order of the control, low, control, medium and high dose groups or in the order of the control, low, medium, control and high dose groups at the end of the dosing period; and alternately the control and high dose groups at the end of the recovery test period), starting with the lowest animal number for all the groups.
- Parameters checked: Red blood cell count (RBC), White blood cell count (WBC), Differential white blood count, Hemoglobin content (Hb), Platelet count, Mean corpuscular volume (MCV), Hematocrit (Ht), Mean corpuscular hemoglobin (MCH), Mean corpuscular hemoglobin concentration (MCHC), Prothrombin time (PT), Activated partial thromboplastin time (APTT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: see HAEMATOLOGY
- Animals fasted: Yes
- How many animals: see HAEMATOLOGY
- Parameters checked: Total protein, Albumin, Total cholesterol, Glucose, Blood urea nitrogen (BUN), Creatinine, ALP activity, GOT (AST) activity, GPT (ALT) activity, gamma-GTP activity, Cholinesterase activity, Triglyceride, Total bilirubin, Inorganic phosphorus, Calcium, A/G ratio, Sodium, Potassium, Chlorine

URINALYSIS: Yes
- Time schedule for collection of urine: At week 4 of administration (immediately after administration) and at week 2 of recovery test (4 hours after administration and at the time when 24 hours elapsed).
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No data
- Parameters checked: Color tone, turbidity, pH, protein, ketone body, bilirubin, sugar, occult blood, urobilinogen, sediment, urine volume, specific gravity

OTHER:

Close clinical observation:
Once a week, approximately 30 minutes after administration during the dosing period and at the time equivalent to approximately 30 minutes after administration during the recovery test period, close clinical observation was carried out for all cases. The observation was performed on the following items by using the scoring method under the condition that information about the doses and animal numbers were kept blind.
Through cage: Body position/posture, locomotor activity, vocalization, tremor, convulsion; At handling: Easiness to take out, easiness to handle, heart beat, body temperature, salivation, fur, skin color, visible mucous membrane, lacrimation, exophthalmos, pupil diameter; Observation on worktable: Posture, body position, exploratory behavior, grooming, vocalization, Straub’s tail reaction, stereotyped behavior, strange behavior, gait, tremor, convulsion, respiratory rate, piloerection, optic fissure, urinary frequency, stool frequency, response to touch, withdrawal reflex, pinna reflex.

Function test
All cases were tested for the following items at week 4 of administration and week 2 of recovery test. (1) Response to auditory stimuli: Startle reaction, (2) Response to visual stimuli: Visual orientation, light reflex (Light stimuli were given alternately to the right and left eye balls to confirm direct and indirect light reflexes (The criteria for judgment were as follows: Normal = score 4, Depressed compared with normal = score 2, No response = score 0.), (3) Response to proprioceptive stimuli: Righting reflex, (4) Grip strength measurement: Grip strengths of forelimb and hindlimb were measured by using a dynamometer (Chatillon, Columbus Instrument), (5) Measurement of locomotor activity: Locomotor activity (the number of times to locomote and that of rearing) 2 hours after administration (at the time equivalent to approximately 2 hours after administration during the recovery test period) was measured by using a locomotor activity monitoring system (SUPER-MEX, Muromachi Kikai Co., Ltd.).

Sacrifice and pathology:

Terminal sacrifice:
Males, on day 29 and 43
Females, on day 29

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

The following organ weights (actual weights) of each animal were measured, and also the respective organ weights were divided by body weight on the autopsy day to obtain the respective weights to body weight (relative weights): Liver, kidneys, adrenals, testes, epididymes, ovaries, thymus, spleen, brain, heart.
Organs and tissues fixed/preserved: Gross lesions, brain, spinal cord, liver, kidneys, adrenals, spleen, heart, stomach, small intestine (duodenum, jejunum, ileum), large intestine (colon, rectum), thymus, thyroid gland, trachea, lungs (including bronchi), gonad (testes, ovaries), accessory reproductive organs (epididymes, prostate, uterus, seminal vesicle, vagina), urinary bladder, lymph nodes (mesenteric lymph nodes, mandibular lymph nodes), sciatic nerves (including gastrocnemius muscle), femora and bone marrow, aortae, tongue, esophagus, pancreas, submaxillary gland, sublingual gland, hypophysis, parathyroid gland.

Statistics:

Quantitative laboratory data: Average value and standard deviation were obtained for each group; the respective laboratory values during and at the end of the dosing period underwent a test for homogeneity of variance (Bartlett method). Then one-way analysis of variance was conducted in the case where variance was homogeneous, and multiple comparison was made (Dunnett method) if there were any significances among the groups. The Kruskal-Wallis rank test was performed when variance was heterogeneous, and multiple comparison was drawn (Dunnett method) if there were significances among the groups. If variance was “0” in any group, Kruskal-Wallis rank test was carried out in place of Bartlett method, and at the same time, multiple comparison was made. The respective laboratory values during and at the end of the recovery test period were subjected to F-test; and Student’s t-test was performed in the case of homogeneous variance, while Aspin-Welch’s t-test was carried out in the case of inhomogeneous variance. If the variance was “0” in either group, t-test was not conducted.
Results of semiquantitative test (test results by using urine test paper, as well as color tone and turbidity of urine): The respective laboratory values obtained at week 4 of administration underwent chi square test; and when any significance was found, multiple comparison was drawn (Dunnett method). Color tone, turbidity, glucose and bilirubin for males and females as well as urobilinogen for males did not undergo the test, because the variance was “0”. The respective laboratory values obtained at week 2 of recovery test were subjected toWilcoxon rank-sum test. For color tone, turbidity, glucose, bilirubinand urobilinogen, the test was not performed, because the whole variance was “0”.
Histopathological findings: Test for significance between the control and each of the other administration groups (Mann-Whitney U test and Fisher’s one-sided exact test, respectively).

Details on results:

CLINICAL SIGNS AND MORTALITY
In the 400 mg/kg bw group, 3 males and 5 females in total died, i.e. 1 female on day 11 of administration, 2 males and 1 female on day 15, 1 male and 2 females on day 18, and 1 female on day 25. There were no dead cases in the other groups including the control. All the dead cases were found on the day of changing the fluid volumes to be administered (the day of body weight measurement).
Clinical signs of intermittent cage-licking and chewing were observed in the 100 and 400 mg/kg bw groups (males and females) after administration on day 1. In many cases, these actions were seen several minutes after administration, and recovery was shown no later than approximately 2 hours after administration. However, for 1 female observed approximately 1 hour and a half after administration on day 9 of administration and for 1 male observed approximately 1 hour and 50 minutes after administration on day 17 of administration, recovery was seen 3 hours after administration.
In the 400 mg/kg bw group, males and females showed clonic or tonic convulsion, spasm, tremor, abnormal phonation and pale skin, and subsequently gasping, slow respiration, deep respiration and prone position, and females occasionally exhibited apathy from day 4 of administration onward. The aforementioned convulsion, spasm, tremor and the like developed approximately 20 - 40 minutes after administration, and recovery was shown no later than approximately 1 hour after administration. Moreover, although gasping, slow respiration, apathy, prone position and the like rapidly subsided afterward, for 1 female showing convulsion approximately 1 hour after administration on day 4 of administration, it was approximately 2 and a half hours after administration that all the symptoms disappeared. Additionally, in the 400 mg/kg bw group, there were crouching and eyelid closure for several males and females, hyperactivity for 1 male, and running, crawling, exophthalmos and reddish tear for 1 to several females; 1 male exhibited gingival bleeding after convulsion, while 1 female showed abnormal respiratory sound (sniffling) before administration. Moribund cases exhibited slow respiration and prone position after convulsion and died during a period approximately 25 - 45 minutes after administration.
For males and females in the 400 mg/kg bw group, salivation was observed at the time of restraint for administration or immediately after administration from week 2 of administration onward. In many cases, salivation disappeared no later than approximately 30 minutes after administration. Salivation was also seen for 1 male in the 100 mg/kg bw group. In the same group, another case of salivation was seen occasionally, which was found not immediately after administration but approximately 30 minutes after administration from day 2 of administration onward.

Close Clinical Observation:
For males and females in the 400 mg/kg bw group, convulsion, vocalization and salivation were reported, which were similar to those found at the time of observing general conditions. Also abnormal gait such as abasia as well as decrease in exploration were observed. At the same time, occasionally, enhanced locomotor activity and mild piloerection were seen for males, while tremor, pale skin, prone position, slow respiration, bradycardia, reddish tear, exophthalmos, decrease in or loss of withdrawal reflex, blunted pinna reflex, reduced or no response to touch and decrease in or loss of locomotor activity were found for females. For males and females in the 100 and 400 mg/kg bw groups, intermittent licking of the inside of cage and chewing-like action were observed, which were similar to those found at the time of observing general conditions. With regard to other items, there were no differences between the control and each of the other administration groups. In addition, 3 females in the 400 mg/kg bw group developed convulsion during close clinical observation, and died from respiratory arrest. Thus the data on the corresponding observation day could not be acquired. According to the results of examination at the recovery test period, there were no differences between the control and each of the other administration groups.

Function Test:
1) Startle reaction, visual orientation, light reflex and righting reflex:
All animals showed normal response throughout all the periods including that of the recovery test.

2) Grip strength measurement:
There were no significant differences between the control and each of the other administration groups throughout all the periods including that of the recovery test.

3) Measurement of locomotor activity:
According to the results of examination at week 4 of administration, when making comparisons on a basis of every 10 minutes, males in the 400 mg/kg bw group showed a significant increase in number of times to locomote for the respective periods of 10 minutes starting from 50 and 100 minutes after administration, and exhibited a significant increase in number of rearing for the period of 10 minutes starting from 100 minutes after administration. Also females in the 100 mg/kg bw group showed a significant increase in number of rearing for the respective periods of 10 minutes starting from 20 and 90 minutes after administration. When making comparisons by dividing up the measuring time into two 60-minute periods as the first and second halves, males in the 400 mg/kg bw group showed a significant increase in number of times to locomote in the second 60 minutes, and also females displayed a tendency toward increase. Additionally, concerning the total quantity of motion for 120 minutes after administration, males in the same group exhibited a significant increase in number of times to locomote. As a result of examination at week 4 of administration, there were no other significant differences between the control and each of the other administration groups.

BODY WEIGHT AND WEIGHT GAIN
Females in the 400 mg/kg bw group showed a significantly low value compared with the control group on day 4 of administration. There were no other significant differences between the control and each of the other administration groups throughout all the periods including recovery.

FOOD CONSUMPTION
For males and females in the 400 mg/kg bw group, food consumptions measured from day 1 to day 2 of administration at week 1 of administration significantly decreased compared with the control group. For both males and females, there were no other significant differences between the control and each of the other administration groups throughout the observation period.

HAEMATOLOGY
No changes due to administration of the test substance were seen.

CLINICAL CHEMISTRY
For the cases autopsied at the end of the dosing period, males and females in the 400 mg/kg bw group showed a significant decrease in chlorine level. In the 400 mg/kg bw group, the sodium level significantly decreased for males, while females developed tendencies toward significantly increased levels of total cholesterol and glucose as well as declined cholinesterase activity. At the same time, females in the 100 mg/kg bw group showed a significant increase in triglyceride level, and also females in the 400 mg/kg bw group had a tendency toward a high value. For the cases autopsied at the end of the recovery test period, there were no other significant changes, comparing the control with each of the other administration groups.

URINALYSIS
No symptoms caused by the administration of the test substance were seen.

ORGAN WEIGHTS and GROSS PATHOLOGY

Organ weights:
For the cases autopsied at the end of the dosing period, males and females in the 400 mg/kg bw group showed a significant increase in relative liver weight. There were also significant increases in relative kidney weight for males as well as in actual and relative adrenal weights for females. There were no other significant differences between the control and each of the other administration groups. For the cases autopsied at the end of the recovery test period, males in the 400 mg/kg bw group showed a significant increase in relative spleen weight. There were no other significant differences between the control and each of the other administration groups.

Autopsy findings:
For the cases autopsied at the end of the dosing period, 1 male in the 400 mg/kg bw group showed an increase in liver size, while 1 female in the 25 mg/kg bw group exhibited a decrease in spleen size. For both males and females in the respective groups including the cases autopsied at the end of the recovery test period, no other changes were observed. As to dead cases consisting of 3 males and 5 females found in the 400 mg/kg bw group, 1 male and 4 females exhibited an increase in liver size; in addition, there were an increase in kidney size for 1 male and 2 females, dark red part in the lung for 1 male, and foamy liquid as contents in the trachea for 1 male.

Histological findings:
(1) Cases autopsied at the end of the dosing period:
For cases sacrificed/autopsied at the end of the dosing period, the following changes were observed in the kidney, liver, adrenal and spleen. Concerning the kidney, in the 400 mg/kg bw group, vacuolation of the epithelial cells of the collecting ducts was observed as a moderate change for all males and as a mild to moderate change for 3 females, and there were significant increases in occurrence frequency and change degree for males as well as in occurrence frequency for females, respectively. With regard to the liver, 2 females in the 400 mg/kg bw group as well as 1 male exhibiting an increase in liver size showed slight centrolobular hypertrophy of hepatocytes, and for 1 female among them, liver cell mitosis was faintly seen. As to the adrenal, 2 females in the 400 mg/kg bw group showed slight hypertrophy of the zona fasciculata. About the spleen, for 1 female in the 25 mg/kg bw group showing a decrease in spleen size at autopsy, there were no findings other than slight extramedullary hematopoiesis and hemosiderosis.

Dead cases (3 males and 5 females in the 400 mg/kg bw group):
For dead cases, the same changes as those autopsied at the end of the dosing period were seen in the kidney, adrenal, and also other changes were observed in the lung, thymus and the like. Regarding the kidney, vacuolation of the epithelial cells of the collecting ducts was found as a moderate change for all males and a slight to moderate change for 3 females. As to the adrenal, 2 females exhibited slight hypertrophy of the zona fasciculata. Concerning the lung, for 1 male exhibiting dark red part in the lung and foamy liquid in the trachea at autopsy, mild pulmonary hemorrhage and slight edema were observed. Moreover, for 2 males and 1 female, slight or mild hemorrhage and hyperemia in the thymus were seen. Additionally, there were no changes in the lung of a size macroscopically increased.

Cases autopsied at the end of the recovery test period:
For the cases autopsied at the end of the recovery test period, no changes caused by the administration of the test substance were found and it was suggested that the effect of administrating the test substance was reversible with 14-day discontinuation of administration.

Dose descriptor:

NOEL

Effect level:

25 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: systemic toxicity

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: systemic toxicity

Dose descriptor:

LOAEL

Effect level:

400 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: mortality, depressed body weight gain, decreased food consumption, variations in clinico-chemical parameters and histological changes in different organs

Critical effects observed:

not specified

Reference 2

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Nov 2018 - Dec 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

25 Jun 2018

GLP compliance:

yes

Limit test:

no

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: ambient
- Stability under storage conditions:
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
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 emulsion

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

Species:

rat

Strain:

Wistar

Details on species / strain selection:

The rat is a frequently used laboratory animal, and there is comprehensive experience with this
animal species. Moreover, the rat has been proposed as a suitable animal species by the OECD
and the EPA for this type of study.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services GmbH, Sulzfeld, Germany
- Age at study initiation: 42 ± 1 days
- Weight at study initiation: males 165.8 - 167.2 / females 131.0 - 134.3
- Housing: The animals were housed together (5 animals per cage and sex) in H-Temp polysulfonate
cages type 2000P supplied by TECNIPLAST, Hohenpeißenberg, Germany
- Diet (e.g. ad libitum): yes
- Water (e.g. ad libitum): yes
- Acclimation period: yes


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

IN-LIFE DATES:
Males Females Phase of study/ Examination /Study day
19 Nov 2018 Study initiation date: signature of study director/-7
20 Nov 2018 Experimental starting date: arrival of the animals and start of the
acclimatization period/-6
23 Nov 2018 Randomization of the animals /3
26 Nov 2018 Ophthalmoscopy/0
26 Nov 2018 Start of the administration period/0
From 28 Jan 2019 onwards Estrous cycle determination From /63
19 Feb 2019 FOB1; MA1 a) /85
20 Feb 2019 FOB2; MA2 b) /86
21 Feb 2019 FOB3; MA3 a) /87
22 Feb 2019 FOB4; MA4 b) /88
female 20 Feb 2019 Urinalysis/86
male 22 Feb 2019 Urinalysis/88
25 Feb 2019 Ophthalmoscopy 91
25 Feb 2019 Last weighing 91
26 Feb 2019 /27 Feb 2019 Blood sampling and necropsy c) /92/93
20 Dec 2019 Experimental completion date; draft report to QAU

a) = First 5 animals of every test group
b) = Second 5 animals of every test group
c) = Fasting period (withdrawal of food) of about 16 to 20 hours
FOB = Functional observational battery
MA = Measurement of motor activity
1..4 = Identification number of the specific examination

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

Dibutylethanolamine was applied as an emulsion. To prepare this emulsion, the appropriate
amount of test substance was weighed out depending on the desired concentration. Then,
corn oil was filled up to the desired volume and subsequently mixed with a magnetic stirrer.
During administration, the test substance preparations were kept homogeneous by stirring
with a magnetic stirrer. The test substance preparations were prepared in such a manner that
the stability was guaranteed. The administration volume was 4 mL/kg body weight.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability of the test substance in corn oil was demonstrated over a period of 7 days at
room temperature. As the mixtures were stored no longer than this time period, the stability
was guaranteed.
Considering the low relative standard deviation in the homogeneity analysis, it can be
concluded that Dibutylethanolamine was distributed homogeneously in corn oil.
The concentration control analyses of test groups 1 to 3 revealed that the values were in the
expected range of the target concentrations, i.e. were always in a range of about 90-110% of
the nominal concentrations at any time point.
Taken together, the results demonstrate the correctness of the concentrations of the test item
in the vehicle for test groups 1 to 3.

Duration of treatment / exposure:

On day of arrival, the animals were subjected to an acclimatization period during which they
received ground diet and drinking water ad libitum. Prior to the first detailed clinical
observation, the animals were distributed according to weight among the individual test
groups, separated by sex. The weight variation of the animals used did not exceed 20 percent
of the mean weight of each sex. The list of randomization instructions was compiled with a
computer.
The test substance was administered daily for 13 weeks. Control animals received only the
vehicle. All remaining animals were sacrificed after a fasting period (withdrawal of food) of at
least 16-20 hours.

Frequency of treatment:

The test substance was administered daily by gavage for 3 months

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Remarks:

control

Dose / conc.:

15 mg/kg bw/day (actual dose received)

Remarks:

low dose

Dose / conc.:

50 mg/kg bw/day (actual dose received)

Remarks:

mid dose

Dose / conc.:

150 mg/kg bw/day (actual dose received)

Remarks:

high dose

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

Rational for dose setting:
Dibutylethanolamine was tested in a 28-day oral toxicity study (OECD 407; including a 2-weeks
recovery period) using Sprague-Dawley rats (Hatano Research Institute, Food and
Drug Safety Center, commissioned by the Ministry of Health, Labor and Welfare, Japan).
Groups of 5 male and 5 female animals were treated at dose levels of 0, 25, 100 and 400
mg/kg bw/d (corn oil served as vehicle) for 4 weeks; another 5 animals per sex were added
to the control and the high-dose groups in view of the recovery test.
In male and female animals treated at a dose level of 400 mg/kg bw/d, severe clinical signs
of toxicity occurred from study day 4 onwards, i.e. clonic or tonic convulsion, spasm, tremor,
abnormal phonation and pale skin, and subsequently gasping, slow respiration, deep
respiration and prone position. Three male and 5 female animals died during the course of
the treatment period, i.e. one female on study day 11, 2 males and 1 female on study day 15,
1 male and 2 females on study day 18, and 1 female on study day 25. During the pathological
examinations, findings occurred in kidneys, liver and adrenal glands (Hatano Research
Institute, 2004).
No relevant changes were observed for male and female animals treated at 25 and 100 mg/kg
bw/d. The same was true for the animals of the recovery groups, i.e. control and high-dose
group.
At BASF SE, another 28-day study to be used as range-finding study in Wistar rats was
performed (BASF project No. 10C0286/05S039). Dibutylethanolamine was administered by
gavage to groups of 5 male and 5 female Wistar rats at dose levels of 0, 100 and 300 mg/kg
bw/d (corn oil served as vehicle). Due to severe clinical findings in male and female animals
treated at 300 mg/kg bw/d, e.g. tremors, tonic/clonic convulsions and the premature deaths
of 1 male and 2 female animals, the dose level was reduced to 200 mg/kg bw/d from study
day 18 onwards. After dose level reduction, findings occurred only for a few more days but
not towards the end of the administration period. No relevant findings were observed for male
and female animals treated at 100 mg/kg bw/d.
Taking the severe findings in the high-dose group during the administration period into
account and, in addition, considering the fact that the animals expected a three-times longer
administration period, the following dose levels were selected for the present study:

150 mg/kg bw/d a) as high dose
50 mg/kg bw/d a) as intermediate dose
15 mg/kg bw/d a) as low dose

a) mg/kg body weight/day

Positive control:

No

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observation: A check for moribund and dead animals was made twice daily on
working days and once daily on Saturdays, Sundays and public holidays. If animals were
in a moribund state, they were sacrificed and necropsied.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily
Detailed clinical observations (DCO) were performed in all animals prior to the administration
period and thereafter at weekly intervals. The findings were ranked according to the degree
of severity, if applicable. The animals were transferred to a standard arena (50 × 37.5 cm
with sides of 25 cm high). The following parameters were examined:
Abnormal behavior when handled, fur, skin, posture, salivation, respiration, activity/arousal level,
tremors, convulsions, abnormal movements, impairment of gait, lacrimation, palpebral closure,
exophthalmos, feces (appearance/ consistency), urine, pupil size

BODY WEIGHT: Yes
- Body weight was determined before the start of the administration period in order to
randomize the animals. During the administration period the body weight was determined on
day 0 (start of the administration period) and thereafter at weekly intervals. The difference
between the body weight on the respective day of weighing and the body weight on day 0
was calculated as body weight change.

FOOD CONSUMPTION:
- Food consumption was determined weekly and calculated as mean food consumption in
grams per animal and day.

WATER CONSUMPTION:
- Drinking water consumption was monitored by daily visual inspection of the water bottles for
any changes in volume.

OPHTHALMOSCOPIC EXAMINATION: Yes / No / Not specified
- Prior to the start of the administration period on day 0 the eyes of all animals and on study
day 91 the eyes of the control and high-dose animals were examined for any changes using
an ophthalmoscope after administration of a mydriatic agent


HAEMATOLOGY: Yes
- The following parameters were determined in blood with EDTA-K3 as anticoagulant using a
particle counter:
leukocyte count (WBC), erythrocyte count (RBC), hemoglobin (HCT), hematocrit (HCT),
mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular
hemoglobin concentration (MCHC), platelet count (PLT), differential blood count, reticulocytes (RET),
prothrombin time (Hepato Quick´s test) (HQT)
Furthermore, blood smears were prepared and stained according to WRIGHT without being
evaluated, because of non-ambiguous results of the differential blood cell counts measured
by the automated instrument.


CLINICAL CHEMISTRY: Yes
alanine aminotransferase (ALT), aspartate aminotransferase (AST), Alkaline phosphate (ALP),
γ-Glutamyltransverase (GGT), sodium (Na), potassium (K), chloride (Cl), inorganic phosphate (INP),
calcium (Ca), urea (UREA),creatine (CREA), glucose (GLUC), total bilirubin (TBIL),
total protein (TPROT), albumin (ALB), globulins (GLOB), triglycerides (TRIG), cholesterol (CHOL),
bite acids (TBA), HDL-cholesterol (HDL-CHOL), LDL-cholesterol (LDL-CHOL)

THYROID HORMONES:
- total triiodothyronine (T3), total thyroxine (T4), thyroid stimulating hormone (TSH)

URINALYSIS: Yes
pH, protein (PRO), glucose (GLU), ketones (KET, urobilinogen (UBG), bilirubin (BIL), blood,
specific gravity (SP,GR), sediment, color, turbidity (COL, TURB), volume (VOL)

NEUROBEHAVIOURAL EXAMINATION: Yes
- A functional observational battery (FOB) was performed in all animals at the end of the
administration period starting at about 10:00 h. At least one hour before the start of the FOB
the rats were transferred to single-animal polycarbonate cages type III (floor area of about
800 cm²). Drinking water was provided ad libitum, but no food was offered during the
measurements. The FOB started with passive observations without disturbing the rats,
followed by removal from the home cage, open field observations in a standard arena and
sensory motor tests as well as reflex tests.

Home cage observations:
The animals were observed in their closed home cages; during this period any disturbing
activities (touching the cage or rack, noise) were avoided during these examinations in order
not to influence the behavior of the rats. Attention was paid to:
posture, tremors, convulsions, abnormal movements, gait, other findings.


Open field observations:
The animals were transferred to a standard arena (50 × 50 cm with sides of 25 cm height)
and observed for at least 2 minutes. The following parameters were examined:
behavior on removal from the cage, fur, skin, salivation, nasal discharge, lacrimation,
eyes/pupil size, posture, palpebral closure, respiration, tremors, convulsions, abnormal
movements/stereotypes, gait abnormalitties, activity/arousal level, feces excreted within
2 minutes (appearance/ consistency), urine excreted within 2 minutes (amount/color),
rearing within 2 minutes, other findings

Sensory motor tests/ reflexes:
The animals were then removed from the open field and subjected to following sensory motor
or reflex tests:
reaction to an object being moved towards the face (approach response),
touch sensitivity (touch response), vision (visual placing response), pupillary reflex, pinna reflex,
audition (auditory startle response), coordination of movements (righting response), behavior during
handling, vocalization, pain perception (tail pinch), grip strength of forelimbs, grip strength of
hindlimbs, landing foot-splay test, other findings.

Motor activity assessment
Motor activity (MA) was also measured from 14:00 h onwards on the same day as the FOB
was performed. The examinations were performed using the TSE Labmaster System
supplied by TSE Systems GmbH (Bad Homburg, Germany). For this purpose, the rats were
placed in new clean polycarbonate cages with a small amount of bedding for the duration of
the measurement. Eighteen beams were allocated per cage. The number of beam interrupts
was counted over 12 intervals for 5 minutes per interval. The sequence in which the rats were
placed in the cages was selected at random. On account of the time needed to place the rats
in the cages, the starting time was "staggered" for each animal. The measurement period
began when the 1st beam was interrupted and finished exactly 1 hour later. No food or water
was offered to the rats during these measurements and the measurement room was
darkened after the transfer of the last rat.

IMMUNOLOGY: No

OTHER:
Estrous cycle determination
Vaginal smears for cycle determination were prepared in the morning and evaluated
according to the timetable for at least 3 weeks until the day of sacrifice.

Sperm parameters
Immediately after necropsy and organ weight determination, the right testis and cauda
epididymis were taken from all male animals.
Sperm motility examinations and the preparation of the specimens for sperm morphology
were carried out in a randomized sequence.
The right testis and right cauda epididymis were deep frozen at -20°C until evaluation of the
sperm head count. Initially, sperm morphology and sperm head count (cauda epididymis and
testis) were evaluated for the control (test group 0) and test group 3, only.
Sperm motility, sperm morphology, sperm head count (cauda epididymis),
sperm head count (testis)

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Necropsy
The animals were sacrificed by decapitation under isoflurane anesthesia. The exsanguinated
animals were necropsied and assessed by gross pathology.

Organ weights
The following weights were determined in all animals sacrificed on schedule:
Anesthetized animals (final body weight), Adrenal glands (fixed), Brain, Epididymides, Heart, Kidneys,
LIver, Ovaries(fixed), Pituitary gland (fixed), Prostate (ventral and dorsolateral part together, fixed),
Spleen, Seminal vesicles with coagulating glands (fixed), Testes, Thymus, Thyroid glands (fixed),
Uterus (with cervix).

Organ/tissue fixation
The following organs or tissues were fixed in in 4% neutral-buffered formaldehyde solution or in
modified Davidson`s solution:
All gross lesions, Adrenal glands, Aorta, Bone marrow (femur), Brain, Cecum, Cervix, Coagulating
glands, Colon, Duodenum, Epididymis, left (modified Davidson`s solution), Esophagus, Extraorbital
lacrimal glands,Eyes with optic nerve (modified Davidson`s solution), Femur with knee joint,
Harderian glands, Heart, Ileum, Jejunum (with Peyer`s patches), Kidneys, Larynx, Liver, Lungs,
Lymph nodes (axillary and mesenteric), Mammary gland (male and female), Nose (nasal cavity),
Ovaries, Oviducts, Pancreas, Parathyroid glands, Pharynx, Pituitary gland, Prostate, Rectum,
Salivary glands (mandibular and sublingual), Sciatic nerve, Seminal vesicles, Skeletal muscle, Skin,
Spinal cord (cervical, thoracic and lumbar cord), Spleen, Sternum with marrow, Stomach
(forestomach and glandular stomach), Testes left (modified Davidson`s solution), Thymus,
Thyroid glands, Trachea, Urinary bladder, Uterus, Vagina.


HISTOPATHOLOGY: Yes
Histopathology
Fixation was followed by histotechnical processing, examination by light microscopy and assessment
of findings. All gross lesion, Adrenal glands, Aorta, Bone marrow (frmur), Brain, Cecum, Cervix,
Coagulating glands, Colon,Duodenum, Epididymidis left, Eyes with optic nerve, Femur with knee
joint, Heart, Ileum, Jejunum, Kidneys, Liver, Lungs, Lymph nodes (axillary and mesenteric),
Mammary gland, ´Ovaries, Pancreas, Parathyroid gland, Peyer`s patches, Pituitary glands, Prostate,
Rectum, Salivary glands (mandibular and sublingual glands), Sciatic nerve, Seminal vesicles,
Skeletal muscle, Skin, Spinal cord (cervical, thoracic, lumbar), Spleen, Stomach (forestomach
and glandular stomach),Testes (left), Thymus, Thyroid glands, Trachea, Urinary bladder, Uterus, Vagina.

Other examinations:

Special stains (Oil-red-O and Sudan Black) were performed exemplarily on kidney sections
of 2 animals
Special attention was given for the synchrony of the morphology of the estrous cycle in
ovaries, uterus, cervix, and vagina.
Special attention was given for the male reproductive organs, especially the stage of
seminiferous tubules.
A correlation between gross lesions and histopathological findings was attempted.

Statistics:

Statistics of clinical examinations
Body weight,body weight change: DUNNETT's test (two-sided)
Rearing, grip strength forelimbs, grip strength hindlimbs, footsplaytest, motor activity:
Non-parametric one-wayanalysis using KRUSKALWALLIS test (two-sided).

Statistics of clinical pathology:
Blood parameters: For parameters with bidirectional changes:
Non-parametric one-way analysis using KRUSKAL-WALLIS test.
If the resulting p-value was equal or less than 0.05, a pairwise
comparison of each dose group with the control group was performed using
WILCOXON-test (two-sided) for the hypothesis of equal medians.
For parameters with unidirectional changes:
Pairwise comparison of each dose group with the control group using the
WILCOXON-test (one-sided) for the hypothesis of equal medians.
Urinalysis parameters: WILCOXON-test (one-sided)
Urine pH, volume and specific gravity: Non-parametric one-way analysis using
KRUSKAL-WALLIS test.
If the resulting p-value was equal or less than 0.05, a pairwise
comparison of each dose group with the control group was performed using
WILCOXON-test (two-sided) for the hypothesis of equal medians.
Sperm analysis parameters:Pairwise comparison of each dose group
with the control group using theWILCOXON-test (one-sided) with
Bonferroni-Holm adjustment for thehypothesis of equal medians; If only control
and one dose group are measured, WILCOXON-test (one-sided) without
adjustment were used.For the percentage of abnormal sperms
(ABNORMAL5_C) values < 5% were set to5 % (cut off 5%)

Statistics of pathology:
Weight parameters;Non-parametric one-way analysis using KRUSKALWALLIS H test
(two-sided). If the resulting p-value was equal or less than 0.05, a pairwise comparison of each
test group with the control group was performed using WILCOXON-test (two-sided)
for the equal medians.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

see attached document: summary clinical observation
All male and all female animals of test group 3 (150 mg/kg bw/d) showed slight salivation
directly after treatment on several days of the application period. Salivation was also
observed in 5 male and 3 female animals of test group 2 (50 mg/kg bw/d) and in 6 male
animals in test group 1 (15 mg/kg bw/d).
From the temporary, short appearance immediately after dosing it was concluded the findings
were induced by a bad taste of the test substance or local affection of the upper digestive
tract. The effect was related to the test substance but assessed as being non-adverse as no
lesions in the upper digestive tract were observed in male and female animals during
pathological examinations.
A protruding right eyeball was observed one in female animal of test group 1 (15 mg/kg bw/d)
between study days 91-93. This finding was considered to be incidental and not related
to treatment.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

see attached document; summary bw_bwc
No test substance-related, adverse effects on body weight development were obtained in any
test group (15, 50 and 150 mg/kg bw/d).
Mean body weights and body weight change values of male and female animals did not show
any significant deviations to the control.

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

no effects observed

Ophthalmological findings:

effects observed, non-treatment-related

Description (incidence and severity):

see attached document: summary ophthalmological findings
No treatment-related findings were observed.
All other apparent findings were assessed as being incidental in nature since they occurred
in control as well as in treated animals and did not show a dose-response relationship
see attached document: ophthalmological findings

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

see attached document: summary haematological and chlinical chemistry
At the end of the administration period, in females of test groups 1 and 2 (15 and 50 mg/kg
bw/d) absolute and relative lymphocyte counts were significantly decreased whereas relative
neutrophil counts were significantly increased. Additionally, in females of test group 2 (50
mg/kg bw/d) relative, large unstained cell (LUC) counts were significantly decreased.
However, all mentioned alterations were not dose-dependent and, therefore, they were
regarded as incidental and not treatment-related.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

see attached document: summary haematological and chlinical chemistry
At the end of the administration period, in males of test group 3 (150 mg/kg bw/d) potassium
levels were significantly increased whereas chloride levels were significantly decreased.
Chloride values were within, potassium values slightly above the historical control range
(males, chloride 96.4-106.1 mmol/L, potassium 4.52-5.04 mmol/L). The unique potassium
increase among these individuals was regarded to be potentially treatment-related but nonadverse
(ECETOC Technical Report No. 85, 2002), whereas the chloride decrease was
regarded as incidental and not treatment-related.

Thyroid hormones
No treatment-related alterations of T3, T4 and TSH levels were observed.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

At the study end, in males of test group 2 (50 mg/kg bw/d) higher incidences of triple
phosphate crystals were observed in the urine sediment. This change was not dosedependent
and, therefore, it was regarded as incidental and not treatment-related.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

see attached document: FOB
Deviations from "zero values" were obtained in several rats. However, as most findings were
equally distributed between test-substance treated groups and controls, were without a doseresponse
relationship or occurred in single rats only, these observations were considered to
have been incidental.
Open field observations: No test substance-related effects were observed.
Sensorimotor tests/reflexes: No test substance-related effects were observed.
Quantitative parameters No test substance-related effects were observed.
In male animals of test group 2 (50 mg/kg bw/d), grip strength of hindlimbs was significantly
increased (+17%), but a dose-response relationship did not occur. The deviation was
assessed as being spontaneous in nature.

Immunological findings:

not examined

Description (incidence and severity):

see attached document: summary organ and body weight
All mean absolute and relative weight parameters did not show significant differences when
compared to the control group 0.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

see attached document: summary gross lesions and microscopic findings
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.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Vacuolation of epithelial cells of the collecting ducts was characterized by macrovesicular
clear vacuoles in the collecting duct epithelium of the inner medulla. The outer part of the
inner medulla was most prominently affected with a gradual decrease towards the tip of
papilla. The vacuoles did not stain with HE, Sudan Black or Oil-Red-O. Vacuolation affected
8 out of 10 males and 1 out of 10 females of test group 3 (150 mg/kg bw/d), with a severity
ranging from minimal to severe in males, the one female animal was only minimally affected.
No signs of necrosis, cellular degeneration, inflammation or edema were present.
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.

Description (incidence and severity):

see attached document: summary motor activiy
Regarding the overall motor activity as well as single intervals, no test substance-related and
relevant deviations to the control animals were noted for male and female animals of test
groups 1-3 (15, 50, and 150 mg/kg bw/d).
In female animals of test group 2 (50 mg/kg bw/d) and test group 3 (150 mg/kg bw/d), the
overall motor activity was significantly higher when compared to the controls. However, a
clear dose-response relationship did not occur, and the changes were assessed to be
incidental and not related to treatment.
Comparing the single intervals with the control groups, significantly higher values were
measured for male animals of test group 1 (15 mg/kg bw/d) at interval No. 8 and for male
animals of test group 3 (150 mg/kg bw/d) at interval Nos. 11 and 12.
Significantly higher values were also obtained for female animals of test groups 2 (50 mg/kg
bw/d) and 3 (150 mg/kg bw/d) at interval No. 5 as well as for female animals of test group 2
at interval No. 6.
All changes were regarded to be incidental and not related to treatment as single intervals
were not changed in a dose-dependent manner.

see attached document: summary estrous cycle
Estrous cycle
No test substance-related, adverse effects on estrous cycles were obtained in any test group
(15, 50 and 150 mg/kg bw/d).

see attached document: summary spermanalysis
Sperm analysis
Concerning motility of the sperms and the incidence of abnormal sperms in the cauda
epididymidis as well as sperm head counts in the testis and in the cauda epididymidis
no treatment-related effects were observed

Key result

Dose descriptor:

NOAEL

Effect level:

50 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

150 mg/kg bw/day (actual dose received)

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

   

Kidneys	Male animals				Female animals
Test group (mg/kg bw/d)	0 (0)	1 (15)	2 (50)	3 (150)	0 (0)	1 (15)	2 (50)	3 (150)
No. of animals	10	10	10	10	10	10	10	10
Vacuolation,collecting ducts	0	0	0	8	0	0	0	1
·      Grade1				4				1
·      Grade2				1
·      Grade3				2
·      Grade4				1

 

Conclusions:

The administration of Dibutylethanolamine by gavage for 3 months to male and female Wistar
rats caused test substance-related findings at a dose level of 150 mg/kg bw/d taking the
vacuolation of the collecting duct epithelium in kidneys into account. Therefore, under the conditions of the present study the NOAEL was 50 mg/kg bw/d for male
and female Wistar rats.

Executive summary:

Dibutylethanolamine was administered orally by gavage to groups of 10 male and 10 female

Wistar rats at dose levels of 0 mg/kg b/day (mg/kg bw/d; test group 0), 15 mg/kg

bw/d (test group 1), 50 mg/kg bw/d (test group 2) and 150 mg/kg bw/d (test group 3) over a

period of 3 months. Corn oil served as vehicle, control animals were dosed daily with the

vehicle only.

With regard to clinical examinations, signs of general systemic toxicity were not observed

even at a dose level of 150 mg/kg bw/d of Dibutylethanolamine. In addition, no test

substance-related effects on estrous cycle length and the number of cycles were obtained

Concerning clinical pathology, no treatment-related, adverse effects were observed up to a

dose of the compound of 150 mg/kg bw/d.

The unique potassium increase observed for male animals of test group 3 (150 mg/kg bw/d)

was considered to be potentially treatment-related but regarded to be non-adverse (ECETOC

Technical Report No. 85, 2002). A relation to the kidney-related findings observed in these

male animals of the test group 3 was excluded.

Regarding pathology, neither treatment-related weight changes nor gross lesions were

observed.

The target organ were the kidneys. In 8 out of 10 males and 1 out of 10 females of test group

3, macrovesicular vacuolation of the epithelial cells of the collecting ducts was observed. No

signs of necrosis, degeneration or inflammation were present.

Therefore, under the conditions of the present study the NOAEL was 50 mg/kg bw/d for male

and female Wistar rats.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

50 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

Available data is reliable and sufficient for assessment.

System:

urinary

Organ:

kidney

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP Guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

(22 Mar 1996; Inhalation exposure is conducted according to OECD 413 (7 September 2009))

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

BASF SE, Experimental Toxicology and Ecology, Ludwigshafen, Germany

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sulzfeld (According to a written statement from the breeder, male and female animals were derived from different litters. This was necessary to rule out the possibility of sibling mating.)
- Age at study initiation: about 10 - 11 weeks
- Housing: individually in Makrolon type M III cages (exceptions: during mating (1 male/1 female per cage), during rearing up to PND 4 (1 dam with her litter); for motor activity (MA) measurements the animals were housed individually in polycarbonate cages
- Diet: Ground Kliba maintenance diet mouse / rat “GLP” (Provimi Kliba SA, Kaiseraugst, Switzerland); ad libitum, but the animals did not have access to feed during the exposure.
- Water: ad libitum, but the animals did not have access to water during the exposure
- Acclimation period: at least 7 days

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

Route of administration:

inhalation: vapour

Type of inhalation exposure:

nose/head only

Vehicle:

air

Remarks on MMAD:

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

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Head / nose inhalation system (Volume ca. 155 L and 90 L, respectively); the snouts of the rats projected into the inhalation chamber to inhale the atmosphere.
- Method of holding animals in test chamber: The rats are restrained in glass exposure tubes.
- Source and rate of air: conditioned supply air: test group 0: 5.7 – 6.3 m³/h, test groups 1 - 3: 4.2 - 4.8 m³/h; compressed air (filtered air pressurized to about 6 bar): 5.1 - 5.7 m³/h
- Method of conditioning air: activated charcoal filtered air conditioned to about 50% ± 20% relative humidity and 22°C ± 2°C.
- System of generating particulates/aerosols: For each concentration, a respective constant amount of the test substance will be supplied to a two component atomizer by means of a metering pump. The aerosol was generated with compressed air into the inhalation system.
- Temperature, humidity, pressure: 21.7 - 24.1°C, 39.3 - 63.8%, positive pressure
- Air change rate: about 67 times / hour

TEST ATMOSPHERE
- Brief description of analytical method used: online by propane-calibrated total hydrocarbon analyzer (FID).
- Samples taken from breathing zone: yes

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentrations of the inhalation atmospheres were analyzed online by propane-calibrated total hydrocarbon analyzer (FID). By means of response factor provided by the manufacture, the measured concentration of propane in each test group less the background concentration, were converted to concentration of the test substance. To verify the correctness of the FID measurement, absorption samples were drawn from the atmospheres. The absorption samples were analyzed by gas chromatography in all test groups. Daily means were calculated based on three measured samples per concentration and exposure. From the daily mean values of each concentration, mean concentrations and standard deviations for the entire study were derived. In these groups, the constancy of concentrations in the inhalation systems in the chambers were continuously monitored using total hydrocarbon analyzers. The control group was analyzed on three days during the exposure period. The analyses were carried out at the Inhalation and Analytical Chemistry Laboratory of the Experimental Toxicology and Ecology of BASF SE.

Duration of treatment / exposure:

6 h /day

Frequency of treatment:

- males: daily (7 days per week); male animals were sacrificed on study day 29 (28 days exposure) after the beginning of the administration, and examined
- females: daily (7 days per week); females were allowed to litter and rear their pups until day 4 after parturition, after sacrifice and necropsy of the pups on PND 4 the parental female animals were exposed to the respective concentrations of test substance for 6 hours a day total 4 exposure days on 4 consecutive days before scheduled killing (study day 51); (exception: no exposure on the day of FOB / MA)

Remarks:

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

Remarks:

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

Remarks:

Doses / Concentrations:
21.5, 86.0, 301.0 mg/m³
Basis:
nominal conc.

No. of animals per sex per dose:

10

Control animals:

other: The control group was exposed to conditioned air

Details on study design:

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

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: The clinical condition of the test animals was recorded once during the pre-exposure period and on non-exposure days and at least 3 times (before, during and after exposure) on exposure days. During exposure only a group wise examination was possible. Abnormalities and changes were documented daily for each animal.
A check for moribund or dead animals was made twice daily on working days or once daily (Saturday, Sunday or on public holidays). If animals were in a moribund state, they were sacrificed and necropsied.


DETAILED CLINICAL OBSERVATIONS: Yes
All animals were subjected to detailed clinical observations outside their cages once before the beginning of the administration period (day 0) and subsequently once a week (in the morning). For observation, the animals were therefore be removed from their cages and placed in a standard arena (50 x 37.5 x 25 cm). The scope of examinations and the scoring of the findings that are observed was based on the current index of findings in PDS ToxData and includes but is not limited to the following parameters listed: abnormal behavior in “handling”, fur, skin, posture, salivation, respiration, activity/arousal level, tremors, convulsions, abnormal movements, gait abnormalities, lacrimation, palpebral closure, exophthalmos, assessment of the feces discharged during the examination (appearance/consistency), assessment of the urine discharged during the examination, pupil size.


BODY WEIGHT: Yes
In general, the body weight of the male and female parental animals was determined once a week at the same time of the day (in the morning) until sacrifice. The body weight change of the animals was calculated from these results. The following exceptions are notable for the female animals: During the mating period the parental females were weighed on the day of positive evidence of sperm (GD 0) and on GD 7, 14 and 20. Females with litter were weighed on the day of parturition (PND 1) and on PND 4. After the pups were sacrificed the females were exposed for 4 consecutive days. The F0 females were weight once before the exposure period and once on the last exposure day.
Females without positive evidence of sperm, without litter or waiting for necropsy, were weighed weekly. These body weight data were solely used for the calculations of the dose volume.


FOOD CONSUMPTION:
Food consumption was determined once a week for male and female parental animals, with the following exceptions:
Food consumption was not determined after the 2nd premating week (male parental animals) and during the mating period (male and female F0 animals). Food consumption of the F0 females with evidence of sperm was determined on gestation days (GD) 0 - 7, 7 - 14, and 14 - 20. Food consumption of F0 females, which gave birth to a litter was determined on PND 1 - 4. Food consumption of the females during the 4 exposure days after necropsy of the pups.
Food consumption was not determined in females without positive evidence of sperm during gestation periods and in females without litter during lactation period.


WATER CONSUMPTION: No


OPHTHALMOSCOPIC EXAMINATION: No


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


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


URINALYSIS: No


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

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
The following weights were determined in all animals sacrificed on schedule: anesthetized animals, epididymides, testes;
The following weights were determined in 5 animals per sex/test group sacrificed on schedule (females with litters only, same animals as used for clinical pathological examinations): adrenal glands, brain, heart, kidneys, liver, lung, spleen, thymus.

HISTOPATHOLOGY: Yes (all gross lesions, adrenal glands, aorta, bone marrow (femur), brain, cecum, cervix, coagulating glands, colon, duodenum, eyes with optic nerve, esophagus, extraorbital lacrimal glands, epididymides, femur with knee joint, heart, ileum, jejunum (with Peyer’s patches), kidneys, larynx, liver, lungs, lymph nodes (tracheobronchial, mediastinal and mesenteric), mammary gland (male and female), nose (nasal cavity), ovaries, oviducts, pancreas, parathyroid glands, pharynx, pituitary gland, prostate gland, rectum, salivary glands (mandibular and sublingual), sciatic nerve, seminal vesicles, skeletal muscle, spinal cord (cervical, thoracic and lumbar cord), spleen, sternum with marrow, stomach (forestomach and glandular stomach), testes, thymus, thyroid glands, trachea, urinary bladder, uterus, vagina; special attention is given on stages of spermatogenesis in the testes)

Statistics:

Food consumption (parental animals), body weight and body weight change (parental animals and pups; for the pup weights, the litter means were used), gestation days, implantation sites, pups delivered, life pups day x: DUNNETT test (two-sided); Male and female mating indices, male and female fertility indices, gestation index, females delivering, females with liveborn pups, females with stillborn pups, females with all stillborn pups: FISHER'S EXACT test (one-sided); Mating days until day 0 pc, viability index: WILCOXON test (one-sided) with BONFERRONI-HOLM adjustment; feces, rearing, grip strength forelimbs, grip strength hind-limbs, landing foot-splay test, motor activity: KRUSKAL-WALLIS and WILCOXON test (one- or two-sided); blood parameters: KRUSKAL-WALLIS and WILCOXON test (two-sided); weight parameters (determined in pathology): KRUSKAL-WALLIS and WILCOXON test (two-sided)

Details on results:

CLINICAL SIGNS AND MORTALITY:
There were no test substance-related or spontaneous mortalities in any of the groups. During the pre-exposure period, the pre-mating and the mating period the animals showed no clinical signs and findings different from normal. During the post-mating day the male animals showed no clinical signs and findings different from normal. During the gestation period six female animals of the control group, eight female animals of the low concentration (25 mg/m³), eight female animals of the mid concentration (75 mg/m³) and seven female animals of the high concentration (225 mg/m³) showed vaginal discharge. As this finding was distributed evenly in the controls and the groups exposed to the test substance, it was considered to be treatment-related (head-nose exposure), but not substance-related. During the lactation period the female animals showed no clinical signs and findings different from normal. During the 4-day exposure period of the females after the pups were sacrificed the animals showed no clinical signs and findings different from normal.
The detailed clinical observations did not reveal any abnormalities in male and female animals of all test groups.


BODY WEIGHT AND WEIGHT GAIN:
Premating period: The mean body weights of the test substance exposed male and female animals were not statistically significantly different from the control group 0.
Mating period: The mean body weights of the test substance exposed male animals were not statistically significantly different from the control group 0, although the mean body weights of the males test group 3 seems to be slightly lower than those of other groups.
Gestation:
The mean body weights of group 3 females were slightly lower than the control on gestation days (GD) 7 and 14 (p < 0.05).
Lactation:
The mean body weights of F0 female animals were not statistically significantly different from the control group 0 during lactation period.
After PND 4:
The mean body weights of F0 female animals after PND 4 were not statistically different to the controls.

Body weight change / during the exposure period:
The body weight change of the F0 male animals of the high concentration group (225 mg/m³) was statistically significantly lower than the controls during premating period (-12.9 g, p < 0.05) at the beginning of the mating period. This effect was diminished in course of the exposure and was not observed any further in the second week of the pre-mating period, as well as during mating period.
During pre-mating period, the mean body weight changes of the female animals were not statistically different when compared with the control.
From gestation day 0 to 7, the mean body weight change of F0 female animals was significantly lower (-37 %, p < 0.01) than the controls. This effect was not observed any further at later time points. The body weight changes were in other test groups not significantly different to the control.


FOOD CONSUMPTION:
In high concentration (225 mg/m³) food consumption during pre-mating was significantly decreased in male animals between study days 0 - 7 (-9 %) and 7 - 14 (-11%) as well as in female animals between study days 0 - 7 (-7%) and 7 - 14 (-5%). These findings were considered to be substance-related. During the gestation the food consumption in the high concentration (225 mg/m³) was significantly decreased in female animals during the whole period (-11%) and in the mid concentration (75 mg/m³) was significantly decreased in female animals between study days 0 - 7 (-9%). No other findings were observed for male and female animals in test group 1 and 2 (25 and 75 mg/m³).

The effects on food consumption and body weight / body weight change might be an indirectly consequence of the local irritation in the nasal cavity. They are therefore considered to be treatment-related.


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


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


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


ORGAN WEIGHTS:
When compared with control group 0 (=100%), the following mean relative weights were significantly increased or decreased in one or more test groups: males: epididymides (group 1: 88%, group 3: 90%), spleen (group 1: 137%), testes (group 2: 93%, group 3: 92%); females: liver (group 3: 92%)
When compared with control group 0 (=100%), the following mean absolute weights were significantly increased or decreased in one or more test groups: males: epididymides (group 1: 88%); females: thymus (group 1: 129%)
Weight changes in thymus, spleen and liver were regarded as incidental as no dose response relationship and/or histopathological correlates were detected. The decrease in epididymides and testes weights was considered to be treatment-related even in the absence of a clear dose response relationship or, in the case of the testes, no changes in relative weights as there was a histopathological correlate (see histopathology).
All other mean absolute or relative weight parameters did not show significant differences when compared to the control group 0.


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


HISTOPATHOLOGY:
Treatment-related findings were observed in level I of larynx, and level I and II of nasal cavity, in males and females as well as in epididymides and testes in male animals:

Larynx: Treatment – related findings in the larynx were epithelial alteration characterized by slight modification of epithelial cells (i.e., three to four cell layers, focally flattened and stratified) indicating beginning metaplastic transformation and minimal to slight squamous metaplasia (characterized by three to four cell layers of flattened, stratified epithelium with no signs of keratinization and only affecting the epiglottis when graded minimal and up to five cell layers of flattened, stratified epithelium, occasionally minimal focal keratinization, with / without focal desquamation of superficial cells when graded slight) (Kaufmann et al., 2009).
Laryngeal epithelial alteration were regarded as non-adverse according to the literature (Kaufmann W. et al., Exp Toxicol Pathol, 2009 Nov, 61(6): 591-603).

Nasal cavity, level I: Degeneration / regeneration of transitional and respiratory epithelium was characterized by variable vacuolation, presence of few apoptotic bodies, minimal infiltrates of inflammatory cells, increased size and basophilia of nuclei and minimal disorganization of cells. Metaplasia, squamous was characterized by flattened epithelial cells with variably present minimal keratinization.
Nasal cavity, level II: One male test group 3 (225 mg/m³) animal (No 134) showed minimal degeneration / regeneration of the olfactory epithelium, in level I it showed degeneration/regeneration of the transitional epithelium).
Degeneration / regeneration of nasal epithelia was regarded as adverse.

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

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

This effect in the testes is likely due to the technical exposure scenario (heat [e.g. Brock WJ et al., 1996] and possibly evading movements by the animals leading to pressing backwards in tubes), rather than being a direct effect of the test substance as this finding is also found in control animals. These findings did generally not affect fertility with one possible exception in one group 1 male animal (No. 17). All findings in testes and epididymides were assessed as treatment - related and adverse but not substance-related.

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


OTHER FINDINGS: see also "Repeated dose toxicity"

Dose descriptor:

NOAEC

Remarks:

(local)

Effect level:

20.6 mg/m³ air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: histopathological lesions of nasal epithelia

Dose descriptor:

NOAEC

Remarks:

(reproductive and developmental parameters)

Effect level:

236.3 mg/m³ air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: highest dose tested, no adverse effects observed

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'

Critical effects observed:

not specified

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

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

236.3 mg/m³

Study duration:

subacute

Species:

rat

Quality of whole database:

Available data is reliable and sufficient for assessment.

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP Guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

(22 Mar 1996; Inhalation exposure is conducted according to OECD 413 (7 September 2009))

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

BASF SE, Experimental Toxicology and Ecology, Ludwigshafen, Germany

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sulzfeld (According to a written statement from the breeder, male and female animals were derived from different litters. This was necessary to rule out the possibility of sibling mating.)
- Age at study initiation: about 10 - 11 weeks
- Housing: individually in Makrolon type M III cages (exceptions: during mating (1 male/1 female per cage), during rearing up to PND 4 (1 dam with her litter); for motor activity (MA) measurements the animals were housed individually in polycarbonate cages
- Diet: Ground Kliba maintenance diet mouse / rat “GLP” (Provimi Kliba SA, Kaiseraugst, Switzerland); ad libitum, but the animals did not have access to feed during the exposure.
- Water: ad libitum, but the animals did not have access to water during the exposure
- Acclimation period: at least 7 days

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

Route of administration:

inhalation: vapour

Type of inhalation exposure:

nose/head only

Vehicle:

air

Remarks on MMAD:

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

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Head / nose inhalation system (Volume ca. 155 L and 90 L, respectively); the snouts of the rats projected into the inhalation chamber to inhale the atmosphere.
- Method of holding animals in test chamber: The rats are restrained in glass exposure tubes.
- Source and rate of air: conditioned supply air: test group 0: 5.7 – 6.3 m³/h, test groups 1 - 3: 4.2 - 4.8 m³/h; compressed air (filtered air pressurized to about 6 bar): 5.1 - 5.7 m³/h
- Method of conditioning air: activated charcoal filtered air conditioned to about 50% ± 20% relative humidity and 22°C ± 2°C.
- System of generating particulates/aerosols: For each concentration, a respective constant amount of the test substance will be supplied to a two component atomizer by means of a metering pump. The aerosol was generated with compressed air into the inhalation system.
- Temperature, humidity, pressure: 21.7 - 24.1°C, 39.3 - 63.8%, positive pressure
- Air change rate: about 67 times / hour

TEST ATMOSPHERE
- Brief description of analytical method used: online by propane-calibrated total hydrocarbon analyzer (FID).
- Samples taken from breathing zone: yes

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentrations of the inhalation atmospheres were analyzed online by propane-calibrated total hydrocarbon analyzer (FID). By means of response factor provided by the manufacture, the measured concentration of propane in each test group less the background concentration, were converted to concentration of the test substance. To verify the correctness of the FID measurement, absorption samples were drawn from the atmospheres. The absorption samples were analyzed by gas chromatography in all test groups. Daily means were calculated based on three measured samples per concentration and exposure. From the daily mean values of each concentration, mean concentrations and standard deviations for the entire study were derived. In these groups, the constancy of concentrations in the inhalation systems in the chambers were continuously monitored using total hydrocarbon analyzers. The control group was analyzed on three days during the exposure period. The analyses were carried out at the Inhalation and Analytical Chemistry Laboratory of the Experimental Toxicology and Ecology of BASF SE.

Duration of treatment / exposure:

6 h /day

Frequency of treatment:

- males: daily (7 days per week); male animals were sacrificed on study day 29 (28 days exposure) after the beginning of the administration, and examined
- females: daily (7 days per week); females were allowed to litter and rear their pups until day 4 after parturition, after sacrifice and necropsy of the pups on PND 4 the parental female animals were exposed to the respective concentrations of test substance for 6 hours a day total 4 exposure days on 4 consecutive days before scheduled killing (study day 51); (exception: no exposure on the day of FOB / MA)

Remarks:

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

Remarks:

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

Remarks:

Doses / Concentrations:
21.5, 86.0, 301.0 mg/m³
Basis:
nominal conc.

No. of animals per sex per dose:

10

Control animals:

other: The control group was exposed to conditioned air

Details on study design:

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

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: The clinical condition of the test animals was recorded once during the pre-exposure period and on non-exposure days and at least 3 times (before, during and after exposure) on exposure days. During exposure only a group wise examination was possible. Abnormalities and changes were documented daily for each animal.
A check for moribund or dead animals was made twice daily on working days or once daily (Saturday, Sunday or on public holidays). If animals were in a moribund state, they were sacrificed and necropsied.


DETAILED CLINICAL OBSERVATIONS: Yes
All animals were subjected to detailed clinical observations outside their cages once before the beginning of the administration period (day 0) and subsequently once a week (in the morning). For observation, the animals were therefore be removed from their cages and placed in a standard arena (50 x 37.5 x 25 cm). The scope of examinations and the scoring of the findings that are observed was based on the current index of findings in PDS ToxData and includes but is not limited to the following parameters listed: abnormal behavior in “handling”, fur, skin, posture, salivation, respiration, activity/arousal level, tremors, convulsions, abnormal movements, gait abnormalities, lacrimation, palpebral closure, exophthalmos, assessment of the feces discharged during the examination (appearance/consistency), assessment of the urine discharged during the examination, pupil size.


BODY WEIGHT: Yes
In general, the body weight of the male and female parental animals was determined once a week at the same time of the day (in the morning) until sacrifice. The body weight change of the animals was calculated from these results. The following exceptions are notable for the female animals: During the mating period the parental females were weighed on the day of positive evidence of sperm (GD 0) and on GD 7, 14 and 20. Females with litter were weighed on the day of parturition (PND 1) and on PND 4. After the pups were sacrificed the females were exposed for 4 consecutive days. The F0 females were weight once before the exposure period and once on the last exposure day.
Females without positive evidence of sperm, without litter or waiting for necropsy, were weighed weekly. These body weight data were solely used for the calculations of the dose volume.


FOOD CONSUMPTION:
Food consumption was determined once a week for male and female parental animals, with the following exceptions:
Food consumption was not determined after the 2nd premating week (male parental animals) and during the mating period (male and female F0 animals). Food consumption of the F0 females with evidence of sperm was determined on gestation days (GD) 0 - 7, 7 - 14, and 14 - 20. Food consumption of F0 females, which gave birth to a litter was determined on PND 1 - 4. Food consumption of the females during the 4 exposure days after necropsy of the pups.
Food consumption was not determined in females without positive evidence of sperm during gestation periods and in females without litter during lactation period.


WATER CONSUMPTION: No


OPHTHALMOSCOPIC EXAMINATION: No


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


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


URINALYSIS: No


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

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
The following weights were determined in all animals sacrificed on schedule: anesthetized animals, epididymides, testes;
The following weights were determined in 5 animals per sex/test group sacrificed on schedule (females with litters only, same animals as used for clinical pathological examinations): adrenal glands, brain, heart, kidneys, liver, lung, spleen, thymus.

HISTOPATHOLOGY: Yes (all gross lesions, adrenal glands, aorta, bone marrow (femur), brain, cecum, cervix, coagulating glands, colon, duodenum, eyes with optic nerve, esophagus, extraorbital lacrimal glands, epididymides, femur with knee joint, heart, ileum, jejunum (with Peyer’s patches), kidneys, larynx, liver, lungs, lymph nodes (tracheobronchial, mediastinal and mesenteric), mammary gland (male and female), nose (nasal cavity), ovaries, oviducts, pancreas, parathyroid glands, pharynx, pituitary gland, prostate gland, rectum, salivary glands (mandibular and sublingual), sciatic nerve, seminal vesicles, skeletal muscle, spinal cord (cervical, thoracic and lumbar cord), spleen, sternum with marrow, stomach (forestomach and glandular stomach), testes, thymus, thyroid glands, trachea, urinary bladder, uterus, vagina; special attention is given on stages of spermatogenesis in the testes)

Statistics:

Food consumption (parental animals), body weight and body weight change (parental animals and pups; for the pup weights, the litter means were used), gestation days, implantation sites, pups delivered, life pups day x: DUNNETT test (two-sided); Male and female mating indices, male and female fertility indices, gestation index, females delivering, females with liveborn pups, females with stillborn pups, females with all stillborn pups: FISHER'S EXACT test (one-sided); Mating days until day 0 pc, viability index: WILCOXON test (one-sided) with BONFERRONI-HOLM adjustment; feces, rearing, grip strength forelimbs, grip strength hind-limbs, landing foot-splay test, motor activity: KRUSKAL-WALLIS and WILCOXON test (one- or two-sided); blood parameters: KRUSKAL-WALLIS and WILCOXON test (two-sided); weight parameters (determined in pathology): KRUSKAL-WALLIS and WILCOXON test (two-sided)

Details on results:

CLINICAL SIGNS AND MORTALITY:
There were no test substance-related or spontaneous mortalities in any of the groups. During the pre-exposure period, the pre-mating and the mating period the animals showed no clinical signs and findings different from normal. During the post-mating day the male animals showed no clinical signs and findings different from normal. During the gestation period six female animals of the control group, eight female animals of the low concentration (25 mg/m³), eight female animals of the mid concentration (75 mg/m³) and seven female animals of the high concentration (225 mg/m³) showed vaginal discharge. As this finding was distributed evenly in the controls and the groups exposed to the test substance, it was considered to be treatment-related (head-nose exposure), but not substance-related. During the lactation period the female animals showed no clinical signs and findings different from normal. During the 4-day exposure period of the females after the pups were sacrificed the animals showed no clinical signs and findings different from normal.
The detailed clinical observations did not reveal any abnormalities in male and female animals of all test groups.


BODY WEIGHT AND WEIGHT GAIN:
Premating period: The mean body weights of the test substance exposed male and female animals were not statistically significantly different from the control group 0.
Mating period: The mean body weights of the test substance exposed male animals were not statistically significantly different from the control group 0, although the mean body weights of the males test group 3 seems to be slightly lower than those of other groups.
Gestation:
The mean body weights of group 3 females were slightly lower than the control on gestation days (GD) 7 and 14 (p < 0.05).
Lactation:
The mean body weights of F0 female animals were not statistically significantly different from the control group 0 during lactation period.
After PND 4:
The mean body weights of F0 female animals after PND 4 were not statistically different to the controls.

Body weight change / during the exposure period:
The body weight change of the F0 male animals of the high concentration group (225 mg/m³) was statistically significantly lower than the controls during premating period (-12.9 g, p < 0.05) at the beginning of the mating period. This effect was diminished in course of the exposure and was not observed any further in the second week of the pre-mating period, as well as during mating period.
During pre-mating period, the mean body weight changes of the female animals were not statistically different when compared with the control.
From gestation day 0 to 7, the mean body weight change of F0 female animals was significantly lower (-37 %, p < 0.01) than the controls. This effect was not observed any further at later time points. The body weight changes were in other test groups not significantly different to the control.


FOOD CONSUMPTION:
In high concentration (225 mg/m³) food consumption during pre-mating was significantly decreased in male animals between study days 0 - 7 (-9 %) and 7 - 14 (-11%) as well as in female animals between study days 0 - 7 (-7%) and 7 - 14 (-5%). These findings were considered to be substance-related. During the gestation the food consumption in the high concentration (225 mg/m³) was significantly decreased in female animals during the whole period (-11%) and in the mid concentration (75 mg/m³) was significantly decreased in female animals between study days 0 - 7 (-9%). No other findings were observed for male and female animals in test group 1 and 2 (25 and 75 mg/m³).

The effects on food consumption and body weight / body weight change might be an indirectly consequence of the local irritation in the nasal cavity. They are therefore considered to be treatment-related.


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


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


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


ORGAN WEIGHTS:
When compared with control group 0 (=100%), the following mean relative weights were significantly increased or decreased in one or more test groups: males: epididymides (group 1: 88%, group 3: 90%), spleen (group 1: 137%), testes (group 2: 93%, group 3: 92%); females: liver (group 3: 92%)
When compared with control group 0 (=100%), the following mean absolute weights were significantly increased or decreased in one or more test groups: males: epididymides (group 1: 88%); females: thymus (group 1: 129%)
Weight changes in thymus, spleen and liver were regarded as incidental as no dose response relationship and/or histopathological correlates were detected. The decrease in epididymides and testes weights was considered to be treatment-related even in the absence of a clear dose response relationship or, in the case of the testes, no changes in relative weights as there was a histopathological correlate (see histopathology).
All other mean absolute or relative weight parameters did not show significant differences when compared to the control group 0.


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


HISTOPATHOLOGY:
Treatment-related findings were observed in level I of larynx, and level I and II of nasal cavity, in males and females as well as in epididymides and testes in male animals:

Larynx: Treatment – related findings in the larynx were epithelial alteration characterized by slight modification of epithelial cells (i.e., three to four cell layers, focally flattened and stratified) indicating beginning metaplastic transformation and minimal to slight squamous metaplasia (characterized by three to four cell layers of flattened, stratified epithelium with no signs of keratinization and only affecting the epiglottis when graded minimal and up to five cell layers of flattened, stratified epithelium, occasionally minimal focal keratinization, with / without focal desquamation of superficial cells when graded slight) (Kaufmann et al., 2009).
Laryngeal epithelial alteration were regarded as non-adverse according to the literature (Kaufmann W. et al., Exp Toxicol Pathol, 2009 Nov, 61(6): 591-603).

Nasal cavity, level I: Degeneration / regeneration of transitional and respiratory epithelium was characterized by variable vacuolation, presence of few apoptotic bodies, minimal infiltrates of inflammatory cells, increased size and basophilia of nuclei and minimal disorganization of cells. Metaplasia, squamous was characterized by flattened epithelial cells with variably present minimal keratinization.
Nasal cavity, level II: One male test group 3 (225 mg/m³) animal (No 134) showed minimal degeneration / regeneration of the olfactory epithelium, in level I it showed degeneration/regeneration of the transitional epithelium).
Degeneration / regeneration of nasal epithelia was regarded as adverse.

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

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

This effect in the testes is likely due to the technical exposure scenario (heat [e.g. Brock WJ et al., 1996] and possibly evading movements by the animals leading to pressing backwards in tubes), rather than being a direct effect of the test substance as this finding is also found in control animals. These findings did generally not affect fertility with one possible exception in one group 1 male animal (No. 17). All findings in testes and epididymides were assessed as treatment - related and adverse but not substance-related.

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


OTHER FINDINGS: see also "Repeated dose toxicity"

Dose descriptor:

NOAEC

Remarks:

(local)

Effect level:

20.6 mg/m³ air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: histopathological lesions of nasal epithelia

Dose descriptor:

NOAEC

Remarks:

(reproductive and developmental parameters)

Effect level:

236.3 mg/m³ air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: highest dose tested, no adverse effects observed

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'

Critical effects observed:

not specified

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

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

20.6 mg/m³

Study duration:

subacute

Species:

rat

Quality of whole database:

Available data is reliable and sufficient for assessment.

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Oral:

 

In a subchronic toxicity study according to OECD 408 (BASF SE, 2020), Dibutylethanolamine was administered orally by gavage to groups of 10 male and 10 female Wistar rats at dose levels of 0, 15, 50 and 150 mg/kg bw/d over a period of 3 months. Regarding clinical examinations, no signs of toxicity were observed up to the highest dose of 150 mg/kg bw/d. No test substance-related effects on estrous cycle length and the number of cycles were obtained. Concerning clinical pathology, no treatment-related, adverse effects were observed. Regarding pathology, neither treatment-related weight changes nor gross lesions were observed. The target organ were the kidneys. In 8 out of 10 males and 1 out of 10 females of the highest dose group, macrovesicular vacuolation of the epithelial cells of the collecting ducts was observed. No signs of necrosis, degeneration or inflammation were present. The vacuolation occurred without changes in urine analysis parameters and was considered treatment-related. Therefore, the NOAEL was 50 mg/kg bw/d for male and female Wistar rats.

A GLP and OECD 407 compliant twenty-eight-day repeated dose oral (gavage) toxicity study with subsequent 14-day recovery with 2-dibutylaminoethanol was conducted by using male and female Sprague-Dawley rats (Ministry of Health, Labour and Welfare, Japan, 2004). Both males and females were divided into 4 groups in total, to which the test substance at doses of 25, 100 and 400 mg/kg bw and vehicle (corn oil) was given, respectively. For both, males and females, 5 each belonged to the respective groups; and another 5 animals each were added to the control and 400 mg/kg bw groups in view of recovery. However, 3 males and 5 females in the 400 mg/kg bw group died during the dosing period, and therefore, the recovery test could not be performed for females. In the 100 and 400 mg/kg bw groups, licking the inside of cage and chewing-like action were observed intermittently after administration. Additionally, for the 400 mg/kg bw group, clonic convulsion, spasm, tremor, abnormal phonation and pale skin, gasping, slow respiration, listlessness and prone position were occasionally found during a period of approximately 20 - 40 minutes after administration; some of the animals exhibiting slow respiration and prone position died. Moreover, in the 400 mg/kg bw group, bradycardia, reddish tear, exophthalmos, mild piloerection, decrease in or loss of withdrawal reflex after convulsion, decrease in exploration and the like were occasionally found by close clinical observation. Furthermore, in the 400 mg/kg bw group, salivation was observed, which appeared at the time of restraint for administration or immediately after administration and disappeared no later than approximately 30 minutes after administration from week 2 of administration onward. Also in the same group, salivation was occasionally found, which appeared not immediately after administration but approximately 30 minutes after administration from day 2 of administration onward. In measurement of locomotor activity at week 4 of administration, males and females in the 400 mg/kg bw group showed increased number of times to locomote for a period of 60 minutes starting from 60 minutes after administration. Males in the same group exhibited increased number of times to locomote for the respective periods of 10 minutes starting from 50 and 100 minutes after administration as well as increased number of times to locomote with respect to the total quantity of motion, and also increased number of rearing for a period of 10 minutes starting from 100 minutes after administration. Additionally, females in the 100 mg/kg bw group showed increased number of rearing for the respective periods of 10 minutes starting from 20 and 90 minutes after administration. In the 400 mg/kg bw group, females showed a low value of body weight on day 4 of administration, and males and females exhibited low food consumptions at week 1 of administration. Blood biochemical examination revealed decreased chlorine level for males and females in the 400 mg/kg bw group as well as decreased sodium level for males at the end of the dosing period. Also females showed tendencies toward increased levels of total cholesterol and glucose as well as decreased cholinesterase activity.

Pathological examination showed an increase in liver weight for the 400 mg/kg bw group, and also slight centrolobular hypertrophy of hepatocytes for some individuals. In the same group, males showed an increase in kidney weight, and mild to moderate vacuolation of the epithelial cells of the renal collecting ducts was seen for males and females. Moreover, females showed an increase in adrenal weight, and slight hypertrophy of the zona fasciculate was observed for some individuals. Pathological examination of dead cases showed increases in liver and kidney sizes. Histologically, vacuolation of the epithelial cells of the renal collecting ducts and slight hypertrophy of the adrenal zona fasciculata were occasionally seen, which were similar to the cases autopsied at the end of the dosing period. Also 1 male exhibited dark red part in the lung and foamy liquid in the trachea, and histologically, mild pulmonary hemorrhage and slight edema were observed. However, there were no findings indicating any damage in the respective organs and tissues, and all the changes possibly caused by administration of the test substance were presumed to be reversible with 14-day discontinuation of administration. From these results, the NOAEL under the conditions of this study was 100 mg/kg bw/day for both males and females and the LOAEL was set to 400 mg/kg bw/day due to mortality and severe findings in nervous system, liver and kidney.

 

Additional data was provided by Cornish et al. (1969). A five week oral toxicity study of 2-dibutylaminoethanol (purity unknown) was undertaken by adding neutralized 2-dibutylaminoethanol to rats’ (Sprague-Dawley) drinking water at concentrations of 4, 2 and 1 g/L (corresponding to 430, 200, 130 mg/kg bw/day for the males and 330, 240, 140 mg/kg bw/day for the females). A control group was maintained on distilled water. Five male and five female rats were maintained on each dose level. At 4 weeks, two male rats from each dose level were sacrificed and examined (hematocrit, red cell counts, white cell counts, blood sugar levels, liver and kidney weights, histopathological examinations of heart, lung, liver, kidney, spleen, adrenal brain and duodenum). For males in the two highest dose levels an initial weight loss was evident over the first few days. Initial water intake was considerably depressed in these two groups. At the highest dose level, this weight loss continued for approximately 2 weeks. In both groups, this was followed by a growth rate paralleling that of the controls and the lowest dosage group. At the lowest dose level, 130 mg/kg bw/day, no initial significant weight loss occurred, although the growth curve remained consistently below that of the control group. For female animals, an initial weight loss at all three dose levels occurred, being greatest at the high dose level. Weight losses at the two intermediate levels were quite comparable. Subsequent growth rates were comparable in all groups, although no group completely overcame its original weight. In animals which were sacrificed at 4 weeks hematocrits, red and white blood cell counts and blood glucose levels were comparable in all groups. In animals which were sacrificed at the end of approximately 5 weeks liver weight to body weight ratios were not different from controls in any fed group. The kidney weight ratios, however, show significant alterations, the ratio generally increasing with increasing dosage. At the high dose level, the ratio is markedly elevated in both male and female rats. Histhopathologic examination of heart, lung, liver, kidney, adrenal, spleen, and duodenum of neutralized test substance were not different from controls. These results show that once an adjustment of water and food intake had been made, animals gained weight at an approximate normal rate over the remainder of the 30-day period. The significant increase in kidney to body weight ratios at the high dose level cannot be directly interpreted as an effect on kidney as histologic examination of the tissues including kidney, indicate that these organs were essentially normal at the 30-day feeding period. The kidney weights of treated and control groups were comparable at the end of the thirty day feeding period, however, treated animals had not regained their initial weight, thus the kidney to body weight ratios remained elevated. Thus, the NOAEL was set to 430 mg/kg bw/day for male animals and to 330 mg/kg bw/day for female animals (highest dose tested).

 

Inhalation:

A GLP-compliant investigation of the toxicological effects resulting from repeated inhalation exposure to Wistar rats was performed following OECD guideline 422 (BASF SE, 2013). Groups of ten male and ten female rats (F0 animals) per test group were exposed nose-only to a dynamic atmosphere of 2-dibutylaminoethanol aerosol 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. In addition, after the lactation period and after necropsy of the pups all parental females were exposed to the test substance on 4 consecutive days. The target concentrations were 25, 75 and 225 mg/m³ (measured concentrations: 20.6, 72.1, 236.3 mg/m³). A concurrent control group was exposed to conditioned air. The test 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. In histopathology lesions of nasal epithelia was 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.

Additional data was provided by Cornish et al. (1969). 50 male Sprague-Dawley rats were exposed (whole body) 6 hours daily for a 6 months period to 2-dibutylaminoethanol vapours (analytical concentration: 22 ppm, corresponding to 156 mg/m³). Animals were sacrificed periodically after 1, 4, 15, 27 and 29 weeks (5 test and 5 control animals each exposure period) and hematocrit, white blood cell count, serum bilirubin, and organ weight ratios determined. Tissues were also taken for histopathological examination. No animal died. No untoward symptoms were noted in the animals during the exposure period. By the end of the 4th week of exposure, total body weight gain was comparable to the controls. Serum bilirubin levels were higher than controls in 3 of 5 rats in the animals that were sacrificed after 4 weeks of exposure. Kidney to body weight ratios were somewhat elevated at the end of the first week on this schedule. No further significant changes were seen in the animals throughout the six month exposure period, and at sacrifice all biological measurements were comparable to those of the control group. Histological sections of the organs from animals exposed for 6 months were not different from the controls. As male rats exposed for 6 months to 22 ppm (156 mg/m³) of 2-dibutylaminoethanol were comparable to controls throughout the exposure period, a NOAEC of 156 mg/m³ was determined.

In the publication of Cornish et al. (1969) 5 male Sprague-Dawley rats were exposed to 2-dibutylaminoethanol vapours (analytical concentrations: 33 and 70 ppm, corresponding to 234 and 495 mg/m³) 6 hours daily for 5 days. Hematocrit, white blood cell count, serum bilirubin, and organ weight ratios determined. Tissues were also taken for histopathological examination. Exposure to high levels (70 ppm, 495 mg/m³) of 2-dibutylaminoethanol produced tremors and convulsive seizures in rats. In addition, visual signs of eye and nasal irritation were evident in those animals exposed to approximately 70 ppm. One animal died and great weight losses occurred in these animals. A one week exposure to 33 ppm (234 mg/m³) resulted in a lack of growth, but no other significant findings. Slightly elevated bilirubin levels in some, but not all rats, were noted at the end of 5 days of exposure to 70 ppm. A similar elevation was not evident in the 33 ppm group. Organ to body weight ratios of livers and kidneys of the 70 ppm animals showed marked elevations due to the great weight loss during this period. Concerning the 33 ppm animals, liver to body weight ratios were normal, while kidney to body weight ratios were slightly elevated. There was no histological evidence of tissue damage in these groups of animals. Due to the results of this range-finding study, a NOAEC of 33 ppm (234 mg/m³) and a LOAEC of 70 ppm (495 mg/m³) was determined.

 

Justification for classification or non-classification

Dangerous Substance Directive (67/548/EEC)

The available experimental test data is reliable and suitable for the purpose of classification under Directive 67/548/EEC. Based on the data, classification for repeated dose toxicity is not warranted.

 

Classification, Labeling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for the purpose of classification under Regulation (EC) No.1272/2008. Based on the data, classification for repeated dose toxicity is not warranted.