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Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key 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).

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2004
Report Date:
2004

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): 2-(Di-n-butylamino)ethanol
- Physical state: light-yellow and clear liquid
- Analytical purity: 99.8%
- Impurities (identity and concentrations): 0.12 % moisture as impurity
- Stability under test conditions: 8-day stability under the condition of cold storage was confirmed. Thus the samples for administration were prepared at least once a week, and the prepared samples were separated into brown bottles and stored in the refrigerator before use.
- Storage condition of test material: Stored in a well-closed container at room temperature, protected from light and high temperatures.

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals 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

Administration / exposure

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
Doses / concentrationsopen allclose all
Dose / conc.:
25 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
400 mg/kg bw/day (actual dose received)
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.)

Examinations

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).

Results and discussion

Results of examinations

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.

Effect levels

open allclose all
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: no effects
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: no adverse effects
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

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
NOAEL: 100 mg/kg bw;
LOAEL: 400 mg/kg bw.
Executive summary:

In the 28-day study (according to OECD 407; Ministry of Health, Labour and Welfare, Japan, 2004), the NOAEL was 100 mg/kg bw/day for both males and females and the LOAEL was set to 400 mg/kg bw due to mortality and severe findings in nervous system, liver and kidney. There were significant increases in relative liver weights in males and females of the highest dose group. There were also significant increases in relative kidney weight for males as well as in actual and relative adrenal weights for females. In animals that died on study and in those that survived until scheduled necropsy, increases in liver and kidney sizes were noted. Microscopically, centrilobular hypertrophy of hepatocytes and vacuolization of the epithelial cells of the collecting ducts of kidneys were observed. The other target organs were adrenals, spleen, lungs and thymus. Concerning parameters of clinical chemistry, 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 in which further data about inhibition of choline esterase activity by butyl ethanolamines are presented) . 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.