2-[2-(dimethylamino)ethoxy]ethanol

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

2-[2-(dimethylamino)ethoxy]ethanol

EC Number:

216-940-1

EC Name:

2-[2-(dimethylamino)ethoxy]ethanol

Cas Number:

1704-62-7

Molecular formula:

C6H15NO2

IUPAC Name:

2-[2-(dimethylamino)ethoxy]ethan-1-ol

Test material form:

liquid

Details on test material:

Test substance No.: 18/0329-1
Batch identification: 83205256P0
Stability: Expiry date: 31 Dec 2019
The stability of the test substance under storage conditions over the test period was guaranteed by the sponsor, and the sponsor holds this responsibility.

Specific details on test material used for the study:

Name of test substance: 2-[2-(dimethylamino)ethoxy]ethanol
Test substance No.: 18/0329-1
Batch identification: 83205256P0
CAS number: 1704-62-7
Purity: 98.3 corr. area-% (GC, RTX-5-Amin capillary); 98.4 corr. area-% (GC, DB-Wax UI capillary)
Identity: Confirmed
Homogeneity: Given
Stability: Expiry date: 31 Dec 2019, The stability of the test substance under storage conditions over the test period was guaranteed by the sponsor, and the sponsor holds this responsibility.

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

In a preliminarily performed range-finding study (BASF project No. 10C0329/18C040), the test substance 2-[2-(dimethylamino)ethoxy]ethanol was administered for 2 weeks to groups of 5 male and 5 female Wistar rats at dose levels of 0, 300, or 1000 mg/kg body weight/day (mg/kg bw/d). At 1000 mg/kg bw/d, findings like poor general condition in males and body weight loss in males as well as in females occurred during the first days of administration. In addition, two males and one female animal showed respiration sounds, and labored respiration was observed in one male. Towards the end of the administration period, one
female animal was found dead. No treatment-related, adverse findings were observed at 300 mg/kg bw/d.
In a subsequently performed Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test (OECD 422; BASF project No. 85R0329/18C038) 2-[2-(dimethylamino)ethoxy]ethanol was administered by gavage at dose levels of 0, 80, 250, and 750 mg/kg bw/d. Treatment-related findings were observed in male and female animals indicated by changed hematology parameters. Based on these results, the following dose levels were selected for the present study:
750 mg/kg bw/d as high-dose level
250 mg/kg bw/d as intermediate-dose level
80 mg/kg bw/d as low-dose level
The oral route was selected since this was proven to be suitable for the detection of a toxicological hazard.

Vehicle:

water

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

90 days

Frequency of treatment:

once daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

750 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

250 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

80 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

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 hours.

Examinations

Observations and examinations performed and frequency:

CLINICAL EXAMINATIONS
Mortality
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.

Clinical observations
All animals were checked daily for any abnormal clinically signs before the administration as well as within 2 hours and between 2 and 5 hours after the administration. Abnormalities and changes were documented for each animal.

Detailed clinical observations
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:
1. Abnormal behavior when handled
2. Fur
3. Skin
4. Posture
5. Salivation
6. Respiration
7. Activity/arousal level
8. Tremors
9. Convulsions
10. Abnormal movements
11. Impairment of gait
12. Lacrimation
13. Palpebral closure
14. Exophthalmus
15. Feces (appearance/ consistency)
16. Urine
17. Pupil size

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

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

Body weight data
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.

Functional observational battery
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. 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. The findings were ranked according to the degree of severity, if applicable. The observations were performed at random.

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:
1. Posture
2. Tremors
3. Convulsions
4. Abnormal movements
5. Gait abnormalities
6. 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:
1. Behavior on removal from the cage
2. Fur
3. Skin
4. Salivation
5. Nasal discharge
6. Lacrimation
7. Eyes/ pupil size
8. Posture
9. Palpebral closure
10. Respiration
11. Tremors
12. Convulsions
13. Abnormal movements/ stereotypes
14. Gait
15. Activity/ arousal level
16. Feces excreted within 2 minutes (consistency/ color)
17. Urine excreted within 2 minutes (amount/ color)
18. Rearing within 2 minutes
19. Other findings

Sensory motor tests/ reflexes:
The animals were then removed from the open field and subjected to following sensory motor or reflex tests:
1. Reaction to an object being moved towards the face (approach response)
2. Touch sensitivity (touch response)
3. Vision (visual placing response)
4. Pupillary reflex
5. Pinna reflex
6. Audition (startle response)
7. Coordination of movements (righting response)
8. Behavior during handling
9. Vocalization
10. Pain perception (tail pinch)
11. Grip strength of forelimbs
12. Grip strength of hindlimbs
13. Landing foot-splay test
14. 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. The program requires a file name for the measured data to be stored. This name consists of the reference number and a serial number.

Ophthalmoscopy
Prior to the start of the administration period on day -2 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 (HEINE OPTOTECHNIK, Herrsching, Germany) after administration of a mydriatic agent (Mydrum, Bausch & Lomb GmbH, Berlin, Germany).

Estrous cycle determination
Vaginal smears for terminal vaginal cytology examinations were prepared in the morning of the day of sacrifice.

CLINICAL PATHOLOGY
In the morning blood was taken from the retro-bulbar venous plexus from fasted animals. The animals were anaesthetized using isoflurane. The blood sampling procedure and subsequent analysis of blood and serum samples were carried out in a randomized sequence. For urinalysis the individual animals were transferred to metabolism cages (withdrawal of food and water) and urine was collected overnight. Urine samples were evaluated in a randomized sequence. The assays of blood and serum parameters were performed under internal laboratory quality control conditions with reference controls to assure reliable test results. The results of clinical pathology examinations were expressed in International System (SI) units. The following examinations were carried out in all surviving animals per test group and sex.

Hematology
The following parameters were determined in blood with EDTA-K3 as anticoagulant using a particle counter (Advia 120 model; Bayer, Fernwald, Germany):
Leukocyte count
Erythrocyte count
Hemoglobin
Hematocrit
Mean corpuscular volume
Mean corpuscular hemoglobin
Mean corpuscular hemoglobin concentration
Platelet count
Reticulocytes
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. (reference: Hematology: Principles and Procedures, 6th Edition, Brown AB, Lea & Febiger, Philadelphia, 1993, page 101). Only evaluated blood smears were archived.
Clotting tests were carried out using a ball coagulometer (AMAX destiny plus model; Trinity biotech, Lemgo, Germany).
Prothrombin time

Clinical chemistry
An automatic analyzer (Cobas c501; Roche, Mannheim, Germany) was used to examine the clinico-chemical parameters.
Alanine aminotransferase
Aspartate aminotransferase
Alkaline phosphatase
γ-Glutamyltransferase
Sodium
Potassium
Chloride
Inorganic phosphate
Calcium
Urea
Creatinine
Glucose
Total bilirubin
Total protein
Albumin
Globulins
Triglycerides
Cholesterol
Bile acids
HDL-Cholesterol
LDL-Cholesterol

Thyroid Hormones
The concentrations of TSH were determined by radio-immuno assay (RIA), using commercially available RIA test kits and a Gamma-Counter (LB 2111, Berthold, Germany). T3 and T4 Elisa was measured with a Sunrise MTP-reader, Tecan AG, Maennedorf, Switzerland, and evaluated with the Magellan-Software of the instrument producer. Parameters and methods:
Total triiodothyronine (T3)
Total thyroxine (T4)
Thyroid stimulating hormone (TSH)

Urinalysis
The dry chemical reactions on test strips (Combur-Test 10 M; Sysmex, Norderstedt, Germany) used to determine urine constituents semi-quantitatively were evaluated with a reflection photometer (CobasU 411; Sysmex, Norderstedt, Germany).
Protein (PRO) tetrabromophenol-phthaleinethylester
Glucose (GLU)
Ketones (KET)
Urobilinogen
Specific gravity
Color, turbidity
Volume (VOL)

Statistics of clinical pathology
Means, medians and standard deviations of each test group were calculated for several parameters. Mean values were rounded, but deviations of means versus control means were calculated with not rounded values. Therefore, slight differences may occur when changes were re-calculated with rounded means.

Sacrifice and pathology:

PATHOLOGY
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:
1. Anesthetized animals (final body weight)
2. Adrenal glands (fixed)
3. Brain
4. Epididymides
5. Heart
6. Kidneys
7. Liver
8. Ovaries (fixed)
9. Pituitary gland (fixed)
10. Prostate (ventral and dorsolateral part together, fixed)
11. Spleen
12. Seminal vesicles including coagulating glands (fixed)
13. Testes
14. Thymus (fixed)
15. Thyroid glands (with parathyroid glands) (fixed)
16. Uterus with cervix
All paired organs were weighed together (left and right).

Organ/tissue fixation
The following organs or tissues were fixed in 4% neutral buffered formaldehyde solution or in modified Davidson’s solution:
1. All gross lesions
2. Adrenal glands
3. Aorta
4. Bone marrow (femur)
5. Brain
6. Cecum
7. Cervix
8. Coagulating glands
9. Colon
10. Duodenum
11. Epididymides (modified Davidson’s solution)
12. Esophagus
13. Extraorbital lacrimal glands
14. Eyes with optic nerve (modified Davidson’s solution)
15. Femur with knee joint
16. Harderian glands
17. Heart
18. Ileum
19. Jejunum (with Peyer’s patches)
20. Kidneys
21. Larynx
22. Liver
23. Lungs
24. Lymph nodes (mesenteric and axillary lymph nodes)
25. Mammary gland (male and female)
26. Nose (nasal cavity)
27. Ovaries
28. Oviducts
29. Pancreas
30. Parathyroid glands
31. Pharynx
32. Pituitary gland
33. Prostate
34. Rectum
35. Salivary glands (mandibular and sublingual glands)
36. Sciatic nerve
37. Seminal vesicles
38. Skeletal muscle
39. Skin
40. Spinal cord (cervical, thoracic and lumbar cord)
41. Spleen
42. Sternum with marrow
43. Stomach (forestomach and glandular stomach)
44. Testes (modified Davidson’s solution)
45. Thymus
46. Thyroid glands
47. Trachea
48. Urinary bladder
49. Uterus
50. Vagina

Histopathology
Fixation was followed by histotechnical processing, examination by light microscopy and assessment of findings according to the table below:
The spleen of some male and female animals were stained exemplarily with a special stain for hemosiderin (Turnbull stain). 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. The organs were trimmed according to the “Revised guides for organ sampling and trimming in rats and mice” (Ruehl-Fehlert et al., 2003; Kittel et al., 2004; Morawietz et al., 2004). A correlation between gross lesions and histopathological findings was attempted.

Peer review
After completion of the histopathological assessment by the study pathologist an internal peer review was performed including the kidneys of all males and the spleen of all males and females in all test and control groups. Results presented in this report reflect the consensus opinion of the study pathologist and the peer review pathologist.

Statistics:

see attachment

Results and discussion

Results of examinations

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

During the administration period, several findings occurred on different study days. No treatment-related, adverse clinical findings were observed for male and female animals of all test groups (80, 250 and 750 mg/kg bw/d).
All male and all female animals of test group 3 (750 mg/kg bw/d) showed slight (sometimes moderate) salivation directly after treatment on several days of the application period. 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.
Respiration sounds were recognized in 3 male animals and 4 female animals of test group 3 (750 mg/kg bw/d) as well as in 1 female animal of test group 2 (250 mg/kg bw/d) on individual days of the administration period. In males, the finding was firstly recognized in one male animal on study day 55 and for the last time in one male animal on study day 69. In females of test group 3 (750 mg/kg bw/d), the finding was firstly recognized in one female animal on study day 16 and for the last time in one female animal on study day 95. In one female animal of test group 2 (250 mg/kg bw/d), the finding occurred on study days 21 and
22, only.
As the finding did not occur continuously and no histopathological lesions in the respiratory tract were observed, the respirations sounds were assessed to be related to treatment but not adverse. Piloerection was found in one female animal of test group 3 (750 mg/kg bw/d) only on study day 47. The reason for the occurrence of this particular finding on a single day of the administration period was assessed to be spontaneous in nature and not related to treatment.
In one male animal of test group 2 (250 mg/kg bw/d) the left testis was not palpable over the entire study period. The finding was assessed to be incidental.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

effects observed, non-treatment-related

Ophthalmological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related findings were observed. All apparent findings were assessed as being incidental in nature since they occurred in control as well as in treated animals. No striking discrepancy were observed between the examined animals of the control group and the concurrent high-dose animals.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

In males and females of test group 3 (750 mg/kg bw/d), absolute and relative eosinophil counts were significantly decreased whereas relative monocyte counts were significantly increased. Additionally, in females of this test group total white blood cell (WBC) counts and absolute lymphocyte counts were significantly decreased, whereas relative neutrophil counts were significantly increased. These alterations were regarded as treatment-related and adverse.
In males of test group 3 (750 mg/kg bw/d), absolute reticulocyte counts were significantly increased. However, the values were only slightly above the historical control range (males, absolute reticulocytes 110.5-159.1 Giga/L), other red blood cell parameters (i.e., red blood cell (RBC) counts, hemoglobin and hematocrit values) were not changed and no histopathological changes in the spleens were observed. Therefore, this change was regarded as potentially treatment-related, but non-adverse (ECETOC Technical Report No. 85, 2002).
The following significant changes were within historical control ranges and, therefore, they were regarded as incidental and not treatment-related: reduced prothrombin time (HQT, Hepatoquick’s test) in males and females of test group 3 (750 mg/kg bw/d) and in females of test group 2 (250 mg/kg bw/d); decreased absolute large unstained cell (LUC) counts in females of test group 3 (HQT, males 35.0-40.4 sec; females 32.5-37.0 sec; absolute LUC, females 0.00-0.01 Giga/L).
In males of test group 1 (80 mg/kg bw/d), absolute and relative basophil counts were significantly increased, but the change was not dose-dependent. Therefore, this alteration was regarded as incidental and not treatment related.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

In males and females of test group 3 (750 mg/kg bw/d), urea levels were significantly increased whereas total protein, albumin and globulin values were significantly decreased. Additionally, in females of this test group triglyceride, potassium and inorganic phosphate levels were significantly increased. Total protein, albumin and globulin levels were already significantly decreased in females of test groups 2 (250 mg/kg bw/d). These changes were regarded as treatment-related and adverse. In males and females of test group 3 (750 mg/kg bw/d), cholesterol were significantly increased whereas sodium and chloride levels were significantly decreased. Chloride values were already significantly lower compared to controls in females of test group 2 (250 mg/kg bw/d). In males of test group 3, potassium levels were significantly increased. However, the values of the mentioned changes in this paragraph were within historical control ranges (males, cholesterol 1.50-2.15 mmol/L; sodium 140.0-149.6 mmol/L; chloride 96.1-106.1 mmol/L; potassium 4.51-5.11 mmol/L; females, cholesterol 1.02-1.76 mmol/L; sodium 140.5-147.3 mmol/L; chloride 97.0-104.4 mmol/L). In male animals of test group 1 (80 mg/kg bw/d), potassium levels were significantly decreased, and in females of the same test group total bilirubin values were significantly increased, but these changes were not dose-dependently altered. Therefore, all mentioned alterations in this paragraph were regarded as incidental and not treatment related.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

In males of test group 3 (750 mg/kg bw/d), total protein and hemoglobin values (i.e. BLOOD_C) in the urine as well as erythrocyte counts in the urine sediment were significantly increased. Additionally, in males and females of test group 3, urine volume was significantly lower and specific gravity of the urine was significantly higher compared to controls. These alterations were regarded as treatment-related and adverse. In males of test group 2 (250 mg/kg bw/d), urine volume was already significantly decreased and specific gravity of the urine was significantly increased. Significantly lower urine volume was also observed in males of test group 1 (80 mg/kg bw/d). However, low urine volume and high urine specific gravity without any other changes of renal parameter indicated normal adaptation of the kidneys towards low fluid income. Therefore, these changes were regarded as potentially treatment-related, but non-adverse.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Deviations from "zero values" were obtained in several animals. However, as most findings were equally distributed between test-substance treated groups and controls, were without a dose-response relationship or occurred in single animals only, these observations were considered to have been incidental. The following examinations were performed during FOB and have to be assessed individually:
Home cage observations
No test substance-related effects were observed.

Open field observations
Respiration sounds were recognized in two female animals of test group 3 (750 mg/kg bw/d).

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 (250 mg/kg bw/d), grip strength of forelimbs was significantly lower when compared to the controls. However, a dose-response relationship did not occur, and the change was assessed to be incidental. In female animals of test groups 2 and 3 (250 and 750 mg/kg bw/d), grip strengths of foreand hindlimbs were significantly increased. The same was true for female animals of test group 1 (80 mg/kg bw/d). These changes were most likely related to the rather low values measured in the control group. A relation to treatment was not considered.

Motor activity measurement
Regarding the overall motor activity as well as single intervals, no test substance-related, relevant deviations to the control animals were noted for male and female animals of test groups 1 to 3 (80, 250 and 750 mg/kg bw/d). In male animals of test group 2 (250 mg/kg bw/d), single interval No. 3 was significantly increased, and in female animals of test groups 1-3 (80, 250, and 750 mg/kg bw/d), single interval No. 5 was significantly increased. Since no dose-response relationship occurred and the overall motor activity was not affected, the changes were assessed as being spontaneous in nature and not related to treatment.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

The significant absolute and relative liver weight increases in female animals of test group 3 (abs. 5.964 g, rel. 2.7%) were both above the historical control range (abs. 4.883-5.840 g,
rel. 2.28-2.49%). Therefore, although no histopathologic correlate was noted, they were considered to be treatment-related but not adverse. The significant relative liver weight increases in males of test groups 2 and 3 (2.232% and 2.272%, respectively) were both within the historical control range (2.045-2.299%) and occurred without a histopathological correlate. Therefore, they were assessed as not treatment-related.
The significant absolute weight increase of the kidneys in female animals of test group 3 (1.664 g) was marginally above the historical control values (1.388-1.659 g), whereas the
significant relative weight increase (0.752%) was within the historical control range (0.6-0.762%). Since no histopathological correlate was observable in these female animals, these
weight changes were not assessed to be treatment-related. The significant absolute and relative kidneys weight increases in test group 2 (abs. 1.574 g, rel. 0.718%) were both within
the historical control values and were, therefore, assessed to be not treatment-related. The significant decrease of mean brain weight in male animals of test group 3 was most likely secondary to the decrease of the final body weight (93%) and was not consistent with any histopathological changes.
The significant absolute and relative spleen weight decreases in male animals of test group 1 occurred without dose-dependency and were regarded as incidental. All other mean relative weight parameters did not show significant differences when compared to the control group 0.

see also attached background material

Gross pathological findings:

effects observed, non-treatment-related

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Treatment-related findings were observed in the kidneys of male animals as well as in thespleen of male and female animals.
The hyperplasia of transitional cells in male animals of test group 3 affected the distal renal pelvis and the start of the ureter and was accompanied by mixed-cell inflammatory cell infiltrates. These were composed predominantly of lympho-histio-plasmocytic cell types including single neutrophilic granulocytes. The cell infiltrates were always subepithelial and sometimes extended into the adjacent fat tissue around the renal pelvis. The erosion/ulceration was seen in the epithelium of the distal renal pelvis. The slight and moderate amounts of basophilic tubules observed in 2 out of 10 male animals occurred exclusively in the areas adjacent to the altered renal pelvis, as being a consequence of the altered renal pelvis and, therefore, differing from the common background basophilic tubules seen in the remaining animals.

In the spleen of males, the extramedullary hematopoiesis did not show a clear dosedependent relationship and was, therefore, not regarded as treatment-related. In females, an apparent minimal increase in test group 3 was noted, however, it was assessed as not relevant and, therefore, not treatment-related. The hemosiderin storage in males was minimally decreased in test group 3, which was regarded to be treatment-related. In females, the hemosiderin storage was not affected by treatment.
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.

see also attached background material

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

Estrous cycle determination
No obvious discrepancies between the examined stage of the estrous cycles and histopathological examinations of female sex organs occurred.

Thyroid hormones
At the end of the administration period, no treatment-related alterations of T3, T4 and TSH levels were observed in males and females of test groups 1, 2 and 3 (80, 250 and 750 mg/kg bw/d).

Details on results:

see also attachment

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

250 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

urinalysis

Target system / organ toxicity

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

750 mg/kg bw/day (nominal)

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Applicant's summary and conclusion

Conclusions:

The administration of 2-[2-(dimethylamino)ethoxy]ethanol by gavage for 3 months to male and female Wistar rats caused test substance-related findings at a dose level of 750 mg/kg bw/d taking changes in clinical pathology parameters as well as histopathological findings in the kidneys into account. Therefore, under the conditions of the present study the NOAEL was 250 mg/kg bw/d for male and female Wistar rats.

Executive summary:

2-[2-(dimethylamino)ethoxy]ethanol was administered by gavage to groups of 10 male and 10 female Wistar rats at dose levels of 0 (test group 0), 80 (test group 1), 250 (test group 2) and 750 mg/kg body weight/day (mg/kg bw/d; test group 3) over a period of 3 months Deionized water served as vehicle.

With regard to clinical examinations, relevant signs of general systemic toxicity were not observed even at a dose level of 750 mg/kg bw/d. All male and all female animals of test group 3 (750 mg/kg bw/d) showed slight (sometimes moderate) salivation directly after treatment on several days of the application period. 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. Respiration sounds were recognized in some male and female animals of test group 3 (750 mg/kg bw/d) as well as in 1 female animal of test group 2 (250 mg/kg bw/d) on individual days of the administration period. As the finding did not occur continuously and no histopathological lesions in the respiratory tract were observed, the respirations sounds were assessed to be related to treatment but not adverse. In addition, no test substance-related effects on estrous cycle length and the number of cycles were obtained.

Regarding clinical pathology, decreased absolute and relative eosinophil counts and increased relative monocyte counts in males and females of test group 3 (750 mg/kg bw/d) were due to an acute-phase reaction, probably stress. Because of the same reason, in females of this test group total white blood cell (WBC) and absolute lymphocyte counts were decreased whereas relative neutrophil counts were increased. Significantly lower total protein, albumin and globulin values and higher urea values in serum of males and females in test group 3 (750 mg/kg bw/d) indicated an increased protein metabolism. In females of test group 2 (250 mg/kg bw/d) the protein fractions were already significantly decreased. Higher serum triglyceride levels in females of the mentioned test group 3 were most probably secondarily due to the increased protein metabolism. In males of test group 3 (750 mg/kg bw/d) higher counts of erythrocytes and higher hemoglobin levels were observed in the urine. These findings were due to a bleeding in the

lower urogenital tract in these individuals.

Regarding pathology, histopathological examinations of the kidneys in male animals of test group 3 (750 mg/kg bw/d) revealed a minimal to moderate hyperplasia of transitional cells in the distal renal pelvis and the start of the ureter, affecting 7 out of 10 animals. This was accompanied by minimal to moderate mixed-cell inflammatory cell infiltrates, erosion/ulceration of the epithelium of the distal renal pelvis (2 out of 10 males) and slight to moderate basophilic tubules in the area around the renal pelvis (2 out of 10 males). All these changes were regarded as treatment-related and adverse. The mean liver weight of females in test group 3 showed a significant increase (absolute +17%, relative +16%). Although no histopathological correlate was noted, the weight increase was minimally above the historical control range. Therefore, these changes were assessed as treatment-related but not adverse. In the spleen, the hemosiderin storage was minimally decreased in males of test group 3, which was regarded to be related to treatment. Since this change was not consistent with signs of anemia in the hematology analysis, it was assessed as treatment-related but not adverse and most likely in relation with the lesions found in the kidneys.

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.