2-[2-(dimethylamino)ethoxy]ethanol

Endpoint summary

Description of key information

There are two repeated dose toxicity studies on 2-[2-(dimethylamino)ethoxy]ethanol available:

- OECD 422 [BASF, 2020]: NOAEL = 250 mg/kg bw

- OECD 408 [BASF, 2020]: NOAEL = 250 mg/kg bw

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

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

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

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.

Species:

rat

Strain:

Wistar

Sex:

male/female

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

Dose / conc.:

750 mg/kg bw/day (nominal)

Dose / conc.:

250 mg/kg bw/day (nominal)

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.

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

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

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

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

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.

Reference 2

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same 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)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

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.

Species:

rat

Strain:

Wistar

Sex:

male/female

Route of administration:

oral: gavage

Vehicle:

water

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

male animals: 30 days; female animals: 61 days

Frequency of treatment:

once daily

Dose / conc.:

750 mg/kg bw/day (nominal)

Dose / conc.:

250 mg/kg bw/day (nominal)

Dose / conc.:

80 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Observations and examinations performed and frequency:

F0 generation parental animals and their progeny
On the day of arrival, the animals were subjected to an acclimatization period during which they received ground diet and drinking water ad libitum. Estrous cycle determination prior to treatment was performed in a pool of up to 50 non-randomized female animals. Only animals with regular estrous cycle were selected for randomization before the start of the treatment period. 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 by a computer. After the acclimatization period, the test substance was administered orally via gavage to the F0 generation parental animals, daily at the same time in the morning (exception: no administration to animals being in labour). The treatment lasted up to one day prior to sacrifice. The animals of the control group were treated in the same way with the vehicle only (deionized water). The volume administered each day was 10 mL/kg body weight. The calculation of the administered volume was generally based on the most recent individual body weights.
Two weeks after the beginning of treatment, the surviving males and females from the same test group were mated overnight in a ratio of 1:1 (for details of pairing see section 3.7.2.).

Mating of F0 generation parental animals
In general, each of the male and female animals was mated overnight in a 1:1 ratio for a maximum of 2 weeks. Throughout the mating period, each female animal was paired with a predetermined male animal from the same test group. The animals were paired by placing the female in the cage of the male mating partner from about 16.00 h until 06.30 - 09.00 h of the following morning. Deviations from the specified times were possible on weekends and public holidays and were reported in the raw data. A vaginal smear was prepared after each mating and examined for the presence of sperm. If sperm was detected, pairing of the animals was discontinued. The day on which sperm was detected was denoted gestation day (GD) 0 and the following day "GD 1". Females that were successfully mated in the first night were directly transferred from study phase “mating” into study phase “gestation” before performing clinical observations. Therefore, the tables clinical observation of females in study phase “mating” show less examined animals than the nominal sample size of 10 females. The clinical observations of successfully mated females were documented in study phase “gestation”.

Standardization of litters (F1 generation pups)
On PND 4, the individual litters were standardized in such a way that, where possible, each litter contained 4 male and 4 female pups (always the first 4 pups/sex and litter were taken for further rearing). If individual litters did not have 4 pups/sex, the litters were processed in such a way that the most evenly distributed 8 pups per litter were present for further rearing (e.g., 5 male and 3 female pups). Standardization of litters was not performed in litters with ≤ 8 pups.

Pups after standardization
On PND 4, as a result of standardization, the surplus pups were sacrificed under isoflurane anesthesia by decapitation. Blood were sampled for possible determination of thyroid hormone concentrations. After sacrifice, the pups were examined externally and eviscerated, and their organs were assessed macroscopically. On PND 13, one selected male and one female pup per litter was sacrificed under isoflurane anesthesia by decapitation. Blood was sampled for determination of thyroid hormone concentrations. Thyroid glands/parathyroid glands were fixed in neutral buffered 4% formaldehyde solution and were transferred to the Pathology Laboratory for possible further processing. The remaining pups were sacrificed under isoflurane anesthesia with CO2. After sacrifice, all pups were examined externally and eviscerated, and their organs were assessed macroscopically. All culled pups, including stillborn pups and those that died during their rearing period, were subjected to a macroscopic (external and visceral) examination. All pups without any notable findings or abnormalities were discarded after their macroscopic evaluation. Animals with notable findings or abnormalities were further evaluated on a case-by-case basis (e.g., histopathological evaluation or special staining), depending on the findings noted.

CLINICAL EXAMINATIONS AND EXAMINATION OF REPRODUCTIVE
PERFORMANCE
Parental animals
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
A cageside examination was conducted at least once daily for any signs of morbidity, pertinent behavioral changes and/or signs of overt toxicity. All animals were checked daily for any abnormal clinical signs before the administration as well as within 2 hours and within 5 hours after the administration. Abnormalities and changes were documented for each animal. The parturition and lactation behavior of the dams was generally be evaluated in the morning in combination with the daily clinical inspection of the dams. Only particular findings (e.g. inhability to deliver or umbilical cord not cut) were documented on an individual dam basis. On weekdays (except Saturdays, Sundays and public holidays) the parturition behavior of the dams was inspected in the afternoons in addition to the evaluations in the mornings. The day of parturition was considered to be the 24-hour period from about 15:00 h of one day until about 15:00 h of the following day.

Food consumption
Generally, food consumption was determined at least 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 parental animals).
• Food consumption of the females with evidence of sperm was determined for GD 0-7, 7-14 and 14-20.
• Food consumption of the females which gave birth to a litter was determined for PND 1-4, 4-7, 7-10 and 10-13.
Food consumption was not determined in females without positive evidence of sperm during the mating and the gestation period and in females without litter during the lactation period.

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 of the male and female parental animals was determined before the start of the administration period in order to randomize the animals. During the administration period body weight was determined on study day 0 (start of the administration period) and thereafter at least once a week at the same time of the day (in the morning). 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 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 0), PNDs 4, 7, 10 and 13. Females without positive evidence of sperm, without litter and females after weaning (PND 13) were weighed weekly. These body weight data were solely used for the calculations of the dose volume.

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 in handling
2. fur
3. skin
4. posture
5. salivation
6. respiration
7. activity/arousal level
8. tremors
9. convulsions
10. abnormal movements
11. gait abnormalities
12. lacrimation
13. palpebral closure
14. exophthalmos (protruding eyeball)
15. assessment of the feces discharged during the examination (appearance/consistency)
16. assessment of the urine discharged during the examination
17. pupil size

Functional observational battery
A functional observational battery (FOB) was performed in the first five parental male animals per test group and the first five surviving females with litter (in order of delivery) of all test groups at the end of the administration period starting at about 10.00 h. 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. 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 animals. Attention was paid to:
1. Posture
2. Tremors
3. Convulsions
4. Abnormal movements
5. Gait
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 (appearance/ consistency)
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 (auditory startle response)
7. coordination of movements (righting response)
8. behavior during handling
9. vocalization
10. pain perception (tail pinch)
11. other findings
12. grip strength of forelimbs
13. grip strength of hindlimbs
14. landing foot-splay test

Motor activity assessment
Motor activity (MA) was also measured from 14:00 h onwards on the same day as the FOB was performed in the first five parental males and the first five surviving females with litter (in order of delivery) per group. The examinations were performed using the TSE Labmaster System supplied by TSE Systems GmbH, Bad Homburg, Germany. For this purpose, the animals 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 numbers of beam interrupts were counted over 12 intervals of 5 minutes per interval. The sequence in which the animals 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 animals during these measurements and the measurement room was darkened after the transfer of the last animal. The program required a file name for the measured data to be stored. This name consisted of the reference number and a serial number.

Estrous cycle
For all females of the pool estrous cycle normality was evaluated before the beginning of the administration. In all parental females in the premating phase, estrous cycle length and normality was evaluated by preparing vaginal smears during a minimum of 2 weeks prior to premating, mating and throughout cohabitation until there is evidence of sperm in the vaginal smear. Additionally, on the day of scheduled sacrifice, the estrous status was also determined in all F0 female animals.

Male reproduction data
The pairing partners, the number of mating days until vaginal sperm was detected in the female animals, and the gestational status of the females were recorded for F0 breeding pairs. For the males, mating and fertility indices were calculated for F1 litters.

The total number of pups delivered and the number of liveborn and stillborn pups were noted, and the live birth index was calculated for F1 litters.

3.8.2. Litter/Pups
Litter data
Pup number and status at delivery
All pups delivered from the F0 parents (F1 litter) were examined as soon as possible on the day of birth to determine the total number of pups, the sex and the number of liveborn and stillborn pups in each litter. At the same time, the pups were also being examined for macroscopically evident changes. Pups, which died before this initial examination, were defined as stillborn pups.

Pup viability/mortality
In general, a check was made for any dead or moribund pups twice daily on workdays (once in the morning and once in the afternoon) or as a rule, only in the morning on Saturdays, Sundays or public holidays. Dead pups were evaluated by the methods, which are described in detail in “Necropsy observations”.
The number and percentage of dead pups on the day of birth (PND 0) and of pups dying between PND 1-4, 5-7 and 8-13 were determined. Pups, which died accidentally or had to be sacrificed due to maternal death, were not included in these calculations. The number of live pups/litter was calculated on the day of birth (PND 0), and on lactation days 4, 7 and 13. Furthermore, viability and survival indices were also calculated.

Sex ratio
On the day of birth (PND 0) the sex of the pups was determined by observing the distance between the anus and the base of the genital tubercle; normally, the anogenital distance is considerably greater in male than in female pups. Later, during the course of lactation, this initial sex determination was followed up by surveying the external appearance of the anogenital region and the mammary line. The sex of the pups was finally confirmed at necropsy. The sex ratio was calculated at PND 0 and PND 13.

Pup clinical observations
The live pups were examined daily for clinical symptoms (including gross-morphological findings) during the clinical inspection of the dams and documented for each pup.

Pup body weight data
The pups were weighed on the day after birth (PND 1) as well as on PND 4 (before standardization), 7 and 13. Pups' body weight change was calculated from these results. The individual weights were always determined at about the same time of the day (in the morning). In the summary tables pup body weights and pup body weight change are listed for males, females and males + females.
“Runts” were defined on the basis of the body weights on PND 1. "Runts" are pups that weigh less than 75% of the mean weight of the respective control pups (separated by sex).

Anogenital distance
Anogenital distance (AGD; defined as the distance from the anus [center of the anal opening] to the base of the genital tubercle) measurements was done blind to treatment in a randomized order, using a measuring ocular, on all live male, female and uncertain pups on PND 1.

Anogenital index
The anogenital index was calculated also.

Nipple/areola anlagen
All surviving male pups were examined for the presence of nipple/areola anlagen on PND 13 of the lactation phase. The number of nipple/areola anlagen was counted.

Pup necropsy observations
On PND 4, as a result of standardization, the surplus pups were sacrificed under isoflurane anesthesia by decapitation. Blood was sampled for determination of thyroid hormone concentrations. After sacrifice, the pups were examined externally and eviscerated, and their organs were assessed macroscopically. On PND 13, one selected male and one female pup per litter was sacrificed under isoflurane anesthesia by decapitation. Blood was sampled for determination of thyroid hormone concentrations. Thyroid glands/parathyroid glands were fixed in neutral buffered 4% formaldehyde solution and were transferred to the Pathology Laboratory for possible further processing. The remaining pups were sacrificed under isoflurane anesthesia with CO2. After sacrifice, all pups were examined externally and eviscerated, and their organs were assessed macroscopically. All stillborn pups and all pups that died before day 13 were examined externally, eviscerated and their organs were assessed macroscopically.
All pups without notable findings or abnormalities were discarded after their macroscopic evaluation. Animals with notable findings or abnormalities were evaluated on a case-by-case basis, depending on the type of finding noted.

Sacrifice and pathology:

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. 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 parameters listed below were examined in the first 5 surviving parental males per group at termination and in the first 5 females with litters (in order of delivery) per group at PND 14.

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.
Only evaluated blood smears were archived. Clotting tests were carried out using a ball coagulometer (AMAX destiny plus model; Trinity biotech, Lemgo, Germany). Parameter and method:
Prothrombin time

Clinical chemistry
An automatic analyzer (Cobas c501; Roche, Mannheim, Germany) was used to examine the clinicochemical parameters Parameters and methods:
Alanine aminotransferase
Aspartate aminotransferase
Alkaline phosphatase
γ-Glutamyltransferase

Blood Chemistry Parameter
Sodium
Potassium
Chloride
Inorganic phosphate
Calcium
Urea
Creatinine
Glucose
Total bilirubin
Total protein
Albumin
Globulins
Triglycerides
Cholesterol
Bile acids

Thyroid Hormones
Blood samples were taken from all surplus pups pups per litter at PND 4 as well as one male and one female pup per litter at PND 13 by decapitation under isoflurane anesthesia. Additionally blood samples from all dams at PND 14/15 and all males at termination were taken by puncturing the retrobulbar venous plexus under isoflurane anesthesia. The adults were fastened before the blood sampling. All generated serum samples were frozen at -80°C until measurement or least until finalization of the report. Blood samples from the adult males and the PND 13 pups were assessed for serum levels for thyroid hormones (T4 and TSH). Samples of the PND 4 pups and the dams were not
measured. (For technical reasons the values of PND 13 pups (males nos 201-240, females nos 301- 340) were listed in the mean and individual tables under test groups 10, 11, 12 and 13 instead of 0, 1, 2 and 3). The concentrations of TSH were determined by radioimmunoassay (RIA), using commercially available RIA test kits and a Gamma-Counter (LB 2111, Berthold, Germany). 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:

Hormone parameter Method (LOQ) References
Total thyroxine
Thyroid stimulating hormone

Statistics of clinical pathology
Means, medians, standard deviations and deviation vs control 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. In these tables “deviation vs control” means x-fold of controls expressed as percentages minus 100%.

PATHOLOGY
Necropsy
All parental animals were sacrificed by decapitation under isoflurane anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology, special attention being given to the reproductive organs. The female animal No. 136 was sacrificed moribund and was 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. Epididymides
3. Ovaries
4. Prostate (ventral and dorsolateral part together, fixed)
5. Seminal vesicles with coagulating glands (fixed)
6. Testes
7. Thyroid glands (with parathyroid glands) (fixed)
8. Uterus with cervix
The following weights were determined in 5 animals per sex/test group sacrificed on schedule (females with litters only, same animals as used for clinical pathological examinations):
1. Adrenal glands (fixed)
2. Brain
3. Heart
4. Kidneys
5. Liver
6. Spleen
7. Thymus (fixed)
All paired organs were weighed together (left and right).

Organ/tissue fixation
The following organs or tissues of all parental animals were fixed in 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. Esophagus
12. Epididymides (modified Davidson’s solution)
13. Extraorbital lacrimal glands
14. Eyes with optic nerve (modified Davidson’s solution)
15. Femur with knee joint
16. Heart
17. Ileum
18. Jejunum (with Peyer’s patches)
19. Kidneys
20. Larynx
21. Liver
22. Lungs
23. Lymph nodes (axillary and mesenteric)
24. Mammary gland (male and female)
25. Nose (nasal cavity)
26. Ovaries (modified Davidson’s solution)
27. Oviducts
28. Pancreas
29. Parathyroid glands
30. Pharynx
31. Pituitary gland
32. Prostate
33. Rectum
34. Salivary glands (mandibular and sublingual)
35. Sciatic nerve
36. Seminal vesicles
37. Skeletal muscle
38. Spinal cord (cervical, thoracic and lumbar cord)
39. Spleen
40. Sternum with marrow
41. Stomach (forestomach and glandular stomach)
42. Testes (modified Davidson’s solution)
43. Thymus
44. Thyroid glands
45. Trachea
46. Urinary bladder
47. Uterus
48. Vagina
The eyes with optic nerve and ovaries of the animals that was sacrificed intercurrently were fixed in 4% neutral buffered formaldehyde solution.

The uteri of all cohabited female F0 parental animals were examined for the presence and number of implantation sites. The uteri of apparently nonpregnant animals or empty uterus horns were placed in 1% ammonium sulfide solutions for about 5 minutes in order to be able to identify early resorptions or implantations (SALEWSKI E, 1964). Then the uteri were rinsed carefully in physiologic salt solution (0.9 % NaCl). When the examinations were completed, the uteri were transferred to the Pathology Laboratory for further processing.

Pups
On PND 13, one selected male and one female pup per litter was sacrificed under isoflurane anesthesia by decapitation. Blood was sampled for determination of thyroid hormone concentrations (see 3.8.2.7.). Thyroid glands/parathyroid glands were fixed in neutral buffered 4% formaldehyde solution, were transferred to the Pathology Laboratory and were archived without further processing.

Histopathology
Fixation was followed by histotechnical processing, examination by light microscopy and assessment of findings
The animal that was sacrificed in a moribund state was processed histotechnically and assessed like control animals. 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. 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. Whenever in the ovary the diagnosis: „no abnormalities detected” was used that implies that all different stages of functional bodies (especially corpora lutea) were present and normal.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Summary clinical observations for males and females
Slight salivation, within 2 hours after administration but not thereafter, was observed in test group 3 (750 mg/kg bw/d) in 6 male and 6 female animals during premating, in 5 male and one female animal during mating phase as well as in two male animals during post-mating phase. Based on the occurrence shortly after the oral administration via gavage but not permanently, this finding was assessed as being related to mild local irritation by or bad taste of the test substance. Thereby, this finding is treatment-related but not adverse. In the control group (0 mg/kg bw/d) one male animal showed a skin lesion at the left shoulder region between mating day 11 to 14 and during post-mating from study day 0 to the day of sacrifice. One male animal of test group 3 (750 mg/kg bw/d) showed slight poor general condition and slight respiration labored during mating phase day 7 to 13. Based on the occurrence of the finding in the high-dose group, it could not be excluded that the observation was treatment related. Since the finding occurred only temporary during mating and was not observed at the end of mating and during post-mating, the finding was assessed as not adverse.
No test substance-related, adverse findings were observed in male and female animals of test groups 1-2 (80 and 250 mg/kg bw/d).

Summary clinical observations for females during gestation
Slight salivation shortly after treatment (<2 hours after treatment) was observed in 4 animals of test groups 3 (750 mg/kg bw/d) during gestation. Based on the occurrence shortly after the oral administration via gavage but not permanently, this finding was assessed as being related to mild local irritation by or bad taste of the test substance. Thereby, this finding is treatment-related but not adverse. In test group 3 (750 mg/kg bw/d) one female was sacrificed in a moribund state after showing labored respiration, respiration sounds and severe poor general condition on GD 1. Based on clinical findings observed within a few hours after gavage administration, the moribund state of the animal was assessed as the consequence of a gavage error and not to be test substance related.

Clinical observations for females during lactation
Slight salivation shortly after treatment (<2 hours after treatment) was observed in eight female animals of test group 3 (750 mg/kg bw/d) during lactation. Based on the occurrence shortly after the oral administration via gavage but not permanently, this finding was assessed as being related to mild local irritation by or bad taste of the test substance. Thereby, this finding is treatment-related but not adverse.

Detailed clinical observations
In the detailed clinical observations on study days 0, 7, 14, 21, 28, 35, 42, 49 and 56 in parental female animals no treatment-related findings were observed. One male animal of the control group (0 mg/kg bw/d) showed a skin lesion at the left shoulder region. One male animal of test group 3 (750 mg/kg bw/d) showed slight poor general condition and slight respiration labored. These findings were also observed in the clinical observations but only temporary. Therefore, these findings were assessed as not adverse. All male animals of test group 1 (80 mg/kg bw/d) and test group 2 (250 mg/kg bw/d) did not show any alterations.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One female animal of test group 3 (750 mg/kg bw/d) was sacrificed in a moribund state after showing respiration labored and sounds and severe poor general condition on gestation day 1. Based on the clinical findings observed within a few hours after gavage administration, the moribund state of the animal was assessed as the consequence of a gavage error and not to be test substance related.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

No test substance-related alterations of mean body weights were observed for male and female animals of test groups 1-3 (80, 250 and 750 mg/kg bw/d) when compared to the control group. Body weight changes varied at a non-statistically significant degree throughout all test groups, partly increased and partly decreased. Since thereby no dose-effect relationship was shown, these alterations were considered to be incidental in nature and not 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):

no effects observed

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

At the end of the administration period in males of test group 3 (750 mg/kg bw/d) hemoglobin, hematocrit and red blood cell (RBC) counts were significantly decreased whereas absolute reticulocyte counts were significantly increased. Only the absolute reticulocyte counts were above the historical control range (males, absolute reticulocytes 99.5-174.4 Giga/L), whereas RBC counts, hemoglobin and hematocrit values were within these ranges (males, RBC 7.91-8.88 Tera/L, hemoglobin 8.5-9.7 mmol/L, hematocrit 0.405- 0.444 L/L). The study control means for RBC counts and hematocrit values were above historical control ranges. However, because all three measured red blood cell parameters (i.e. RBC, hemoglobin and hematocrit) in males of test group 3 were significantly decreased and the absolute reticulocyte counts were increased above the historical control range, a treatment-related, adverse effect is assumed. In contrast, in males of test groups 1 and 2 (80 and 250 mg/kg bw/d) RBC counts and hematocrit values were decreased (hematocrit values in males of test group 2 not statistically significantly) within their historical control ranges, and the absolute reticulocyte counts were not changed. Therefore, these alterations were regarded as incidental and not treatment-related. In males of test group 1 (80 mg/kg bw/d) platelet counts were significantly decreased, but the change was not dose-dependent. Therefore, it was regarded as incidental and not treatment-related. In rats of both sexes of test group 3 (750 mg/kg bw/d), absolute and relative eosinophil counts were significantly decreased. Additionally, in females of this test group total white blood cell (WBC) counts as well as absolute and relative lymphocyte counts were decreased (WBC not statistically significant). In contrast, relative neutrophil counts were significantly increased in these individuals. These alterations were regarded as treatment-related and adverse. In males of test groups 1 and 3 (80 and 750 mg/kg bw/d) absolute and relative, large unstained cell (LUC) counts were significantly increased. However, absolute LUC values were within, those of the relative LUC counts in test group 1 within, whereas in test group 3 marginally above the historical control range (males, absolute LUC 0.01-0.03 Giga/L; relative LUC 0.20-0.50 %). Because absolute LUC counts were within the normal range, both alterations were regarded as incidental and not treatment-related.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

No treatment-related, adverse changes among clinical chemistry parameters were observed. At the end of the administration period, in males of test group 3 (750 mg/kg bw/d) total protein and albumin levels were significantly decreased. Total protein values were marginally below, albumin levels within the historical control range (males, total protein 59.24-65.10 g/L; albumin 34.48-37.97 g/L). In females of the same test group total bile acids were significantly increased. In each sex of this test group only one clinical chemistry parameter was changed. Therefore, these changes were regarded as maybe treatmentrelated but non-adverse (ECETOC Technical Report No. 85, 2002). In males of test groups 1, 2 and 3 (80, 250 and 750 mg/kg bw/d) alanine aminotransferase (ALT) activities were significantly decreased, and in males of test group 3 (750 mg/kg bw/d) inorganic phosphate levels were significantly increased. However, all mentioned values were within historical control ranges (males, ALT 0.53-0.87 μkat/L; inorganic phosphate 1.43-2.06 mmol/L). Therefore, these alterations were regarded as incidental and not treatment-related.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Deviations from "zero values" were obtained in several rats. However, as most findings were equally distributed between test-substance treated groups and controls, without a doseresponse relationship or occurred in single animals only, these observations were considered as incidental. The following examinations were performed during FOB and are assessed individually:
Home cage observations
No test substance-related effects were observed.
Open field observations
No test substance-related effects were observed.
Sensorimotor tests/reflexes
No test substance-related effects were observed.

Quantitative Parameters
Grip strength of forelimbs were significantly decreased in female animals of test group 3 (750 mg/kg bw/d, 10.1 Newton versus 11.0 Newton the current control). The minor change of less than 10% was assessed as being spontaneous in nature and not related to treatment. The value was well within the historical control data.

Motor activity measurement
Regarding the overall motor activity as well as the individual intervals of observations, no test substance-related deviations were noted for male and female animals. No treatment-related changes on motor activity data (summation of all intervals) were observed in all male and female animals of all dose groups in comparison to the concurrent control group. Overall, the shape of the habituations curves in both sexes of all dose groups were comparable to control.
In female animals of test group 2 and 3 (250 and 750 mg/kg bw/d) a significantly decrease of beam interruptions were recorded for interval 5. These isolated single findings in both test groups without any effect on the respective sum of all intervals, were assessed as incidental and spontaneous in nature.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

The significant absolute weight increases of the brain in females of test groups 2 (5.4%) and 3 (3.3%) showed neither a histopathological correlate nor a dose-dependent relationship and were therefore considered incidental. The significant relative liver weight increase (2.84%) in females of test group 3 was within the historical control range (2.4 – 3.312%) and showed no histopathological correlate. Therefore, the relative weight increase (+17.0%) was assessed as incidental.

see also attached background material

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Description (incidence and severity):

Treatment-related findings were observed in the thyroid glands of males. Compared to the control group, the hypertrophy/hyperplasia of the follicular epithelium was minimal and similarly increased in its incidence and grading in test group 2 and 3. This finding was accompanied by altered colloid which was minimally increased in test group 2 and 3. 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. The stages of spermatogenesis in the testes of males of the high dose test group were comparable to those of the controls. In high dose females the different stages of functional bodies in the ovaries were present and comparable to the control animals. The animal sacrificed moribund showed in the thymus a severe decreased cellularity. This single histopathological finding might have contributed but does not fully explain the moribund state of this animal. All other histopathological findings were not relevant.

see also attached background material

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Thyroid hormones
In parental males (test groups 1, 2 and 3; 80, 250 and 750 mg/kg bw/d) and in female pups at PND13 (test groups 11, 12 and 13; 80, 250 and 750 mg/kg bw/d), no treatment-related alterations of T4 and TSH levels were observed. In PND 13 male pups T4 values were significantly increased. The values were slightly above the historical control range, whereas the corresponding TSH mean was within its historical control range (male PND13 pups, T4 46.18-76.60 nmol/L; TSH 3.00-5.34 μg/L). Therefore, this isolated T4 increase only in male pups at PND13 was regarded as maybe treatment-related but non-adverse.

Key result

Dose descriptor:

NOAEL

Effect level:

250 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

haematology

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

750 mg/kg bw/day (nominal)

System:

haematopoietic

Organ:

blood

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Conclusions:

Under the conditions of this Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, the oral administration by gavage of 2-[2-(dimethylamino)ethoxy]ethanol to Wistar rats revealed signs of systemic toxicity in
parental animals at 750 mg/kg bw/d manifested in clinical pathology. Thus, the no observed adverse effect level (NOAEL) for general systemic toxicity was 250 mg/kg bw/d in both sexes of parental animals.

Executive summary:

In this Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test in Wistar Rats 2-[2-(dimethylamino)ethoxy]ethanol was administered orally by gavage to groups of 10 male and 10 female Wistar rats (F0 animals) at dose levels of 0 (test group 0, vehicle control), 80 (test group 1), 250 (test group 2) and 750 mg/kg bw/d (test group 3).

The duration of treatment covered a 2 weeks pre-mating period, 2 weeks mating period in both sexes, up to 7 days post-mating in males, the entire gestation period as well as up to 13 days of the lactation period in females, and up to one day prior to the day of schedule sacrifice of the animals. Analyses confirmed the overall accuracy of the prepared concentrations of the test substance in the vehicle. The stability of these preparations was demonstrated over a period of 5 days under ambient conditions.

Regarding clinical examinations including determination of food consumption and body weight parameters, during pre-mating and mating in males and females of all test groups as well as during gestation and lactation in females no treatment-related findings were observed. In the subsequent investigations including the detailed clinical observation (DCO), the functional observational battery (FOB) and measurement of motor activity (MA) no treatment related, adverse differences to control were observed at any dose level.

Concerning clinical pathology, in males of test group 3 (750 mg/kg bw/d) decreased red blood cell (RBC) counts, hemoglobin and hematocrit values and increased absolute reticulocyte counts without any significant changes of the red blood cell indices (i.e., MCH, MCHCH and MCV) indicated a regenerative, normochromic, normocytic anemia. Decreased eosinophil counts in male and female rats of test group 3 (750 mg/kg bw/d) as well as decreased total white blood cell (WBC) counts and absolute lymphocyte counts in females of this test group were most probably due to a stress situation for these individuals.

Regarding pathology, there were neither treatment-related organ weight changes nor gross lesions. The thyroid gland of male animals in test groups 2 (250 mg/kg bw/d) and 3 (750 mg/kg bw/d) showed hypertrophy/hyperplasia of the follicular epithelium, which was minimal and similarly increased in both test groups 2 and 3, when compared to the control group. This finding was accompanied by altered colloid, which was minimally increased in test group 2 and 3. Since these morphological changes were not consistent with hormonal deviations they were regarded as possibly treatment-related but not adverse. 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.

Thus, the no observed adverse effect level (NOAEL) for general systemic toxicity was 250 mg/kg bw/d in both sexes of parental animals.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

250 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

A modern subchronic toxicity study (OECD 408) is available for this endpoint.

System:

urinary

Organ:

kidney

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

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

In a Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test in Wistar Rats 2-[2-(dimethylamino)ethoxy]ethanol was administered orally by gavage to groups of 10 male and 10 female Wistar rats (F0 animals) at dose levels of 0 (test group 0, vehicle control), 80 (test group 1), 250 (test group 2) and 750 mg/kg bw/d (test group 3). The duration of treatment covered a 2 weeks pre-mating period, 2 weeks mating period in both sexes, up to 7 days post-mating in males, the entire gestation period as well as up to 13 days of the lactation period in females, and up to one day prior to the day of schedule sacrifice of the animals. Under the conditions of the test, the test substance induced signs of systemic toxicity in parental animals at 750 mg/kg bw/d manifested in clinical pathology:

In males of test group 3 (750 mg/kg bw/d) decreased red blood cell (RBC) counts, hemoglobin and hematocrit values and increased absolute reticulocyte counts without any significant changes of the red blood cell indices (i.e., MCH, MCHCH and MCV) were observed, indicating a regenerative, normochromic, normocytic anemia. Based on that results, the no observed adverse effect level (NOAEL) for general systemic toxicity was 250 mg/kg bw/d in both sexes of parental animals. [BASF, 2020]

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. The administration of the test substance caused 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. 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. Therefore, considering the effects in the kidney, the NOAEL was set to 250 mg/kg bw/d for male and female Wistar rats. [BASF, 2020]

Both studies showed toxic effects after repeated exposure at a high dosage of 750 mg/kg bw. In the OECD 422, the most critical effect was observed in the hematopoetic system of the animals indicating a regenerative, normochromic, normocytic anemia. In the subsequent OECD 408, the anemia was not reproducible. The dominating effect was some kind of kidney toxicity. Since the effects of the OECD 422 were not seen in the OECD 408, where the exposure period is much longer, it is concluded that the most critical effect of repeated exposure to 2-[2-(dimethylamino)ethoxy]ethanol is kidney toxicity.

Justification for classification or non-classification

After subchronic repeated expopsure to rats, 2-[2-(dimethylamino)ethoxy]ethanol affected the kidneys of the animals in terms of changes in clinical pathology parameters as well as histopathological findings. The effects were observed at dosages of 750 mg/kg bw, which are far beyond the C&L threshold of 100 mg/kg bw for target organ toxicity. Therefore, no classification is warranted according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008).