2-[2-(dimethylamino)ethoxy]ethanol

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Reference

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.

Cross-reference 2

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

screening for reproductive / developmental toxicity

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

Parental animals: Observations and examinations:

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.

Oestrous cyclicity (parental animals):

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.

Litter observations:

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.

Postmortem examinations (parental animals):

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.

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.

Postmortem examinations (offspring):

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.

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.

Dermal irritation (if dermal study):

not examined

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

Ophthalmological findings:

not examined

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.

Urinalysis findings:

not specified

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

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

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.

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Estrous cycle data revealed regular cycles in the rearing F1 females of all test groups including the control. The mean estrous cycle duration in the different test groups (0-3) ranged from 3.9 to 4.0 days.

Reproductive function: sperm measures:

not examined

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

Male reproduction data
Male mating index
The male mating index calculated after the mating period for F1 litter was 100% in all test groups.

Male fertility index
Fertility was proven for nearly all of the F0 parental males within the scheduled mating interval to produce F1 litter, with exception of one male of the control group. This male of control group did not generate implants in utero. For one male animal of test group 1 and one male of test group 3 no sperm could be detected in vaginal smear during the 14 days of mating, however, the females were pregnant and delivered pups .
These values reflected the normal range of biological variation inherent in the strain of rats used for this study as all respective values were within the range of the historical control data. The male fertility index was 100% in test groups 1, 2 and 3. The index was 90% in control group.

see also attached background material

Female reproduction and delivery data
Female mating index
The female mating index calculated after the mating period for F1 litter was 100% in all test groups. The mean duration until sperm was detected (GD 0) was 2.5 days for test group 0, 2.4 days for test group 1 (80 mg/kg bw/d), 2.8 days for test group 2 (250 mg/kg bw/d), and 2.4 days for test group 3 (750 mg/kg bw/d). These values reflected the normal range of biological variation inherent in the strain of rats used for this study as all respective values were within the range of the historical control data.

Female fertility index
All sperm positive rats got pregnant with one exception in the control group. One female animal (test group 0) which was mated did get sperm in vaginal smear but showed no implants. One additional female animal (test group 3) had sperm in the vaginal smear but the female animal was sacrificed in a moribund state on GD1. Since the implantation of rat embryos occurs around gestational day 4, no embryos/implantations sites are detectable. Therefore, no gestation could be determined based on the scheduled observation methods in this study type. The necropsy of this female detected alterations in the thymus but not on the sex organs. Therefore, no reason was given that this female would not be pregnant after being successfully mated within two days. The animal was assessed to be sacrificed moribund after a gavage error. Therefore, this animal was excluded from the mating assessment. The female fertility index was 90% in test groups 0 and 100% in test group 1, 2 and 3.

see also attached background material

The mean duration of gestation was comparable in all test groups, i.e. 22.1 days (test group 0 and 1), 22.2 days (test group 2) and 22.8 days (test group 3). Implantation was not affected by the treatment since the mean number of implantation sites was comparable between all test substance-treated groups and the control, taking normal biological variation into account (13.4 / 13.2 / 12.1 and 11.3 implants/dam in test groups 0 - 3, respectively).

Gestation index
The gestation index was 100% in all test groups.

Live birth indices
The rate live birth indices were 100% in all test groups.

Postimplantation loss
The postimplantation loss was 14.6% in test group 0 (control), 6.8% in test group 1 (80 mg/kg bw/d), 3.9% in test group 2 (250 mg/kg bw/d) and 5.4% in test group 3 (750 mg/kg bw/d). The incidences of postimplantation loss in all test groups were within the historical control range 0.9 – 16.8% and were assessed as incidental and not treatment-related.

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

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

The surviving F1 pups of test groups 0-3 did not show adverse clinical signs up to scheduled sacrifice on PND 4, resp. PND 13.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

The viability index indicating pup mortality during PND 0-4 varied between 100% in test group 0 (control), test group 1 (80 mg/kg bw/d) and test group 2 (250 mg/kg bw/d), 96.4% in test group 3 (750 mg/kg bw/d). These values reflected the normal range of biological variation inherent in the strain of rats used for this study as all respective values were within the range of the historical control data (86.6-100.0%). The survival index indicating pup mortality on PND 4 – 13 was 100% in all test groups.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Mean body weight and body weight changes of pups in all test groups were comparable to the control group with one exception. Mean body weights of male pups (11.5 g) in test group 3 (750 mg/kg bw/d) were statistically significantly increased on PND 4 in comparison to the current control (9.9 g), but was within the range of the historical control data (9.5-11.8 g). On PND 7 no different to the current control was observed. Therefore, this isolated finding was assessed as incidental and not treatment-related. The statistically significantly increased body weight change in the high-dose male pups during PND 1 - 4 was considered as spontaneous in nature and not treatment related as no dose-response relationship occurred. On PND 1, respectively one female runt was seen in the control group (0 mg/kg bw/d), in test group 1 (80 mg/kg bw/d) and in test group 2 (250 mg/kg bw/d), but not in test group 3 (750 mg/kg bw/d). Thereby, no treatment-related alteration was observed.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

effects observed, non-treatment-related

Description (incidence and severity):

Anogenital distance (3.14 mm) and index (1.64) was significantly increased in male pups of test group 3 (750 mg/kg bw/d) in comparison to the current control (2.75 mm and 1.48, respectively). Thereby, it has to be considered that the body weight of male pups on postnatal day 1 was 11.5% higher in the test group 3 in comparison to the current control (nonstatistically significant). Since it is known that the anogenital distance is related to the body size/weight the anogenital index should correct the influence of the body weight. However, the index can only reduce but not eliminate the influence of the body weight. Because the anogenital distance and the anogenital index were in the range of historical control data (2.77 – 3.36 mm and 1.42 – 1.75, respectively), the findings were regarded as incidental and not related to treatment in this study.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

The apparent number and percentage of male pups having nipple/areolae was not influenced by the test substance when examined on PND 13.

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

A few F1 pups showed spontaneous findings at gross necropsy, such as post mortem autolysis, testis discolored and small testis. These findings occurred without any relation to dosing or were observed in a single pup only and considered to be spontaneous in nature. All other F1 pups of any test groups (0-3) did not show adverse findings during necropsy.

Histopathological findings:

not examined

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Sex ratio
The sex distribution and sex ratios of live F1 pups on the day of birth and PND 13 did not show substantial differences between the control and the test substance-treated groups; slight differences were regarded to be spontaneous in nature.

see also attached background material

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

750 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

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. The no observed adverse effect level (NOAEL) for fertility and reproductive performance was 750 mg/kg bw/d in both sexes of parental animals. The NOAEL for developmental toxicity in the F1 progeny was 750 mg/kg bw/d.

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.

The fertility and reproductive performance were not impaired in male or female parental animals of all test groups (80, 250, and 750 mg/kg bw/d).

Regarding developmental toxicity, no adverse effects were observed up to the highest dose tested 750 mg/kg bw/d.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD 422

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

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Duration of treatment / exposure:

male animals: 30 days; female animals: 61 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

Examinations

Maternal examinations:

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.
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.

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.

Ovaries and uterine content:

see below

Fetal examinations:

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.
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.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

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.

Dermal irritation (if dermal study):

not examined

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

Ophthalmological findings:

not examined

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.

Urinalysis findings:

not specified

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

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

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

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.

Other effects:

effects observed, treatment-related

Maternal developmental toxicity

Number of abortions:

no effects observed

Pre- and post-implantation loss:

effects observed, non-treatment-related

Description (incidence and severity):

Postimplantation loss
The postimplantation loss was 14.6% in test group 0 (control), 6.8% in test group 1 (80 mg/kg bw/d), 3.9% in test group 2 (250 mg/kg bw/d) and 5.4% in test group 3 (750 mg/kg bw/d). The incidences of postimplantation loss in all test groups were within the historical control range 0.9 – 16.8% and were assessed as incidental and not treatment-related.

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Description (incidence and severity):

Implantation was not affected by the treatment since the mean number of implantation sites was comparable between all test substance-treated groups and the control, taking normal biological variation into account (13.4 / 13.2 / 12.1 and 11.3 implants/dam in test groups 0 - 3, respectively).

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

The mean duration of gestation was comparable in all test groups, i.e. 22.1 days (test group 0 and 1), 22.2 days (test group 2) and 22.8 days (test group 3).

Changes in number of pregnant:

no effects observed

Other effects:

no effects observed

Effect levels (maternal animals)

Key result

Dose descriptor:

NOAEL

Effect level:

250 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

haematology

Results (fetuses)

Fetal body weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Mean body weight and body weight changes of pups in all test groups were comparable to the control group with one exception. Mean body weights of male pups (11.5 g) in test group 3 (750 mg/kg bw/d) were statistically significantly increased on PND 4 in comparison to the current control (9.9 g), but was within the range of the historical control data (9.5-11.8 g). On PND 7 no different to the current control was observed. Therefore, this isolated finding was assessed as incidental and not treatment-related. The statistically significantly increased body weight change in the high-dose male pups during PND 1 - 4 was considered as spontaneous in nature and not treatment related as no dose-response relationship occurred. On PND 1, respectively one female runt was seen in the control group (0 mg/kg bw/d), in test group 1 (80 mg/kg bw/d) and in test group 2 (250 mg/kg bw/d), but not in test group 3 (750 mg/kg bw/d). Thereby, no treatment-related alteration was observed.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

The viability index indicating pup mortality during PND 0-4 varied between 100% in test group 0 (control), test group 1 (80 mg/kg bw/d) and test group 2 (250 mg/kg bw/d), 96.4% in test group 3 (750 mg/kg bw/d). These values reflected the normal range of biological variation inherent in the strain of rats used for this study as all respective values were within the range of the historical control data (86.6-100.0%). The survival index indicating pup mortality on PND 4 – 13 was 100% in all test groups.

Changes in sex ratio:

effects observed, non-treatment-related

Description (incidence and severity):

Sex ratio
The sex distribution and sex ratios of live F1 pups on the day of birth and PND 13 did not show substantial differences between the control and the test substance-treated groups; slight differences were regarded to be spontaneous in nature.

see also attached background material

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

no effects observed

Description (incidence and severity):

The survival index indicating pup mortality on PND 4 – 13 was 100% in all test groups.

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

A few F1 pups showed spontaneous findings at gross necropsy, such as post mortem autolysis, testis discolored and small testis. These findings occurred without any relation to dosing or were observed in a single pup only and considered to be spontaneous in nature. All other F1 pups of any test groups (0-3) did not show adverse findings during necropsy.

Skeletal malformations:

not examined

Visceral malformations:

not examined

Other effects:

no effects observed

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

750 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Fetal abnormalities

Key result

Abnormalities:

no effects observed

Overall developmental toxicity

Key result

Developmental effects observed:

no

Applicant's summary and conclusion

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. The NOAEL for developmental toxicity in the F1 progeny was 750 mg/kg bw/d.

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.

Regarding developmental toxicity, no adverse effects were observed up to the highest dose tested 750 mg/kg bw/d.