2-[[2-(dimethylamino)ethyl]methylamino]ethanol

Repeated dose toxicity: oral

Administrative data

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

Cross-reference

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)

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

other: Commission Regulation EC 440/2008

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3650

Qualifier:

according to guideline

Guideline:

EU Method B.34 (One-Generation Reproduction Toxicity Test)

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

Product name: DABCO T Catalyst
CAS number: 2212-32-0
Appearance: Colourless to pale yellow liquid
Batch: 2096108
Purity: 99.4%
Manufacture date: 08 February 2016
Retest date: 08 February 2018
Storage conditions: Room temperature protected from light; in a tightly closed container; in a dry cool place; kept away from sources of heat, ignition and oxidisers

Species:

rat

Strain:

Wistar

Remarks:

Wistar Hannover RccHan:WIST

Sex:

male/female

Details on test animals or test system and environmental conditions:

Supplier: Envigo RMS
Age (at delivery): 10-11 weeks (male and females)
Body weight (at start of treatment): 347-432 g (males); 189-240 g (females)
Randomisation: Method based on similarity of mean body weights among groups
Acclimatisation period: 28 days prior to treatment
Vetinary examinations: During acclimatisation and before treatment. Additional inspections in some animals in the highest dose group during the first six days of treatment.
Air changes: 15-20 air changes per hour
Room temperature: 20-24 °C
Humidity: 30-70%
Photoperiod: 12 hours light/dark
Caging: standard sawdust bedding; 5 animals/cage during premating; 1 male and 1 female/cage during mating; individually during postmating
Diet: Standard Teklad 2014C rat/mouse maintenance diet; ad libitum
Water: Tap water; ad libitum
Enrichment: material specific for species supplied

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

Initially males dosed first then females. Starting order was alternated between males and females for each day of treatment.
Amount administered based on the most recent body weight recorded. Administration volume was 4 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentration of dose formulation was determined twice during the study and once for homogeneity (top/middle/bottom) in samples taken from the formulation administered to Groups 1 to 4. The formulations were quantified following a validated analytical procedure. On each occasion, duplicate samples of the dosing solution (10 mL each) were transferred to labeled vials. One aliquot was used for analysis. The test item was used as analytical standard. The analytical procedure was successfully validated with respect to specificity of chromatographic analysis, linearity of detector response, method accuracy and precision. The homogeneity and stability was confirmed for the test item in vehicle formulations at nominal concentrations of 3.75 mg/mL and 250 mg/mL when stored at ambient (6 hours), refrigerated and frozen condition for 5 days. The mean concentrations of test item in test formulations analyzed for the study were within 100 ± 11 % of nominal concentrations, confirming accurate formulation.

Duration of treatment / exposure:

Males: 2 weeks prior to mating up to and including the day before sacrifice (Days 44 to 47); at least 30 days
Females: 2 weeks prior to mating up to and including the day before sacrifice (Day 4 to Day 7 post partum).

Frequency of treatment:

Daily

Details on study schedule:

On day 15 of treatment, the mating period started while the test item was still being administered.
During the mating period the females were housed with males within each dose group (one male : one female) until evidence of copulation was observed for a period of 18 or 24 hours.
The females were removed and housed individually when:
a) The daily vaginal smear was sperm-positive, and/or
b) A copulation plug was observed.
The day of mating was designated day 0 post coitum.
In dose group 1, female no. 156, which was paired with male no. 8 but failed to mate during the 14-day mating period, was paired with another male (no. 3) from the same group that had previously mated successfully.

Dose / conc.:

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

Remarks:

Vehicle control

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

Remarks:

This group was administered 400 mg/kg bw/day for Days 1 to 6 of treatment. The dose was reduced to 250 mg/kg bw/day from Day 7 to the end of the study.

No. of animals per sex per dose:

Twelve

Control animals:

yes, concurrent vehicle

Details on study design:

Three groups of twelve male and twelve female rats received 2-[[2-(Dimethylamino) ethyl] methylamino] ethanol at the doses of 25, 100 and 400/250 mg/kg bw/d. Males were treated continuously for two weeks before pairing up to necropsy, after a minimum of 48 consecutive days. Females were treated continuously for two weeks before pairing, throughout pairing and gestation, and until Day 7 of lactation at the most, i.e. the day before sacrifice. Females were allowed to litter and rear their offspring, and litters were killed on Day 4/7 of lactation. F1 generation received no direct administration of the test item; any exposure was in utero or via the milk. A similarly constituted control group received the vehicle, corn oil. During the study, mortality, clinical signs, sensory reactivity observations, grip strength motor activity, body weight, food consumption, haematology and coagulation, blood biochemistry, pre-coital interval, mating performance, fertility, gestation length, organ weight and macroscopic examination were evaluated Clinical signs, behaviour assessment, litter size and survival, sex ratio, body weight and macropathology were also assessed for all offspring.

Positive control:

None; not required

Parental animals: Observations and examinations:

Morbidity/mortality and cage-side observations were conducted twice daily. In Group 4, females no. 190 and 194, which were found dead, and females no. 185 and 191 and males no. 39 and 46, which were sacrificed due to animal welfare reasons, were subjected to macroscopic examination. Food consumption was assessed in males twice at pretest, once weekly during the pre-pairing/treatment period, and during the two weeks of the postpairing period and in females twice at pretest, once weekly during the pre-pairing/treatment period, on days 0-7, 7-14, 14-21 post coitum and on days 1-4 postpartum. Food consumption was not examined during the mating period.

Body weight was recorded in males three times during pretest and twice weekly during the pre-pairing, pairing and postpairing periods and in females three times during pretest, twice weekly during the
pre-pairing and pairing periods and daily from day 0 of gestation until days 4-8 postpartum.

Detailed clinical observations were performed on all test item treated and control animals before the first exposure to the test item, once weekly thereafter and one day before sacrifice6. These observations were performed outside the home cage, in a standard arena, between 1-3 hours after dosing to ensure that any transient effects of treatment are identified. In view of the clinical signs and body-weight loss observed in some Group-4 animals, additional clinical observations were done to record the health condition of the animals from day 6 of treatment onwards. After reducing the dose, salivation was recorded daily in some of the animals from this group.

Grip strength (fore- and hind limbs) and motor activity were measured in five randomly selected males per group once during the final week of treatment and at least one hour after dosing. Five randomly selected females per group were similarly evaluated on day 4 postpartum.

Sensory reactivity to different stimuli (as follows) was evaluated in the animals mentioned above:
- Blink reflex
- Pinna reflex
- Iridic light / Pupil closure reflex
- Proprioception(right leg) / pushoff (hind legs)
- Pain response / Tail punch response
- Startle / hearing
- Righting reflex in the air

From day 20 post coitum, the females were examined twice daily for signs of parturition. The duration of gestation (days) was recorded.

The females that gave birth were checked to observe whether they nursed their offspring.

Blood samples (1.8 mL) were drawn from the retro-orbital plexus of animals selected for functional observations (five animals/sex/group) under light isoflurane anesthesia. Given poor condition of the blood sample obtained in female no. 151, it was necessary to obtain a sample in female no. 149 (not selected for functional observations) in order to achieve the sample size in Group 1 (control). The animals were fasted before blood sampling but allowed access to water ad libitum for 3-4 hours. The samples were collected early in the working day to reduce biological variation caused by circadian rhythms.
Blood sampling performed as follows:
Males: Once on day 44 of the study (on the day of sacrifice)
Females: Once on day 5 or 6 postpartum (on the day of sacrifice)

The following paramenters were determined in samples collected for haematology: erythrocyte count, haemoglobin, haematocrit, mean corpuscular volume, red cell volume distribution width, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, haemoglobin concentration distribution width, reticulocyte count, reticulocyte maturity index, platelet count, total leukocyte count and differential leukocyte count (neutrophils, lymphocytes, monocytes, eosinophils, basophils and large unstained cells).

The following parameters were determined in samples collected for coagulation: prothrombin time and activated partial thromoplastin time.

The following parameters were determined in samples collected for clinical biochemistry:glucose, urea, creatinine, total bilirubin, total cholesterol, triglycerides, aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, gamma-glutamyl transferase, creatine kinase, inorganic phosphorus, sodium, potassium, calcium, chloride, total protein, albumin, globulin (calculated from total protein and albumin), albumin/globulin ratio and bile acids.

The following organs were weighed: adrenal glands, brain (including sections of medulla/pons, cerebral and cerebellar cortex), epididymides, heart (with papillary muscle), kidneys, liver, lungs (with bronchi and bronchioles), pituitary gland, prostate gland and seminal vesicles, salivary glands (mandibular and sublingual), spleen,thymus, testes, thyroid (including parathyroid) and uterus (including cervix and oviducts).

The following organs were collected and examined for histopathological analysis: adrenal glands, aorta, bone (sternum, including bone marrow), bone (femur, including joint), brain (including sections of medulla/pons, cerebral and cerebellar cortex), epididymides, eyes (with optic nerve), heart (with papillary muscle), large intestine (cecum, colon and rectum), small intestine (duodenum, jejunum and ileum), kidneys, larynx, liver, lungs (with bronchi and bronchioles), mammary gland area, peyer's patches, pituitary gland, prostate gland and seminal vesicles, salivary glands (mandibular and sublingual), salivary glands (parotid), sciatic nerve, skeletal muscle, skin (abdominal), spinal cord (cervical, midthoracic and lumbar), spleen, stomach (glandular and nonglandular), thymus, testes, thyroid (including parathyroid), tongue, trachea, urinary bladder, uterus (including cervix and oviducts) and vagina. In addition, bone marrow from the femur (air dried) was collected but not examined. Tissues were fixed with 10% formalin, with the exception of epididymides and the eyes which were fixed with modified Davidson's fixative or Davidson's fixative, respectively.

Oestrous cyclicity (parental animals):

Vaginal smears were collected from all females for evaluation of the estrus cycle during the mating period. Smearing of individual females was discontinued when sperm was found.

Sperm parameters (parental animals):

Not assessed

Litter observations:

The number of stillbirths and of live and dead pups and any macroscopic anomalies were recorded for each litter within 24 hours of parturition (day 0 or 1 postpartum).
When parturition ended before 7 am, that day was considered day 1 postpartum; when it ended after 7 am it was considered day 0 postpartum. The size of the litters was recorded daily.
The sex of the pups was recorded on days 1 and 4 postpartum.
Morbidity/mortality was observed twice daily. Any rat sacrificed or found dead during the study was subjected to macroscopic examination.
Animals were observed once daily for clinical signs.
Body weight was recorded on Days 1 and 4 postpartum.
Behaviour test (surface righting reflex) was assess on Day 1 postpartum.

Postmortem examinations (parental animals):

Animals that died (Group 4: females no. 190 and 194) or were sacrificed in extremis (Group 4: female no. 185 and 191; males no. 39 and 46) were necropsied. Their organs were extracted and fixed but they were not weighed.

From days 5 to 8 postpartum, all females were sacrificed by intraperitoneal injection of sodium pentobarbital and immediately exsanguinated by excision of the axillary vessels and aorta and necropsied. The mated females that did not give birth or did not show signs of pregnancy were sacrificed after 25-28 days post coitum. The external surface of the body, all orifices and cranial, thoracic and abdominal cavities and their contents were examined postmortem with emphasis on the uterus, number of corpora lutea and implantation sites. Organs were observed in situ and all macroscopic abnormalities were described.
When no implantation sites were evident, the uterus was placed in an aqueous solution of ammonium sulfide (Salewski, 1964) to accentuate possible hemorrhagic areas of implantation sites.

From days 44 to 47 of the study all males were sacrificed by intraperitoneal injection of sodium pentobarbital and immediately exsanguinated by excision of the axillary vessels and aorta and necropsied, subject to confirmation of successful mating.
The external surface of the body, all orifices and cranial, thoracic and abdominal cavities and their contents were examined postmortem. Organs were observed in situ and all macroscopic abnormalities were described.

The organs weights were recorded on the scheduled necropsy dates for all surviving parental animals and their organ-to-terminal-body-weight ratios and organ-to-brainweight ratios were determined.

Samples of tissues and organs as well as specimens of abnormal tissue were collected from all animals at necropsy and fixed in neutral phosphate buffered 4% formaldehyde solution (10% formalin) unless otherwise indicated. All organ and tissue samples (except for the nose) of selected animals from groups 1 to 4 (5 animals/sex) to be examined by the histopathologist were processed, embedded and cut at an approximate thickness of 2-4 micrometers and stained with hematoxylin and eosin. Where possible, the microscopic findings were correlated with the gross observations. The bone marrow smears were stained using the May Grünwald-Giemsa method. In addition, the reproductive organs of all control and high dose animals were also examined.
A qualitative staging of spermatogenesis and histopathology evaluation of interstitial cells of all males from the control and high-dose groups was performed by the histopathologist. Stomach, larynx and trachea of the selected animals from Groups 2 and 3 (5 animals/sex) were processed as indicated above and examined by the histopathologist. Full tissues from animals no. 4, 38, 45, 152, 187, 193 and 196, all stomachs, all female larynxes and all Group 2 and 3 stomach, larynxes and tracheas were peer reviewed.

Postmortem examinations (offspring):

From days 4 to 7 postpartum, all pups were sacrificed by intraperitoneal injection of sodium pentobarbital and subjected to macroscopic examination.
All offspring (including those that died during the study) were examined externally and necropsied, except those excessively cannibalized. Samples of organs and tissues with
macroscopic alterations were taken and preserved in neutral phosphate buffered 4% formaldehyde solution for possible microscopic examination.

Statistics:

All statistical analyses were carried out separately for males and females. Data relating to food consumption were analyzed on a cage basis. For all other parameters, the analyses were carried out using the individual animal as the basic experimental unit.
The following data types were analyzed at each time point separately:
Grip strength and motor activity
Body weight
Food consumption, over appropriate study periods
Hematology and coagulation
Clinical biochemistry
Organ weights, absolute and adjusted for terminal body weight
Pathological findings, for the number of animals with and without each finding
Pre/post-implantation loss, sex ratio and litter size

Reproductive indices:

The following parameters were calculated from the individual data during the mating period of the parental generation:
i) Pre-coital time
Calculated as the time elapsing between initial pairing and the observation of positive evidence of mating.

ii) Fertility Indices
The following indices were calculated for each group:
Percentage Mating (%) = Number of females mated/Number of females paired x 100
Conception rate (%) = Number of females achieving pregnancy/Number of females mated x 100
Fertility index (%) = Number of females achieving pregnancy/Number of females paired x 100

iii) Implantation Losses (%)
Group mean percentile pre-implantation and post-implantation loss was calculated for each
female/litter as follows:
Pre–implantation loss = (Number of corpora lutea - number of implantation sites)/ Number of corpora luteax 100

Post–implantation loss =(Number of implantation sites - total number of offspring born)/ Number of implantation sites x 100

The following parameters were calculated for individual data during the gestation and parturition period of the parental generation.
i) Gestation Length
Calculated as the number of days of gestation including the day for observation of mating and the start of parturition.
ii) Gestation Index
The following index was calculated for each group:
Gestation Index (%) = Number of females with living pups/Number of females pregnant x 100

Offspring viability indices:

The standard unit of assessment was considered the litter; therefore, values were first calculated for each litter and the group mean was calculated using the individual litter values.
Group mean values included all litters reared to termination (Day 4 of age).
Viability Index (%) = Number of alive pups on Day 4/ Number of offspring alive on Day 1 x 100
Live birth index (%) = Number of offspring alive on Day 1/ Number of offspring delivered x 100

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

There were no other clinical signs other than salivation that were considered to indicate an immediate or delayed reaction to treatment once the high dose was reduced to 250 mg/kg/ bwd.
Salivation at the high dose may be related to the test item palatability. Before decreasing the dose from 400 to 250 mg/kg bw/d, the female sacrificed for animal welfare reasons (no. 185) showed irregular breathing, piloerection and hunched posture. In males administered at 400/250 mg/kg/day also sacrificed for welfare reasons (nos. 39 and 46), piloerection, labored breathing, reduced body tone and pallor were recorded, in addition to hunched posture in one of them. These clinical signs seem to be related with toxicity at the dose of 400 mg/kg bw/d as they were not present after reducing the dose. In female no. 191 administered at 400/250 mg/kg bw/d, gasping breathing and pallor were observed after delivery (lactation day 1). As these signs were not observed in the other females at this dose they cannot be considered a treatment-related effect.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

In Group 4 (400/250 mg/kg bw/d), two females (nos. 194 and 190, respectively) were found dead on days 3 and 7 of the treatment period, and another female (no. 185) was sacrificed for welfare reasons on day 6. Although the cause of death could not be determined, it was considered to be related with the test item and thus it was necessary to decrease the high dose. Moreover, due to the probable effects caused by the test item at this dose, two males (no. 39 and 46) were sacrificed for animal welfare reasons on day 8 of treatment. Female no. 191, administered at 400/250 mg/kg bw/d, was sacrificed for animal welfare reasons after parturition (on day 1 of lactation).

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There was no adverse effect on mean body-weight in males throughout the study or in females before pairing, during gestation or during lactation. Mean body weight recorded on day 8 of treatment in males receiving 400/250 mg/kg bw/d was lower than that recorded in the control group. However, this did not attain significance. There were no effects after decreasing the dose.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There was no indication of an effect on food consumption in males throughout the study or in females before pairing, during gestation or during lactation. However, despite there being no statistically significant differences, lower food consumption was observed in week 1 of treatment mainly in males administered at 400/250 mg/kg bw/d when compared to control. This effect correlates with the dose reduction in this group.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

Hematological evaluation at the end of treatment revealed no statistical differences with the control group in males. In treated females (25, 100 and 400/250 mg/kg bw/d), mean eosinophil values were 50% lower than in the control group. However, this difference could not be considered of any relevance, taking into account the normal values observed in rats of this strain and age.
No differences were observed in coagulation when comparing the test-item administered groups with the control.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Clinical biochemistry at the end of treatment showed statistically higher creatinine mean values in females at 100 and 400/250 mg/kg bw/d and in males at 100 mg/kg/day. However, given the magnitude of change and in the absence of any dose relation, it cannot be considered to be treatment-related. All other differences from control (statistically significant differences in potassium, chloride and total proteins) were minor, lacked dose relationship or were confined to one sex and were therefore attributed to normal biological variation.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Despite the fact that lower mean values in forelimb grip strength were observed in males at 400/250 mg/kg bw/d and in females at 100 and 400/250 mg/kg bw/d when compared to the control group, they cannot be considered to be of toxicological relevance given the magnitude of change or because no dose response was observed. Therefore, there were no indications of any adverse effect of the test item on grip strength assessment. No noteworthy changes were recorded in the motor activity examination. The statistically significant differences observed in females receiving 400/250 mg/kg bw/d at 10 and 50 minutes and in females receiving 100 mg/kg bw/d at 50 minutes are considered to be of no relevance as they were not part of a dose-related trend and/or this effect was not present in males. There was no indication of any test item effect on sensory activity.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

In the testes, seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and the integrity of the various cell types present within the different stages. No cell or stage specific abnormalities were noted in males treated at 400/250 mg/kg bw/d. Interstitial cells were also assessed qualitatively, and no treatment related alterations were seen.
In the animals administered at 400/250 mg/kg bw/d, the following alterations were recorded:

Stomach:
- Minimal to slight focal areas of epithelial hyperplasia of the squamous epithelium, with underlying mucosal/submucosal subacute to chronic inflammation in three males. Occasionally, these were associated with minimal focal hyperkeratosis, minimal to moderate mucosal/submucosal edema, or granulation tissue formation that extended down the serosa.
- Similar but milder changes in the forestomach in two females. In one of them, mucosal/submucosal edema was seen both in the nonglandular and, to a milder degree, glandular regions.
- Minimal focal area of mucosal necrosis and erosion in the pylorus in one female.

Trachea:
- Focal to extensive areas of epithelial regeneration and/or hyperplasia in a few males and females where gastric changes were also detected.

Larynx:
- Minimal to moderate areas of hyperplasia of the respiratory epithelium, with squamous metaplasia in the arytenoid cartilage region, sometimes along with underlying minimal to slight subacute to chronic inflammation and focal areas of flattened regenerative epithelium in some males and females.

All other histopathological findings were considered to be incidental and unrelated to the test item.

In the pathology extension, some punctual effects at 25 and 100 mg/kg bw/d in larynx (hyperplasia/metaplasia of the epithelium) and only at 100 mg/kg/day in stomach (edema, hyperplasia and inflammation) and in trachea (regeneration hyperplasia in epithelium) were observed.

Histopathological findings: neoplastic:

no effects observed

Other effects:

not examined

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Reproductive parameters of pre-coital interval, fertility or gestation length or index were unaffected by treatment

Key result

Dose descriptor:

NOAEL

Remarks:

Systemic toxicity

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Key result

Dose descriptor:

NOAEL

Remarks:

Fertility and reproductive performance

Effect level:

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

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Gross pathological findings:

not examined

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

not examined

Clinical signs:

no effects observed

Description (incidence and severity):

There were no clinical signs indicative of a reaction to the test item.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

No relevant differences were observed between the test-item administered groups and Control in post implantation survival index or live birth index. However, the viability index on day 4 was reduced at 400/250 mg/kg bw/d.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There was no effect of the test item on body weight.

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

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no macroscopic findings noted at the end of the exposure period that could be attributed to the test item.

Histopathological findings:

not examined

Other effects:

effects observed, treatment-related

Description (incidence and severity):

There was no effect on litter size on day 1 between the test item administered animals and the control group. However, litter size was reduced on day 4 in the treated groups compared to the Control group (maximum effect 86% of control). Regarding the sex ratio of live offspring, a lower percentage of male pups was observed in the treated groups when compared to the Control group, including those that died prior to the designated day 1 (maximum effect 73% of control at 400/250 mg/kg/day, which was statistically significant).

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

There were no macroscopic findings noted at the end of the exposure period that could be attributed to the test item.

Developmental immunotoxicity:

not examined

Dose descriptor:

NOAEL

Remarks:

Reproductive/developmental toxicity

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Reproductive effects observed:

no

Clinical signs

	Males				Females
Dose (mg/kg bw/day)	0	25	100	250/400	0	25	100	250/400
Mortality^a	0/12	0/12	0/12	2/12	0/12	0/12	0/12	4/12
Salivation	0/12	0/12	0/12	10/12	0/12	0/12	0/12	8/12
Changes to breathing^bor rales	0/12	0/12	0/12	5/12	0/12	0/12	0/12	3/12
Piloerection	0/12	0/12	0/12	3/12	0/12	0/12	0/12	2/12
Reduced muscle tone	0/12	0/12	0/12	2/12	0/12	0/12	0/12	0/12
Haunched posture	0/12	0/12	0/12	2/12	0/12	0/12	0/12	1/12
Hair loss	0/12	0/12	0/12	0/12	0/12	0/12	1/12	1/12
Pallor, whole body	0/12	0/12	0/12	2/12	0/12	0/12	0/12	1/12

 a            Including animals euthanised for welfare reasons
b            Including gasping, irregular, slow and laboured breathing

Organ weights

	Males				Females
Dose (mg/kg bw/day)	0	25	100	250/400	0	25	100	250/400
Adjusted mean lung and bronchi weight (g)	1.635	1.534	1.486*	1.492*	1.184	1.152	1.171	1.169
Adjusted mean brain weight (g)	2.109	2.112	2.064	2.038*	1.883	1.914	1.837	1.904
Adjusted mean adrenals weight (g)	0.069	0.069	0.069	0.070	0.075	0.081	0.080	0.084

 

 

 

* Statistically significant

Treatment related findings in the stomach

	Males				Females
Dose (mg/kg bw/day)	0	25	100	250/400	0	25	100	250/400
Number of tissues examined	5	5	5	5	5	5	5	5
Edema, Mucosa/submucosa, nonglandular region
Minimal	0	0	1	1	0	0	0	1
Moderate	0	0	0	1	0	0	0	1
Total	0	0	1	2	0	0	0	2
Hyperkeratosis, nonglandular
Minimal	0	0	0	1	0	0	0	0
Total	0	0	0	1	0	0	0	0
Hyperplasia, squamous cell non glandular region
Minimal	0	0	1	2	0	0	0	2
Slight	0	0	0	1	0	0	0	0
Total	0	0	1	3	0	0	0	2
Inflammation, mucosa/submucosa, nonglandular region
Minimal	0	0	1	2	0	0	0	0
Slight	0	0	0	1	0	0	0	0
Total	0	0	1	3	0	0	0	0

  

 Treatment related findings in the larynx

	Males				Females
Dose (mg/kg bw/day)	0	25	100	250/400	0	25	100	250/400
Number of tissues examined	5	5	5	5	5	5	5	5
Hyperplasia/Metaplasia respiratory epithelium
Minimal	0	0	1	1	0	1	2	1
Slight	0	0	0	1	0	0	0	3
Moderate	0	0	0	0	0	0	0	1
Total	0	0	1	2	0	1	2	5
Inflammation, respiratory epithelium
Minimal	0	0	0	2	0	0	0	0
Slight	0	0	0	0	0	0	0	1
Total	0	0	0	2	0	0	0	1
Regeneration, respiratory epithelium
Minimal	0	0	0	1	0	0	0	3
Total	0	0	0	1	0	0	0	3

  

Treatment related findings in the trachea

	Males				Females
Dose (mg/kg bw/day)	0	25	100	250/400	0	25	100	250/400
Number of tissues examined	5	5	5	5	5	5	5	5
Regeneration/Hyperplasia, respiratory epithelium
Minimal	0	0	1	0	0	0	0	0
Slight	0	0	1	1	0	0	0	1
Marked	0	0	0	0	0	0	0	1
Total	0	0	2	1	0	0	0	2

  

Litter data

Dose (mg/kg bw/day)	0	25	100	250/400
Number mated	12	12	12	9
Non-pregnant	1	3	0	0
Total litter loss	0	1	0	0
Litters examined	11	8	12	9
Fertility index (%)	91.67	75.00	100.0	100.0
Implantations	13.5	12.1	12.3	12.2
Live birth index (%)	97.0	86.9	100.0	99.1
Viability index on Day 4 (%)	98.5	100.0	98.0	87.6
Pre implantation loss (%)	16.25	4.46	10.53	12.97
Post implantation loss (%)	9.79	13.51	9.07	6.84
F1 body weight (g) at 1 day old (M/F)	5.8/5.5	6.3/6.0	6.0/5.8	5.8/5.5
Number of live pups on Day 0-1 post partum (M/F)	6.8/4.9	5.0/4.8	4.9*/6.3	5.3/6.0
Number of still born pups on Day 0-1 post partum (M/F)	0.1/0.0	0.2/0.2	0.0/0.0	0.0/0.3
Number of live pups on Day 4 post partum (M/F)	6.7/4.8	5.0/4.8	4.8*/6.3	4.8/5.0

Conclusions:

In conclusion, oral administration (by gavage) of DABCO®T Catalyst (2-[[2-(Dimethylamino) ethyl] methylamino] ethanol) to Wistar rats at 25, 100 and 400/250 mg/kg bw/d for two weeks prior to mating and up to the day before sacrifice inclusive (males) or up to days 4-7 postpartum (females) was associated with mortality of parental generation when the animals were administered at 400 mg/kg bw/d. Given the changes in the stomach, larynx and trachea observed in both sexes in the microscopic examination at the dose of 400/250 mg/kg bw/d and as no relevant test item effect was observed at 25 or 100 mg/kg bw/d, the no-observed-adverse-effect-level (NOAEL) for systemic toxicity could be established at 100 mg/kg bw/d.

The no-observed-adverse-effect-level (NOAEL) for fertility and reproductive performance was considered to be 250 mg/kg bw/d.

The no-observed-adverse-effect-level (NOAEL) for reproductive/developmental toxicity was considered to be 250 mg/kg bw/d.

Executive summary:

A screening for reproductive/developmental toxicity has been conducted with 2-[[2-(dimethylamino)ethyl]methylamino]ethanol in rats according to OECD guideline 422. Animals were initially dosed with 25, 100 and 400 mg/kg bw/d. Based on mortality observed in the high dose group, the dose level was reduced from 400 mg/kg bw/d after 6 days of treatment to 250 mg/kg bw/d. Twelve animals/sex were dosed orally by gavage for each dose group. A vehicle control group comprising corn oil was also included. Males were treated continuously for two weeks before pairing up to necropsy, after a minimum of 48 consecutive days. Females were treated continuously for two weeks before pairing, throughout pairing and gestation, and until Day 7 of lactation at the most, i.e. the day before sacrifice. Females were allowed to litter and rear their offspring, and litters were killed on Day 4/7 of lactation. F1 generation received no direct administration of the test item; any exposure was in utero or via the milk. During the study, mortality, clinical signs, sensory reactivity observations, grip strength motor activity, body weight, food consumption, haematology and coagulation, blood biochemistry, pre-coital interval, mating performance, fertility, gestation length, organ weight and macroscopic examination were evaluated. Clinical signs, behaviour assessment, litter size and survival, sex ratio, body weight and macropathology were also assessed for all offspring. Following the reduction in the high dose level, there was no mortality or clinical signs, with the exception of salivation which could be attributed to treatment. There were no effects on body weight, food consumption, haematology or coagulation which were attributed to treatment. Higher creatinine levels were noted in males and females dosed with 100 mg/kg bw/d and females dosed with 250/400 mg/kg bw/d. There were no treatment related macroscopic findings. In males there was a decrease in the weight of the brain and lungs and bronchi and in the females there was an increase in the weight of the adrenals when compared to the concurrent vehicle control treated groups. Histopathological changes were noted in the stomach, larynx and trachea of males and females treated with 250/400 mg/kg bw/d. There were no changes to the reproductive parameters (pre-coital interval fertility or gestation length or index) and no adverse effects on offspring (survival, litter size, sex ratio, growth, clinical or necropsy signs, or body weight). The no observed adverse effect level (NOAEL) is considered to be 250 mg/kg bw d for fertility and reproductive performance and reproductive/developmental toxicity. The NOAEL was considered to be 100 mg/kg bw/d for systemic toxicity.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1996

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

2-[[2-(dimethylamino)ethyl]methylamino]ethanol

EC Number:

218-658-4

EC Name:

2-[[2-(dimethylamino)ethyl]methylamino]ethanol

Cas Number:

2212-32-0

Molecular formula:

C7H18N2O

IUPAC Name:

2-{[2-(dimethylamino)ethyl](methyl)amino}ethan-1-ol

Test material form:

liquid

Specific details on test material used for the study:

Product name: DABCO T Catalyst
CAS number: 2212-32-0
Appearance: Colourless to pale yellow liquid
Batch: 2096108
Purity: 99.4%
Manufacture date: 08 February 2016
Retest date: 08 February 2018
Storage conditions: Room temperature protected from light; in a tightly closed container; in a dry cool place; kept away from sources of heat, ignition and oxidisers

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Wistar Hannover RccHan:WIST

Sex:

male/female

Details on test animals or test system and environmental conditions:

Supplier: Envigo RMS
Age (at delivery): 10-11 weeks (male and females)
Body weight (at start of treatment): 347-432 g (males); 189-240 g (females)
Randomisation: Method based on similarity of mean body weights among groups
Acclimatisation period: 28 days prior to treatment
Vetinary examinations: During acclimatisation and before treatment. Additional inspections in some animals in the highest dose group during the first six days of treatment.
Air changes: 15-20 air changes per hour
Room temperature: 20-24 °C
Humidity: 30-70%
Photoperiod: 12 hours light/dark
Caging: standard sawdust bedding; 5 animals/cage during premating; 1 male and 1 female/cage during mating; individually during postmating
Diet: Standard Teklad 2014C rat/mouse maintenance diet; ad libitum
Water: Tap water; ad libitum
Enrichment: material specific for species supplied

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

Initially males dosed first then females. Starting order was alternated between males and females for each day of treatment.
Amount administered based on the most recent body weight recorded. Administration volume was 4 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentration of dose formulation was determined twice during the study and once for homogeneity (top/middle/bottom) in samples taken from the formulation administered to Groups 1 to 4. The formulations were quantified following a validated analytical procedure. On each occasion, duplicate samples of the dosing solution (10 mL each) were transferred to labeled vials. One aliquot was used for analysis. The test item was used as analytical standard. The analytical procedure was successfully validated with respect to specificity of chromatographic analysis, linearity of detector response, method accuracy and precision. The homogeneity and stability was confirmed for the test item in vehicle formulations at nominal concentrations of 3.75 mg/mL and 250 mg/mL when stored at ambient (6 hours), refrigerated and frozen condition for 5 days. The mean concentrations of test item in test formulations analyzed for the study were within 100 ± 11 % of nominal concentrations, confirming accurate formulation.

Duration of treatment / exposure:

Males: 2 weeks prior to mating up to and including the day before sacrifice (Days 44 to 47); at least 30 days
Females: 2 weeks prior to mating up to and including the day before sacrifice (Day 4 to Day 7 post partum).

Frequency of treatment:

Daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Remarks:

Vehicle control

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

Doses / concentrations 4

Dose / conc.:

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

Remarks:

This group was administered 400 mg/kg bw/day for Days 1 to 6 of treatment. The dose was reduced to 250 mg/kg bw/day from Day 7 to the end of the study.

No. of animals per sex per dose:

Twelve

Control animals:

yes, concurrent vehicle

Details on study design:

Three groups of twelve male and twelve female rats received 2-[[2-(Dimethylamino) ethyl] methylamino] ethanol at the doses of 25, 100 and 400/250 mg/kg bw/d. Males were treated continuously for two weeks before pairing up to necropsy, after a minimum of 48 consecutive days. Females were treated continuously for two weeks before pairing, throughout pairing and gestation, and until Day 7 of lactation at the most, i.e. the day before sacrifice. Females were allowed to litter and rear their offspring. A similarly constituted control group received the vehicle, corn oil. During the study, mortality, clinical signs, sensory reactivity observations, grip strength motor activity, body weight, food consumption, haematology and coagulation, blood biochemistry, pre-coital interval, mating performance, fertility, gestation length, organ weight and macroscopic examination were evaluated.

Positive control:

None; not required.

Examinations

Observations and examinations performed and frequency:

Morbidity/mortality and cage-side observations were conducted twice daily. In Group 4, females no. 190 and 194, which were found dead, and females no. 185 and 191 and males no. 39 and 46, which were sacrificed due to animal welfare reasons, were subjected to macroscopic examination. Food consumption was assessed in males twice at pretest, once weekly during the pre-pairing/treatment period, and during the two weeks of the postpairing period and in females twice at pretest, once weekly during the pre-pairing/treatment period, on days 0-7, 7-14, 14-21 post coitum and on days 1-4 postpartum. Food consumption was not examined during the mating period.

Body weight was recorded in males three times during pretest and twice weekly during the pre-pairing, pairing and postpairing periods and in females three times during pretest, twice weekly during the pre-pairing and pairing periods and daily from day 0 of gestation until days 4-8 postpartum.

Detailed clinical observations were performed on all test item treated and control animals before the first exposure to the test item, once weekly thereafter and one day before sacrifice. These observations were performed outside the home cage, in a standard arena, between 1-3 hours after dosing to ensure that any transient effects of treatment are identified. In view of the clinical signs and body-weight loss observed in some Group-4 animals, additional clinical observations were done to record the health condition of the animals from day 6 of treatment onwards. After reducing the dose, salivation was recorded daily in some of the animals from this group.

Grip strength (fore- and hind limbs) and motor activity were measured in five randomly selected males per group once during the final week of treatment and at least one hour after dosing. Five randomly selected females per group were similarly evaluated on day 4 postpartum.

Sensory reactivity to different stimuli (as follows) was evaluated in the animals mentioned above:
- Blink reflex
- Pinna reflex
- Iridic light / Pupil closure reflex
- Proprioception(right leg) / pushoff (hind legs)
- Pain response / Tail punch response
- Startle / hearing
- Righting reflex in the air

From day 20 post coitum, the females were examined twice daily for signs of parturition. The duration of gestation (days) was recorded.

The females that gave birth were checked to observe whether they nursed their offspring.

Blood samples (1.8 mL) were drawn from the retro-orbital plexus of animals selected for functional observations (five animals/sex/group) under light isoflurane anesthesia. Given poor condition of the blood sample obtained in female no. 151, it was necessary to obtain a sample in female no. 149 (not selected for functional observations) in order to achieve the sample size in Group 1 (control). The animals were fasted before blood sampling but allowed access to water ad libitum for 3-4 hours. The samples were collected early in the working day to reduce biological variation caused by circadian rhythms.
Blood sampling performed as follows:
Males: Once on day 44 of the study (on the day of sacrifice)
Females: Once on day 5 or 6 postpartum (on the day of sacrifice)

The following paramenters were determined in samples collected for haematology: erythrocyte count, haemoglobin, haematocrit, mean corpuscular volume, red cell volume distribution width, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, haemoglobin concentration distribution width, reticulocyte count, reticulocyte maturity index, platelet count, total leukocyte count and differential leukocyte count (neutrophils, lymphocytes, monocytes, eosinophils, basophils and large unstained cells).

The following parameters were determined in samples collected for coagulation: prothrombin time and activated partial thromoplastin time.

The following parameters were determined in samples collected for clinical biochemistry:glucose, urea, creatinine, total bilirubin, total cholesterol, triglycerides, aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, gamma-glutamyl transferase, creatine kinase, inorganic phosphorus, sodium, potassium, calcium, chloride, total protein, albumin, globulin (calculated from total protein and albumin), albumin/globulin ratio and bile acids.

The following organs were weighed: adrenal glands, brain (including sections of medulla/pons, cerebral and cerebellar cortex), epididymides, heart (with papillary muscle), kidneys, liver, lungs (with bronchi and bronchioles), pituitary gland, prostate gland and seminal vesicles, salivary glands (mandibular and sublingual), spleen,thymus, testes, thyroid (including parathyroid) and uterus (including cervix and oviducts).

The following organs were collected and examined for histopathological analysis: adrenal glands, aorta, bone (sternum, including bone marrow), bone (femur, including joint), brain (including sections of medulla/pons, cerebral and cerebellar cortex), epididymides, eyes (with optic nerve), heart (with papillary muscle), large intestine (cecum, colon and rectum), small intestine (duodenum, jejunum and ileum), kidneys, larynx, liver, lungs (with bronchi and bronchioles), mammary gland area, peyer's patches, pituitary gland, prostate gland and seminal vesicles, salivary glands (mandibular and sublingual), salivary glands (parotid), sciatic nerve, skeletal muscle, skin (abdominal), spinal cord (cervical, midthoracic and lumbar), spleen, stomach (glandular and nonglandular), thymus, testes, thyroid (including parathyroid), tongue, trachea, urinary bladder, uterus (including cervix and oviducts) and vagina. In addition, bone marrow from the femur (air dried) was collected but not examined. Tissues were fixed with 10% formalin, with the exception of epididymides and the eyes which were fixed with modified Davidson's fixative or Davidson's fixative, respectively.

Sacrifice and pathology:

Animals that died (Group 4: females no. 190 and 194) or were sacrificed in extremis (Group 4: female no. 185 and 191; males no. 39 and 46) were necropsied. Their organs were extracted and fixed but they were not weighed.

From days 5 to 8 postpartum, all females were sacrificed by intraperitoneal injection of sodium pentobarbital and immediately exsanguinated by excision of the axillary vessels and aorta and necropsied. The mated females that did not give birth or did not show signs of pregnancy were sacrificed after 25-28 days post coitum. The external surface of the body, all orifices and cranial, thoracic and abdominal cavities and their contents were examined postmortem with emphasis on the uterus, number of corpora lutea and implantation sites. Organs were observed in situ and all macroscopic abnormalities were described.
When no implantation sites were evident, the uterus was placed in an aqueous solution of ammonium sulfide (Salewski, 1964) to accentuate possible hemorrhagic areas of implantation sites.

From days 44 to 47 of the study all males were sacrificed by intraperitoneal injection of sodium pentobarbital and immediately exsanguinated by excision of the axillary vessels and aorta and necropsied, subject to confirmation of successful mating.
The external surface of the body, all orifices and cranial, thoracic and abdominal cavities and their contents were examined postmortem. Organs were observed in situ and all macroscopic abnormalities were described.
The organs weights were recorded on the scheduled necropsy dates for all surviving parental animals and their organ-to-terminal-body-weight ratios and organ-to-brainweight ratios were determined.

Samples of tissues and organs as well as specimens of abnormal tissue were collected from all animals at necropsy and fixed in neutral phosphate buffered 4% formaldehyde solution (10% formalin) unless otherwise indicated. All organ and tissue samples (except for the nose) of selected animals from groups 1 to 4 (5 animals/sex) to be examined by the histopathologist were processed, embedded and cut at an approximate thickness of 2-4 micrometers and stained with hematoxylin and eosin. Where possible, the microscopic findings were correlated with the gross observations. The bone marrow smears were stained using the May Grünwald-Giemsa method. In addition, the reproductive organs of all control and high dose animals were also examined.
A qualitative staging of spermatogenesis and histopathology evaluation of interstitial cells of all males from the control and high-dose groups was performed by the histopathologist. Stomach, larynx and trachea of the selected animals from Groups 2 and 3 (5 animals/sex) were processed as indicated above and examined by the histopathologist. Full tissues from animal no. 4, 38, 45, 152, 187, 193 and 196, all stomachs, all female larynxes and all Group 2 and 3 stomach, larynxes and tracheas were peer reviewed.

Other examinations:

None

Statistics:

All statistical analyses were carried out separately for males and females. Data relating to food consumption were analyzed on a cage basis. For all other parameters, the analyses were carried out using the individual animal as the basic experimental unit.
The following data types were analyzed at each time point separately:
Grip strength and motor activity
Body weight
Food consumption, over appropriate study periods
Hematology and coagulation
Clinical biochemistry
Organ weights, absolute and adjusted for terminal body weight
Pathological findings, for the number of animals with and without each finding

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

There were no other clinical signs other than salivation that were considered to indicate an immediate or delayed reaction to treatment once the high dose was reduced to 250 mg/kg/ bwd. Salivation at the high dose may be related to the test item palatability. Before decreasing the dose from 400 to 250 mg/kg bw/d, the female sacrificed for animal welfare reasons (no. 185) showed irregular breathing, piloerection and hunched posture. In males administered at 400/250 mg/kg/day also sacrificed for welfare reasons (nos. 39 and 46), piloerection, labored breathing, reduced body tone and pallor were recorded, in addition to hunched posture in one of them. These clinical signs seem to be related with toxicity at the dose of 400 mg/kg bw/d as they were not present after reducing the dose. In female no. 191 administered at 400/250 mg/kg bw/d, gasping breathing and pallor were observed after delivery (lactation day 1). As these signs were not observed in the other females at this dose they cannot be considered a treatment-related effect.

Mortality:

mortality observed, treatment-related

Description (incidence):

There were no other clinical signs other than salivation that were considered to indicate an immediate or delayed reaction to treatment once the high dose was reduced to 250 mg/kg/ bwd.
Salivation at the high dose may be related to the test item palatability. Before decreasing the dose from 400 to 250 mg/kg bw/d, the female sacrificed for animal welfare reasons (no. 185) showed irregular breathing, piloerection and hunched posture. In males administered at 400/250 mg/kg/day also sacrificed for welfare reasons (nos. 39 and 46), piloerection, labored breathing, reduced body tone and pallor were recorded, in addition to hunched posture in one of them. These clinical signs seem to be related with toxicity at the dose of 400 mg/kg bw/d as they were not present after reducing the dose. In female no. 191 administered at 400/250 mg/kg bw/d, gasping breathing and pallor were observed after delivery (lactation day 1). As these signs were not observed in the other females at this dose they cannot be considered a treatment-related effect.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There was no adverse effect on mean bodyweight in males throughout the study or in females before pairing, during gestation or during lactation. Mean body weight recorded on day 8 of treatment in males receiving 400/250 mg/kg bw/d was lower than that recorded in the control group. However, this did not attain significance. There were no effects after decreasing the dose.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There were no effects after decreasing the dose.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

Hematological evaluation at the end of treatment revealed no statistical differences with the control group in males. In treated females (25, 100 and 400/250 mg/kg bw/d), mean eosinophil values were 50% lower than in the control group. However, this difference could not be considered of any relevance, taking into account the normal values observed in rats of this strain and age. No differences were observed in coagulation when comparing the test-item administered groups with the control.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Clinical biochemistry at the end of treatment showed statistically higher creatinine mean values in females at 100 and 400/250 mg/kg bw/d and in males at 100 mg/kg/day. However, given the magnitude of change and in the absence of any dose relation, it cannot be considered to be treatment-related. All other differences from control (statistically significant differences in potassium, chloride and total proteins) were minor, lacked dose relationship or were confined to one sex and were therefore attributed to normal biological variation.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Despite the fact that lower mean values in forelimb grip strength were observed in males at 400/250 mg/kg bw/d and in females at 100 and 400/250 mg/kg bw/d when compared to the control group, they cannot be considered to be of toxicological relevance given the magnitude of change or because no dose response was observed. Therefore, there were no indications of any adverse effect of the test item on grip strength assessment. No noteworthy changes were recorded in the motor activity examination. The statistically significant differences observed in females receiving 400/250 mg/kg bw/d at 10 and 50 minutes and in females receiving 100 mg/kg bw/d at 50 minutes are considered to be of no relevance as they were not part of a dose-related trend and/or this effect was not present in males. There was no indication of any test item effect on sensory activity.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

In males, lower lung and bronchi weights were recorded in treated groups (maximum effect 91% of control at 100 mg/kg bw/d). There was a decrease in brain weight in the treated groups when compared to control, which was statistically significant at 400/250 mg/kg bw/d. In females, higher adrenal weights were recorded in treated groups compared to the control group (maximum effect 112% of control).

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment-related macroscopic findings noted at the end of the study. The isolated findings observed were considered of no toxicological relevance as they were consistent with findings normally observed in rats of this strain and age.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

In the testes, seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and the integrity of the various cell types present within the different stages. No cell or stage specific abnormalities were noted in males treated at 400/250 mg/kg bw/d. Interstitial cells were also assessed qualitatively, and no treatment related alterations were seen.
In the animals administered at 400/250 mg/kg bw/d, the following alterations were recorded:

Stomach:
- Minimal to slight focal areas of epithelial hyperplasia of the squamous epithelium, with underlying mucosal/submucosal subacute to chronic inflammation in three males. Occasionally, these were associated with minimal focal hyperkeratosis, minimal to moderate mucosal/submucosal edema, or granulation tissue formation that extended down the serosa.
- Similar but milder changes in the forestomach in two females. In one of them, mucosal/submucosal edema was seen both in the nonglandular and, to a milder degree, glandular regions.
- Minimal focal area of mucosal necrosis and erosion in the pylorus in one female.

Trachea:
- Focal to extensive areas of epithelial regeneration and/or hyperplasia in a few males and females where gastric changes were also detected.

Larynx:
- Minimal to moderate areas of hyperplasia of the respiratory epithelium, with squamous metaplasia in the arytenoid cartilage region, sometimes along with underlying minimal to slight subacute to chronic inflammation and focal areas of flattened regenerative epithelium in some males and females.

All other histopathological findings were considered to be incidental and unrelated to the test item.

In the pathology extension, some punctual effects at 25 and 100 mg/kg bw/d in larynx (hyperplasia/metaplasia of the epithelium) and only at 100 mg/kg/ bwd in the stomach (edema, hyperplasia and inflammation) and in trachea (regeneration hyperplasia in epithelium) were observed.

Histopathological findings: neoplastic:

no effects observed

Other effects:

not examined

Details on results:

At 400/250 mg/kg bw/d, during the first 7 days of the treatment period, two females were found dead, and another had to be sacrificed for welfare reasons. At the same dose, on day 8 of treatment, two males were also sacrificed for welfare reasons. As this was associated with toxicity at 400 mg/kg bw/d, it was considered necessary to decrease the dose to 250 mg/kg bw/d. One female administered at 400/250 mg/kg/ bwd was also sacrificed for welfare reasons after parturition, on day 1 of lactation. After reducing the dose, there was no mortality nor were there any clinical signs other than salivation that could be attributable to treatment. No effects on bodyweight or food consumption were observed after reduction of the dose. No noteworthy differences were observed in haematology or coagulation in the treated groups when compared to controls. Despite the fact that no relevant differences were observed in clinical biochemistry, statistically higher creatinine mean values were recorded at 100 mg/kg bw/d in males and females and at 400/250 mg/kg bw/d in females. There were no treatment related macroscopic findings noted at the end of the treatment period. In males, brain and lungs and bronchi weights appeared to decrease in treated groups when compared to control. In females, adrenal mean weights were also higher than in the control group. Changes that were considered related to treatment were seen in
the stomach, larynx and trachea of males and females treated at 400/250 mg/kg bw/d.

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Target system / organ toxicity

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

100 mg/kg bw/day (nominal)

System:

gastrointestinal tract

Organ:

stomach

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

no

Any other information on results incl. tables

Clinical signs

	Males				Females
Dose (mg/kg bw/d)	0	25	100	250/400	0	25	100	250/400
Mortality^a	0/12	0/12	0/12	2/12	0/12	0/12	0/12	4/12
Salivation	0/12	0/12	0/12	10/12	0/12	0/12	0/12	8/12
Changes to breathing^bor rales	0/12	0/12	0/12	5/12	0/12	0/12	0/12	3/12
Piloerection	0/12	0/12	0/12	3/12	0/12	0/12	0/12	2/12
Reduced muscle tone	0/12	0/12	0/12	2/12	0/12	0/12	0/12	0/12
Haunched posture	0/12	0/12	0/12	2/12	0/12	0/12	0/12	1/12
Hair loss	0/12	0/12	0/12	0/12	0/12	0/12	1/12	1/12
Pallor, whole body	0/12	0/12	0/12	2/12	0/12	0/12	0/12	1/12

a            Including animals euthanised for welfare reasons
b            Including gasping, irregular, slow and laboured breathing

Organ weights

	Males				Females
Dose (mg/kg bw/d)	0	25	100	250/400	0	25	100	250/400
Adjusted mean lung and bronchi weight (g)	1.635	1.534	1.486*	1.492*	1.184	1.152	1.171	1.169
Adjusted mean brain weight (g)	2.109	2.112	2.064	2.038*	1.883	1.914	1.837	1.904
Adjusted mean adrenals weight (g)	0.069	0.069	0.069	0.070	0.075	0.081	0.080	0.084

* Statistically significant

Treatment related findings in the stomach

	Males				Females
Dose (mg/kg bw/d)	0	25	100	250/400	0	25	100	250/400
Number of tissues examined	5	5	5	5	5	5	5	5
Edema, Mucosa/submucosa, nonglandular region
Minimal	0	0	1	1	0	0	0	1
Moderate	0	0	0	1	0	0	0	1
Total	0	0	1	2	0	0	0	2
Hyperkeratosis, nonglandular
Minimal	0	0	0	1	0	0	0	0
Total	0	0	0	1	0	0	0	0
Hyperplasia, squamous cell non glandular region
Minimal	0	0	1	2	0	0	0	2
Slight	0	0	0	1	0	0	0	0
Total	0	0	1	3	0	0	0	2
Inflammation, mucosa/submucosa, nonglandular region
Minimal	0	0	1	2	0	0	0	0
Slight	0	0	0	1	0	0	0	0
Total	0	0	1	3	0	0	0	0

Treatment related findings in the larynx

	Males				Females
Dose (mg/kg bw/d)	0	25	100	250/400	0	25	100	250/400
Number of tissues examined	5	5	5	5	5	5	5	5
Hyperplasia/Metaplasia respiratory epithelium
Minimal	0	0	1	1	0	1	2	1
Slight	0	0	0	1	0	0	0	3
Moderate	0	0	0	0	0	0	0	1
Total	0	0	1	2	0	1	2	5
Inflammation, respiratory epithelium
Minimal	0	0	0	2	0	0	0	0
Slight	0	0	0	0	0	0	0	1
Total	0	0	0	2	0	0	0	1
Regeneration, respiratory epithelium
Minimal	0	0	0	1	0	0	0	3
Total	0	0	0	1	0	0	0	3

 

Treatment related findings in the trachea

	Males				Females
Dose (mg/kg bw/d)	0	25	100	250/400	0	25	100	250/400
Number of tissues examined	5	5	5	5	5	5	5	5
Regeneration/Hyperplasia, respiratory epithelium
Minimal	0	0	1	0	0	0	0	0
Slight	0	0	1	1	0	0	0	1
Marked	0	0	0	0	0	0	0	1
Total	0	0	2	1	0	0	0	2

 

Applicant's summary and conclusion

Conclusions:

Oral administration (by gavage) of DABCO®T Catalyst (2-[[2-(Dimethylamino) ethyl] methylamino] ethanol) to Wistar rats at 25, 100 and 400/250 mg/kg bw/d for two weeks prior to mating and up to the day before sacrifice inclusive (males) or up to days 4-7 postpartum (females) was associated with mortality when the animals were administered at 400 mg/kg bw/d. Given the changes in the stomach, larynx and trachea observed in both sexes in the microscopic examination at the dose of 400/250 mg/kg bw/d and as no relevant test item effect was observed at 25 or 100 mg/kg bw/d, the no-observed-adverse-effect-level (NOAEL) for systemic toxicity could be established at 100 mg/kg bw/d.

Executive summary:

A screening for reproductive/developmental toxicity has been conducted with 2-[[2-(dimethylamino)ethyl]methylamino]ethanol in rats according to OECD guideline 422. Animals were initially dosed with 25, 100 and 400 mg/kg bw/d. Based on mortality observed in the high dose group, the dose level was reduced from 400 mg/kg bw/d after 6 days of treatment to 250 mg/kg bw/d. Twelve animals/sex were dosed orally by gavage for each dose group. A vehicle control group comprising corn oil was also included. Males were treated continuously for two weeks before pairing up to necropsy, after a minimum of 48 consecutive days. Females were treated continuously for two weeks before pairing, throughout pairing and gestation, and until Day 7 of lactation at the most, i.e. the day before sacrifice. Females were allowed to litter and rear their offspring. During the study, mortality, clinical signs, sensory reactivity observations, grip strength motor activity, body weight, food consumption, haematology and coagulation, blood biochemistry, pre-coital interval, mating performance, fertility, gestation length, organ weight and macroscopic examination were evaluated. Following the reduction in the high dose level, there was no mortality or clinical signs, with the exception of salivation which could be attributed to treatment. There were no effects on body weight, food consumption, haematology or coagulation which were attributed to treatment. Higher creatinine levels were noted in males and females dosed with 100 mg/kg bw/d and females dosed with 250/400 mg/kg bw/d. There were no treatment related macroscopic findings. In males there was a decrease in the weight of the brain and lungs and bronchi and in the females there was an increase in the weight of the adrenals when compared to the concurrent vehicle control treated groups. Histopathological changes were noted in the stomach, larynx and trachea of males and females treated with 250/400 mg/kg bw/d. The NOAEL was considered to be 100 mg/kg bw/d for systemic toxicity.