Registration Dossier

Administrative data

Description of key information

A screening study (OECD 422) and a 14 -day dose finding toxicity study are available for the submission substance.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
10 December 2015 to 30 May 2016
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Screening study
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Qualifier:
according to
Guideline:
other: Commission Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH)
GLP compliance:
yes (incl. certificate)
Limit test:
no
Specific details on test material used for the study:
CAS number: 2212-32-0
Appearance: colourless to pale yellow liquid
Batch number: 1721964
Purity: 99%
Manufacture date: 08 April 2014
Retest date: 08 April 2016
Storage conditions: Room temperature protected from light. Tightly closed container in a dry cool place kept away from heat, sources of ignition and oxidizers.
Species:
rat
Strain:
other: Hannover Wistar
Details on species / strain selection:
Species is recognised by international guidelines as a a recommended test method.
Sex:
male/female
Details on test animals and environmental conditions:
Supplied by Charles River Laboratories, France
Breed by Charles River Laboratories, Germany
Age at start of treatment: 12 weeks
Body weight range at start of treatment: 325-342 g (males); 194-217 g (females)
Randomisation: method based on the similarity of mean body weights among groups.
Allocation: Shortly before the start of treatment the animals were allocated at random to the four treatment groups. The spare animals were allocated to group 5 and assigned numbers 13-15 for males and 28-30 for females. Then the animals were weighed and the data obtained was reviewed and the allocation adjusted where considered appropriate using the spare animals and/or exchange of animals. The rejected animals took no further part in the study after start of treatment and were removed from it. Only data from animals assigned to the study are reported.
Acclimitisation: Seven days after arrival
Temperature: 21-23 °C target
Humidity: 35-50% target
Air changes: Minimum of 15-20 air changes per hour
Photoperiod: 12 hour light/dark cycle
Caging: Makrolon type IV cages with sawdust bedding
Grouping: Groups of five of the same sex duribng acclimitisation and groups of three of the ssame sex during treatment
Diet: Pelleted standard Teklad 2014C rat/mouse diet; ad libitum; batch number 061915MA
Water: Tap water; ad libitum
Enrichment: Paper wool
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
For each dose group the administration started all males first and then all females. Each day of treatment the starting order was alternated between males and females, except fourth day that males were repeated as start.
Administration volume: 4 mL/kg bw
Formulations stored refrigerated at 2-8 °C protected from light for five days of at room temperature for 5 hours.
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
14 consecutive days
Frequency of treatment:
Daily
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Vehicle control
Dose / conc.:
500 mg/kg bw/day (nominal)
Dose / conc.:
700 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
Three
Control animals:
yes, concurrent vehicle
Details on study design:
Animals were dosed based on the most recent body weight recorded. Control animals were administered with the vehicle following the same dosing regimen as that used for animals treated with the test material.
Positive control:
Not tested.
Observations and examinations performed and frequency:
Viability, mortality and cage-side observations recorded twice daily.
Clinical signs recorded twice daily from Days 5 to 14 with detailed clinical signs once on Days 2 and 4.
Food consumption recorded twice weekly
Body weight recorded once during acclimitisation and on Days 1 and 4 and daily from seventh day until sacrifice.
Blood samples taken on Day 15 (1.3 mL from retro-orbital plexus) for all animals under light isoflurane anesthia following 4-5 hours fasting (water provided ad libitum). Collection early in the morning.
- 0.5 mL collected into Tri-potassium-EDTA to determine 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, differential leukocyte count (neutrophils, lymphocytes, monocytes, eosinophils, basophils and large unstained cells).
-0.8 mL collected into lithium heparin tubes to determine the following parameters: glucose, urea, creatinine, total bilirubin, total cholesterol, triglycerides, aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, gamma-glutamyl transferase, creatine kinase, inorganic phosphorus, sodium, potassium, chloride, total protein, albumin, globulin (calculated from total protein and albumin), albumin/globulin ratio and bile acids.






Sacrifice and pathology:
All the animals surviving to the end of the observation period were deprived of food and deeply anesthetized with sodium pentobarbital administered intraperitoneally and killed by exsanguination.
A full necropsy was performed on all rats. The necropsy included the examination of the external surface of the body, all orifices, cranial, thoracic and abdominal cavities and the
observation of the organs both in situ and after evisceration. Descriptions of all macroscopic abnormalities were recorded, and in animals that died the cause of death was determined. Sample of tissues and organs were weighed from surviving animals. Samples of tissues and organs were collected at necropsy and fixed in 10% formalin or for the epididymides, eyes (with optic nerve) testes in Davidson's solution. The following organs were collected: adrenal glands, aorta, bone (sternum, including bone marrow), 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. 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).
No histology was performed.
Other examinations:
None.
Statistics:
The following statistical methods were used to analyze body weight, clinical laboratory data and organ weights and ratios.
If the variables were assumed to follow a normal distribution, the Dunnett test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
The Cochran and Cox test was applied instead of the Dunnett test when the data was not assumed to follow a normal distribution.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Salivation was recorded in a test material treated groups in both sexes. Laboured/irregular breathing, arched back, decreased motor activty, partially closed eyelids and piloerection were recorded occasionally in few animals at the end of the treatment period.
Mortality:
mortality observed, treatment-related
Description (incidence):
At 500 mg/kg bw/d, no mortality was recorded. At 700 mg/kg bw/d, male no. 8 was found dead on day 6 of study after receiving 5 administrations. This animal showed up of the product after the second administration. At 1000 mg/kg bw/d, two animals died. Male no. 11 was found dead on day 6 of study after receiving 5 administrations and this animal presented the day before its dead decreased motor activity and partially closed eyelids. Male no. 12 died four hours after the 13th administration after showing laboured breathing, piloerection, arched back.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Mean body weight gain decreased (-200% on day 13 of study) was recorded in males treated at 1000 mg/kg bw/d from day 4 onwards. Statistically significant differences were recorded with respect to Control animals. At the dose of 700 mg/kg bw/d a slight lower mean body weight gain (ca. 28%) was recorded in males from day 9 onwards. No statistically significant differences were observed. At 500 mg/kg bw/d, the body weight gain of males was similar to the control except on days 13 onwards due to the animal no. 6 that presented clinical signs. A slight lower body weight gain was observed occasionally in females in treated groups. The differences observed were not dose level dependent and lower than 5%.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Lower mean food consumption was recorded in males treated at 1000 mg/kg/ bw/d (~ 50%) and in males at 700 mg/kg bw/d (~15%) compared to the Control group. At 500 mg/kg bw/d, lower food consumption was observed in males only during days 11-14 of treatment. This was attributable to the lower intake recorded in male no. 6. Food consumption in females from treated groups were lower than Control group in some periods but not presented a dose level dependency pattern.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
At 1000 and 700 mg/kg bw/d, higher leucocytes values were recorded in both sexes with respect to the Control group, as well as males at 500 mg/kg bw/d. Statistically significant differences were observed in some of the parameters of the differential leukocyte count. No other relevant differences were recorded.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Lower albumin and protein total values were observed in males and females treated with the test material. Furthermore lower Alanine aminotransferase (ALT) and Creatinine kinase values were recorded in some of the groups. Statistically significant differences were recorded in some of the parameters mainly in the high dose group. Other differences could be not considered relevant in the absence of a dose-response relationship
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Higher adrenal weights relative to brain were observed at all dose levels in both sexes. These differences were not statistically significant compared to Controls.
In females, a dose-dependent increase in liver weights at 500, 700 and 1000 mg/kg bw/d and a decrease in pituitary weights were observed (absolute and relative) at 700 and 1000 mg/kg bw/d. Liver values were statistically significant at 700 and 1000 mg/kg bw/d. Few additional statistically significant differences were recorded in some organs with respect to the Control group: lower weights of thymus and uterus in female treatment groups could be related to the high organ weight recorded in female no. 16. Furthermore male no. 6 treated at 700 mg/kg bw/d and male no. 10 treated at 1000 mg/kg bw/d had low thymus weights correlated with the gross macroscopic findings.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
At 1000 mg/kg bw/d dark areas and adhesions were observed on the stomach of the survived male no. 10, as well as pale spleen and small thymus. All the females had thickened gastric mucosa. Moreover, one of them had pale gastric mucosa and another one brown foci and serosa mucosa adhered to the diaphragm. Males found dead, no 11 and 12 showed autolysis and reddish organs at necropsy respectively. At 700 mg/kg bw/d thickened gastric mucosa was observed in two males (no. 7 and 9) and one female (no. 22). This finding was accompanied in the male no. 9 by pale gastric mucosa and in female no. 22 by reddish gastric mucosa. Animal no. 8 that was found dead on day 6 of treatment showed reddish organs at necropsy. At 500 mg/kg bw/d male no. 5 had stomach with dark areas and male no. 6 had dilation of small and gross intestine and small thymus. Female no. 21 had thickened stomach and reddish-dark thymus. No noteworthy findings were recorded in the Controls.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Details on results:
The oral administration of the test item to Wistar Hannover rats once a day for 14 days at the doses of 500, 700 and 1000 mg/kg bw/d and a volume of 4 mL/kg resulted in mortality in one male and two males at 700 and 1000 mg/kg bw/d, respectively. A dose-related effect was recorded at 1000 and 700 mg/kg bw/d. Relevant clinical signs such as labored breath, arched back and piloerection recorded in both sexes as well as a decrease in food consumption. Mean body weight decreased was recorded in males treated at 1000 mg/kg bw/d and lower increase was observed at 700 mg/kg bw/d. No changes were recorded in body weight infemales. Some hematological and biochemical changes were recorded, such as higher leukocytes values and lower albumin values in both sexes. Moreover, increases in liver weights (in females) and adrenals (in both sexes) were recorded. Treatment-related macroscopic alterations were observed in the stomach and consisted of thickened wall and reddish foci/dark areas in mucosa in both sexes. Treatment at 500 mg/kg bw/d caused similar clinical signs recorded at the other doses. No relevant differences were recorded in food consumption, body weight or clinical pathology. At necropsy, thickened gastric mucosa with reddish foci and an increase in adrenal weight was observed in both sexes and an increase in liver weight was observed in females.
Key result
Dose descriptor:
other:
Remarks:
Maximum concentration to which longer term studies should be dosed.
Effect level:
< 500 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
haematology
clinical biochemistry
organ weights and organ / body weight ratios
gross pathology
Remarks on result:
not measured/tested
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
500 mg/kg bw/day (nominal)
System:
gastrointestinal tract
Organ:
stomach
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
no
Conclusions:
The overall conclusion of the study was that the highest dose levels for longer term oral toxicity studies should not exceed 500 mg/kg bw/d.
Executive summary:

A 14-day repeated dose toxicity study was conducted with Wistar Hannover rats as part of a range-finder study for a longer term study. The rats were dosed by oral gavage at doses of 500, 700 and 1000 mg/kg bw/d for 14 days. The animals were assessed for mortality, clinical signs, body weight gain, food consumption, haematology parameters, clinical biochemistry and were subject to gross pathology. Organs were also weighed at neropsy. Mortality was observed in one male at 700 mg/kg bw/d and two males at 1000 mg/kg bw/d. Relevant clinical signs such as laboured breathing, arched back and piloerection recorded in both sexes at 1000 and 700 mg/kg bw/d as well as a decrease in food consumption. Mean body weight loss was recorded in males treated at 1000 mg/kg bw/d and lower weight gain was observed at 700 mg/kg bw/d. No changes were recorded in body weight in females. Some haematological and biochemical changes were recorded, such as higher leukocyte values and lower albumin values in both sexes. Moreover, increases in liver weights (in females) and adrenals (in both sexes) were recorded. Test-item-related macroscopic alterations were observed in the stomach and consisted of thickened wall and reddish foci/dark areas in mucosa in both sexes. Treatment at 500 mg/kg bw/d caused similar clinical signs recorded at the other doses. No relevant differences were recorded in food consumption, body weight or clinical pathology. At necropsy, thickened gastric mucosa with reddish foci and an increase in adrenal weight was observed in both sexes and an increase in liver weight was observed in females. The overall conclusion of the study was that the highest dose levels of test article for longer term oral toxicity studies should not exceed 500 mg/kg bw/d.

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:
reference to same study
Qualifier:
according to
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. certificate)
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 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 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
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. 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.
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
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.
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
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

Clinical signs

 

Males

Females

Dose (mg/kg bw/d)

0

25

100

250/400

0

25

100

250/400

Mortalitya

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 breathingbor 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

 

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.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
100 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
A modern, GLP-compliant screening study (OECD 422) is available for the submission substance and is supported by a 14 -day dose finding toxicity study.
System:
gastrointestinal tract
Organ:
stomach

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Mode of Action Analysis / Human Relevance Framework

The OECD 422 screening study reports excessive toxicity (including mortality) at the initial high dose level of 400 mg/kg bw/d. Following reduction of the high dose level to 250 mg/kg bw/d, signs of toxicity were limited to post-dose salivation consistent with the gavage administration of an irritant or unpalatable material. There was no evidence of systemic toxicity in this study; the results of the 14-day range-finding study show slightly elevated liver weight at high dose levels. In the screening study, treatment-related pathological findings were limited to the gastrointestinal tract and are consistent with local irritation. Findings are not of clear relevance to the human risk assessment; however a conservative NOAEL of 100 mg/kg bw/d is proposed for this study.

Additional information

Range-finding study

A 14-day repeated dose toxicity study was conducted with Wistar Hannover rats as part of a range-finder study for a longer term study. The rats were dosed by oral gavage at doses of 500, 700 and 1000 mg/kg bw/d for 14 days. The animals were assessed for mortality, clinical signs, body weight gain, food consumption, haematology parameters, clinical biochemistry and were subject to gross pathology. Organs were also weighed at neropsy. Mortality was observed in one male at 700 mg/kg bw/d and two males at 1000 mg/kg bw/d. Relevant clinical signs such as laboured breathing, arched back and piloerection recorded in both sexes at 1000 and 700 mg/kg bw/d as well as a decrease in food consumption. Mean body weight loss was recorded in males treated at 1000 mg/kg bw/d and lower weight gain was observed at 700 mg/kg bw/d. No changes were recorded in body weight in females. Some haematological and biochemical changes were recorded, such as higher leukocyte values and lower albumin values in both sexes. Moreover, increases in liver weights (in females) and adrenals (in both sexes) were recorded. Test-item-related macroscopic alterations were observed in the stomach and consisted of thickened wall and reddish foci/dark areas in mucosa in both sexes. Treatment at 500 mg/kg bw/d caused similar clinical signs recorded at the other doses. No relevant differences were recorded in food consumption, body weight or clinical pathology. At necropsy, thickened gastric mucosa with reddish foci and an increase in adrenal weight was observed in both sexes and an increase in liver weight was observed in females. The overall conclusion of the study was that the highest dose levels of test article for longer term oral toxicity studies should not exceed 500 mg/kg bw/d.

Screening study

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.

Justification for classification or non-classification

No relevant toxicity was observed in the available studies at dose levels that would trigger classification for STOT-RE under the CLP Regulation. Findings in the critical study are limited to local effects on the gastrointestinal tract consistent with the gavage administration of a corrosive substance.