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Key value for chemical safety assessment

Effects on fertility

Description of key information

Key study, BASF, 2019. Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test (OECD 422). Wistar rat, oral: drinking water: 0, 200, 1000, 5000 ppm.

Key study, BASF 2008. Pre- and Postnatal Developmental Toxicity Screening test (Modified Developmental Toxicity Screening test; according to OECD 414; similar to OECD 421 postnatal part). Wistar rat, oral: gavage: 0, 300, 600 mg/kg bw/day

Supp. study, Leung et al., 1996. Developmental Toxicity Study in Fischer 344 Rats by Whole-body Exposure to N,N-Dimethylethanolamine Vapor. Journal of Applied Toxicology. Vol. 16(6), 533-538; Fischer 344 Rats, Inhalation, 0, 10, 30 and 100 ppm nominal.

Link to relevant study records

Referenceopen allclose all

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 Feb 2018 (study initiation) To:
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
29 Jul 2016
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
12. to 14.09.2016
Limit test:
no
Justification for study design:
SPECIFICATION OF STUDY DESIGN FOR EXTENDED ONE-GENERATION REPRODUCTION TOXICITY STUDY WITH JUSTIFICATIONS:

- Premating exposure duration for parental (P0) animals : 13 days
- Basis for dose level selection : selected by request of the sponsor
- Route of administration : via drinking water
- Other considerations: vehicle and number of animals. The test guideline requires the rat to be used as the animal species. This rat strain was selected since extensive historical control data are available for Wistar rats.
Species:
rat
Strain:
Wistar
Remarks:
Crl:WI(Han)
Details on species / strain selection:
The test guideline requires the rat to be used as the animal species. This rat strain was selected since extensive historical control data are available for Wistar rats.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH,
- Females nulliparous and non-pregnant: yes
- Age at study initiation: (P) 11-12 wks (male), 10 wks (female)
- Weight at study initiation: (P) Males: 364.8 ± 14.9 to 366.8 ± 15.0 g; Females: 222.4 ± 9.4 to 225.8 ± 10.1 g;
- Housing: During pretreatment of the study period, the rats were housed together (up to 5 animals per sex and cage) in Polysulfonate cages Typ 2000P (H-Temp) supplied by TECHNIPLAST, Hohenpeißenberg, Germany. During the study period, the rats were housed individually in Polycarbonate cages type III supplied by TECHNIPLAST, Hohenpeißenberg, Germany and Becker & Co., Castrop-Rauxel, Germany, with the following exceptions: During overnight mating, male and female mating partners were housed together in Polycarbonate cages type III and pregnant animals and their litters were housed together until PND 13 in Polycarbonate cages type III. Pregnant females were provided with nesting material (cellulose wadding) toward the end of gestation. For enrichment wooden gnawing blocks (Typ Lignocel® block large, J. Rettenmaier & Söhne GmbH + Co KG, Rosenberg, Germany) were added. In addition in Polysulfonate cages large play tunnels (Art. 14153; supplied by PLEXX B.V., Elst, Netherlands) were added. The cages with the test animals were arranged on the racks in such a way that uniform experimental conditions (ventilation and light) were ensured.
- Diet (e.g. ad libitum): The food used was ground Kliba maintenance diet mouse/rat “GLP” meal, supplied by Provimi Kliba SA (new name Garanovit AG), Kaiseraugst, Switzerland, which was available to the animals ad libitum throughout the study (from the day of supply to the day before necropsy).
- Water (e.g. ad libitum): Drinking water was supplied from water bottles (ad libitum).
- Acclimation period: 3 wks
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 45-65%
- Air changes (per hr): The air change rate was 15 times per hour
- Photoperiod (hrs dark / hrs light): 12 hours light from 6.00 h to 18.00 h and 12 hours darkness from 18.00 h to 6.00 h
IN-LIFE DATES: From: 19 Feb 2018 (study initiation) To: 12 Apr 2018 (Male parental animals) and 09 May 2018 (Female parental animals)
Route of administration:
oral: drinking water
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test substance solutions in drinking water were prepared in intervals, which took into account the analytical results of the stability verification. For the preparation of the administration solutions the test substance was weighed in a calibrated beaker depending on the dose group, topped up with deionized water, were then titrated with aqueous HCl until ph7 was reached and intensely mixed with a magnetic stirrer until it was completely homogeneous and dissolved. For supply of the animals with drinking water and aqueous N,N-Dimethylaminoethanol solutions polycarbonate drinking bottles supplied by Bioscape EBECO GmbH., Castrop- Rauxel, FRG, with the capacity of 300 mL were used. The bottles had a stainless steel cap with a silicone sealing ring and a hole of diameter 0.6 mm, and each was placed in a recess in the cage lid. The animals obtained water or aqueous N,N-Dimethylaminoethanol solutions by licking the drops from the hole in the cap. Their special design substantially prevented the drinking bottles from uncontrolled emptying or leakage.

VEHICLE
- Purity: The drinking water is regularly assayed for chemical contaminants as well as for the presence of (pathogenic) microorganisms by the municipal authorities of Frankenthal and the Environmental Analytics Water/Steam Monitoring of BASF SE.
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: In general, each of the male and female animals was mated overnight in a 1:1 ratio for a maximum of 2 weeks.
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged: individually
- Any other deviations from standard protocol: Deviations from the specified times were possible on weekends and public holidays and were reported in the raw data.
Analytical verification of doses or concentrations:
yes
Remarks:
HPLC-MS
Details on analytical verification of doses or concentrations:
Based on the analytical results it is concluded that N,N-Dimethylaminoethanol is stable in deionized water adjusted to pH = 7 with hydrochloric acid over a period of 10 days at room temperature. All determined concentrations were in the range of 90 % - 110 % of the nominal concentration.
Duration of treatment / exposure:
2 weeks before mating and male and female animals were sacrificed 31 and 58 days, respectively, after the beginning of the administration
Frequency of treatment:
continuously
Details on study schedule:
After the acclimatization period, the test substance was administered to the parental animals as addition to the drinking water continuously throughout the entire study. The animals of the control group were treated in the same way, with the vehicle (drinking water only). Males and females from the same dose group were mated, after two weeks of treatment, overnight at a ratio of 1:1.
The females were allowed to deliver and rear their pups until PND 4 (standardization) or PND 13. On study day 27, a functional observational battery and motor activity measurement were carried out in five male animals per group. On study day 55, a functional observational battery and motor activity measurement were carried out in five female animals (with litter) per group. The male and female animals were sacrificed 31 and 58 days, respectively, after the beginning of the administration, and examined.
Dose / conc.:
200 ppm (nominal)
Remarks:
During the lactation period test substance concentrations in the drinking water of the F0 females were reduced to 50% (= 100 ppm) due to increased water consumption during this period
Dose / conc.:
1 000 ppm (nominal)
Remarks:
During the lactation period test substance concentrations in the drinking water of the F0 females were reduced to 50% (= 500 ppm) due to increased water consumption during this period
Dose / conc.:
5 000 ppm (nominal)
Remarks:
During the lactation period test substance concentrations in the drinking water of the F0 females were reduced to 50% (2500 ppm) due to increased water consumption during this period
No. of animals per sex per dose:
10/sex/ dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: on sponsors request
- Fasting period before blood sampling for clinical biochemistry: not specified
Positive control:
none
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule:
- Cage side observations checked were included: see "any other information on materials & methods"

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once prior to the first administration (day 0) and at weekly intervals during the administration period

BODY WEIGHT: Yes
- Time schedule for examinations: once a week at the same time of the day (in the morning)

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: daily

OTHER: For detailed information on: Open field observations, Functional observation battery, Sensory motor tests/Reflexes, Motor activity measurement, Estrous cycle determinations, Male reproduction data, Female reproduction and delivery data, please refer to "any other information on material & methods"
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in [F1] offspring:
Pup number and status at delivery, Pup viability/mortality, Sex ratio, Pup clinical observations, Pup body weight data, Anogenital distance, Anogenital index, Nipple/areola anlagen, Pup necropsy observations, Choline Determination, Thyroid hormones,

GROSS EXAMINATION OF DEAD PUPS:
yes, all stillborn pups and all pups that died before weaning were examined externally, eviscerated and their organs were assessed macroscopically. All pups without notable findings or abnormalities were discarded after their macroscopic evaluation. Animals with notable findings or abnormalities were evaluated on a case-by-case basis, depending on the type of finding noted.
Postmortem examinations (parental animals):
SACRIFICE
- Maternal animals: All surviving animals. The male and female animals were sacrificed 31 and 58 days, respectively, after the beginning of the administration, and examined.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical and thoracic viscera

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in "any other information on material & methods" were prepared for microscopic examination and weighed, respectively.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring were sacrificed at 13 days of age.
On PND 13, one selected male and one female pup per litter was sacrificed under isoflurane anesthesia by decapitation. Blood was sampled for determination of thyroid hormone concentrations. Thyroid glands/parathyroid glands were fixed in neutral buffered 4% formaldehyde solution and were transferred to the Pathology Laboratory for possible further processing. After sacrifice, all pups were examined externally and eviscerated, and their organs were assessed macroscopically. All stillborn pups and all pups that died before weaning were examined externally, eviscerated and their organs were assessed macroscopically. All pups without notable findings or abnormalities were discarded after their macroscopic evaluation. Animals with notable findings or abnormalities were evaluated on a case-by-case basis, depending on the type of finding noted.
Statistics:
DUNNETT-test (two-sided): Water consumption, food consumption (parental animals), body weight and body weight change (parental animals and pups; for the pup weights, the litter means were used), gestation days, anogenital distance, anogenital index
FISHER'S EXACT test (one-sided): Male and female mating indices, male and female fertility indices, females mated, females delivering, gestation index (females with liveborn pups), females with stillborn pups, females with all stillborn pups
WILCOXON test (one-sided+) with BONFERRONI-HOLM: Mating days until day 0 pc, %postimplantation loss, pups stillborn, %perinatal loss, nipple development
WILCOXON test (one-sided-) with BONFERRONI-HOLM: Implantation sites, pups delivered, pups liveborn, live pups day x, viability Index, survival index
WILCOXON test (two-sided): % live male day x, %live female day x
KRUSKAL-WALLIS test (two-sided): Number of cycles and Cycle Length, rearing, grip strength of forelimbs and hindlimbs, landing foot-splay test, motor activity
Reproductive indices:
Female mating index (%), Female fertility index (%), Gestation index (%), Live birth index (%), Postimplantation loss (%), Male fertility index (%), Male mating index (%)
Offspring viability indices:
Male mating index (%), Postimplantation loss (%)
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
No clinical signs or changes of general behavior, which may be attributed to the test substance, were detected in any of the male F0 parental animals in any of the groups and in any of the female F0 parental animals of test groups 1 - 2 during the entire study period. All clinical signs of the high-dose dams were only observed during lactation. Five out of nine high-dose females (Nos. 131, 133, 136, 138 and 139) showed piloerection. Four high-dose females (Nos. 131, 134, 136 and 138) showed tonic-clonic convulsions (grade: slight to severe) during several parts of the lactation period. During detailed clinical observation (DCO, see below), three high-dose females (Nos. 134, 135 and 137) showed tonic-clonic convulsions (grade: slight to moderate) on study days 42 and 56. In the open field observation (FOB, see below), high-dose females Nos. 134 and 135 showed slight tremors and slight to severe convulsions. The above-mentioned findings were assessed as treatment-related and adverse.
Mortality:
mortality observed, treatment-related
Description (incidence):
There were no test substance-related mortalities in test groups 1-2 in females and all test groups in males. On high-dose female (No. 131 - 5000 ppm) was found dead on PND 17. The female showed piloerection and moderate tonic-clonic convulsions beforehand on PND 7. A relation to treatment cannot be excluded.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Mean body weights of all males and females of test group 1-2 and body weight change of all male parental animals in all test substance-treated groups were comparable to the concurrent control values during the entire study period. A slight decrease of body weight development in high-dose females was apparent during gestation as the body weight change was statistically significantly below the concurrent control values (GD 0- 20: about 15%). It was assessed as treatment-related and adverse. The body weight change of the low- and mid-dose females was comparable to the concurrent control values during the entire study.
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
In the high-dose F0 males (5000 ppm), mean water consumption was statistically significantly below the concurrent control values during premating days 3 - 7 and 10 - 13 (about 24% and 23%, respectively). During mating, water consumption of the high-dose males was decreased without statistical significance (up to 25% below control). In the high-dose F0 females, water consumption was statistically significantly below the concurrent control values during premating days 3 – 13 (up to 32%), during several parts of the gestation period (up to 28%) and during lactation (PND 4 – 5: up to 24% below control, without statistical significance: up to 30%). The decrease in water consumption in high-dose males and females was most probably due to the bad taste/smell of the test substance related to the administration of the test substance in the drinking water. It was assessed as treatment-related.
Water consumption of the mid- and low-dose males and females was comparable to the concurrent control values during the entire study.
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related changes among hematological parameters were observed. At the end of the administration period, in males of test group 2 (1000 ppm) absolute reticulocyte counts were significantly decreased, but the change was not dose-dependent. Therefore, this alteration was regarded as incidental and not treatment-related.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
No treatment-related changes among clinical chemistry parameters were observed. At day 50, in dams of test group 3 (5000 ppm) triglyceride values were significantly decreased. The mean was below the historical control range (dams, triglycerides 1.19-3.20 mmol/L), but this was an isolated changed parameter among these individuals and, therefore, this alteration was regarded as treatment-related but non-adverse.
Urinalysis findings:
not specified
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
No changes of general behavior, which may be attributed to the test substance, were detected in any of the male F0 parental animals in any of the groups and in any of the female F0 parental animals of test groups 1 - 2 during the entire study period.
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Adverse, neurological deficits may be explained by the findings observed in pathology: the brain, cervical and thoracic spinal cord were the target organs in female animals. Axonal degeneration in the cerebellum, medulla oblongata, cervical and thoracic spinal cord were observed. This was regarded to be treatment-related and adverse.
Other effects:
no effects observed
Description (incidence and severity):
In parental males (test groups 1, 2 and 3; 200, 1000 and 5000 ppm) no treatment-related alterations of T4 and TSH levels were observed.
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
Estrous cycle data, generated during the last 2 weeks prior to mating for the F1 litter, revealed regular cycles in the females of all test groups 0 - 3. The mean estrous cycle duration was similar: 4.0 / 4.1 / 4.0 and 4.0 days in test groups 0-3, respectively.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
The stages of spermatogenesis in the testes of males of the high dose test group were comparable to those of the controls. In high dose females the different stages of functional bodies in the ovaries were present and comparable to the control animals.
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
The male animal No. 21 revealed severe diffuse degeneration of seminiferous tubules in the testes resulting in aspermia in the epididymides which was regarded to be the cause of the missing offspring. The female animal No. 122 showed a neutrophilic inflammation in the uterus which was regarded to be the reason for not becoming pregnant. The female animals (Nos. 109, 121, 123 and 137), which were not pregnant as well as the male mating partners (Nos. 9, 22, 23 and 37) did not show relevant histopathological findings.
Thus, the male fertility index ranged between 70.0 % and 100 % without showing any relation to dosing. This reflects the normal range of biological variation inherent in the strain of rats used for this study. Also the fertility index varied between 77.8 % and 100 % without showing any relation to the dose level. This reflects the normal range of biological variation inherent in the strain of rats used for this study.
Regarding fertility and reproductive performance, no adverse signs of toxicity were observed in male or female parental animals of test groups 1-3 (200, 1000 and 5000 ppm) during the entire study.
Almost all F0 parental animals proved to be fertile. Mating behavior, conception, implantation and gestation were not influenced.
Dose descriptor:
NOAEL
Effect level:
1 000 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
water consumption and compound intake
Remarks on result:
not determinable due to absence of adverse toxic effects
Dose descriptor:
NOAEL
Effect level:
5 000 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive performance
Dose descriptor:
LOAEL
Effect level:
200 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
organ weights and organ / body weight ratios
Dose descriptor:
LOAEL
Effect level:
5 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
water consumption and compound intake
Clinical signs:
not specified
Description (incidence and severity):
There were no test substance-related adverse clinical signs observed in any of the F1 generation pups of the different test groups.
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
Viability/mortality was adversely affected by the test substance at the highest test group 3 (5000 ppm). This was based on an increased postimplantation loss (25.5 % vs 0.9 % in control) leading to a decrease in mean number of F1 pups delivered per dam. A higher rate of stillborn pups (n=25, 28.7 % vs. 7.3 % in control) led to a decreased rate of liveborn pups (71.3 % vs. 92.7 % in control). Furthermore, postnatal death was indicated by higher numbers of found dead (16.1 % vs 2.7 % in control) and cannibalized high-dose pups (n=5) leading to a viability index of 69.6 % from PND 0-4.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The mean body weight (PND 1) and body weight change (PND 4 - 7) of the high-dose male pups were statistically significantly below to the concurrent control values (about 13 % and 18 %, respectively). The value of the high-dose pup weights (6.3 g) was slightly below the historical control range (Supplement, HCD, pup weights, day 1, males, range: 6.4 – 7.3 g). In females and in both sexes combined, body weights of test group 3 were also decreased (-8.7 and -12 %, respectively, without statistical significance) compared to control after birth (PND 1).
Towards PND 13, high-dose F1 males showed a slight recovery in body weight (-10% below control). The mean body weight of the high-dose F1 females recovered to values comparable to control. The decrease in pup body weights of test group 3 was assessed as treatment-related and adverse.
The mean body weights and body weight change of all low- and mid-dose male and female pups were comparable to the concurrent control values throughout the entire study. One male runt was seen in control, two male runts were seen in test group 2 and four male and two female runts were seen in test group 3. The higher number of runts in test group 3 was assessed as treatment-related and adverse.
Anogenital distance (AGD):
no effects observed
Description (incidence and severity):
The anogenital distance and anogenital index of all test substance treated male pups was comparable to the concurrent control values. The anogenital index of the high-dose female pups was statistically significantly above the concurrent control values (0.87 vs. 0.80 in control). However, the value was within the historical control range (AG index, range 0.72 – 0.87) and the parameter AG distance was not affected. Therefore, it was not assessed as treatment-related, adverse finding. The anogenital distance of all test substance treated female pups and anogenital index of all female pups of test groups 1 - 2 was comparable to the concurrent control values.
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
The apparent number and percentage of male pups having areolae was not influenced by the test substance when examined on PND 13.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
A few pups showed spontaneous findings at gross necropsy, such as discolored testis (red), dilated renal pelvis, post mortem autolysis and discolored thymus (red). These findings occurred without any relation to dosing and/or can be found in the historical control data at comparable or even higher incidences. Thus, all these findings were not considered to be associated to the test substance.
Regarding developmental toxicity, pup number, status at delivery, viability/mortality and pup body weight was adversely affected by the test substance at the highest test group 3 (5000°ppm). This was based on an increased postimplantation loss (25.5% vs 0.9% in control) leading to a decrease in mean number of F1 pups delivered per dam. A higher rate of stillborn pups (n=25, 28.7% vs. 7.3% in control) led to a decreased rate of liveborn pups (71.3% vs. 92.7% in control). Furthermore, postnatal death was indicated by higher numbers of found dead (16.1% vs 2.7% in control) and cannibalized high-dose pups (n=5) leading to a viability index of 69.6% from PND 0-4. Consequently, the effect on sex ratio/distribution at birth (females: 31.2%, males: 68.8%) and on PND13 (females: 23.9%, males 76.1%) was assessed as secondary. A decrease in F1 high-dose pup body weight was observed after birth (PND1) which recovered partially towards PND13.
Dose descriptor:
LOAEL
Generation:
F1
Effect level:
>= 5 000 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
mortality
body weight and weight gain
other: developmental toxicity
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
2 500 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
mortality
body weight and weight gain
other: developmental toxicity
Reproductive effects observed:
no
Lowest effective dose / conc.:
5 000 ppm (nominal)
Treatment related:
no

Female fetility:

The gestation index was 100% in control and test groups 1 - 2 and 88.9% in test group 3. The value of 88.9% is within the historical control range (gestation index: 87.5 – 100 %) and is, therefore, not assessed as treatment-related. Implantation was not affected by the treatment since the mean number of implantation sites was comparable between all test substance-treated groups and the control, taking normal biological variation into account (12.3 / 13.4 / 13.1 and 13.0 implants/dam in test groups 0 - 3, respectively). The postimplantation loss was 0.9% / 4.4% / 3.2% and 25.5%** (**:p0.01) in test groups 0-3, respectively. The mean value of test group 3 was outside the historical control range (postimplantation loss, range of 0.0 – 18.12 %). The finding was assessed as treatment-related and adverse. The mean number of F1 pups delivered per dam was 12.2 / 12.8 / 12.7 and 9.7* (*:p0.05) pups/dam in test groups 0-3, respectively. The value of test group 3 was within the historical control range (pups delivered, mean: 9.3 – 13.9). The low number of delivered pups is caused by the high post-implantation loss in the high-dose dams and is, therefore, assessed as treatment-related and adverse. The number of females with stillborn pups was increased in test group 3 (5 vs. 1 in control). The value was outside the historical control range (females with stillborn pups, range of 0-3). Furthermore, the percentage of stillborn pups was 7.3%, 0.8%, 1.1%, 28.7% in test groups 0-3, respectively. The value of test group 3 was outside the historical control range (pups stillborn, 0.0 - 8.3%). Consequently, the rate of liveborn pups indicated by live birth indices was 92.7% / 99.2% / 98.9% and 71.3%** (**:p0.01) in test groups 0 -3, respectively. The value of test group 3 was outside the historical control range (pups liveborn, range of 91.7 – 100 %). The changes in all three parameters were assessed as treatment-related and adverse. One high-dose female (No. 138) had 6 pups and all pups were stillborn. One further high-dose female (No. 139) had a complete litter loss on PND 2 (9 stillborns, 1 found dead, 2 cannibalized). Since higher numbers of stillborn pups and postnatal death occurred in the highdose group, the findings in the two high-dose dams were assessed as treatment-related and adverse.

Regarding developmental toxicity, pup number, status at delivery, viability/mortality and pup body weight was adversely affected by the test substance at the highest test group 3 (5000°ppm). This was based on an increased postimplantation loss (25.5% vs 0.9% in control) leading to a decrease in mean number of F1 pups delivered per dam. A higher rate of stillborn pups (n=25, 28.7% vs. 7.3% in control) led to a decreased rate of liveborn pups (71.3% vs. 92.7% in control). Furthermore, postnatal death was indicated by higher numbers of found dead (16.1% vs 2.7% in control) and cannibalized high-dose pups (n=5) leading to a viability index of 69.6% from PND 0 -4. Consequently, the effect on sex ratio/distribution at birth (females: 31.2%, males: 68.8%) and on PND13 (females: 23.9%, males 76.1%) was assessed as secondary.

A decrease in F1 high-dose pup body weight was observed after birth (PND1) which recovered partially towards PND13.

Conclusions:
Under the conditions of the present OECD 422 combined repeated dose toxicity study with the reproductive/developmental screening test in Wistar rats N,N-Dimethylaminoethanol caused signs of systemic toxicity in females consisting of decreased food consumption and body weight, clinically apparent signs (mortality, tremors and convulsions) together with axonal degeneration in cerebellum, medulla oblongata, cervical and thoracic spinal cord at a concentration of 5000 ppm in drinking water. Males of the same concentration group showed no adverse findings. Thus, the no observed adverse effect level (NOAEL) for general systemic toxicity was 5000 ppm (approx. 257 mg/kg bw/d) for males and 1000 ppm (approx. 89 mg/kg bw/d) for female Wistar rats. The NOAEL for fertility and reproductive performance was 5000 ppm (approx. 257 and 355 mg/kg bw/d, respectively) for male and female rats, the highest concentration tested. The NOAEL for developmental toxicity in the offspring was 1000 ppm based on adverse effects on pup number, status at delivery, viability/mortality and pup body weight at 5000 ppm.
Executive summary:

In the OECD 422 study, the test compound N,N-Dimethylaminoethanol was administered to groups of 10 male and 10 female healthy young Wistar rats (F0 animals) as a solution to the drinking water in different concentrations, i.e. 0 ppm (test group 0), 200 ppm (test group 1), 1000 ppm (test group 2) and 5000 ppm (test group 3). The duration of treatment covered a 2-weeks premating period and mating in both sexes (mating pairs were from the same test group), 3 days postmating in males and approximately 4-weeks postmating in two females (for sperm negative females) as well as the entire gestation and approximately 3 weeks of lactation period in females up to the day of scheduled sacrifice of the animals.

After 2 weeks of premating treatment the F0 animals were mated to produce F1 generation pups. Mating pairs were from the same test group. Mating was discontinued as soon as sperm were detected in the vaginal smear. F0 animals were examined for their reproductive performance including determination of the number of implantation sites and the calculation of postimplantation loss for all F0 females. A detailed clinical observation (DCO) was performed in all animals before the start of the administration period and, as a rule, thereafter at weekly intervals. Water consumption of the F0 parents was determined twice a week. However, during gestation and lactation water consumption of the F0 females were determined on gestation days (GD) 0-1, 4-5, 7-8, 10-11, 14-15, 17-18 and 19-20 and on postnatal days (PND) 1-2, 4-5, 7-8, 10-11 and 12-13. Food consumption of the F0 parents was determined once a week during premating. In dams food consumption was determined for GD 0-7, 7-14, 14-20 and PND 1-4, 4-7, 7-10 and 10-13. Body weights of F0 parents were determined once a week, in males throughout the study and in females during premating. During gestation and lactation period, F0 females were weighed on GD 0, 4, 7, 10, 14, 17 and 20, on the day after parturition (PND 1) and on PND 4, 7, 10 and 13. Estrous cycle data were evaluated for F0 generation females over a two weeks period prior to mating until evidence of mating occurred. Moreover, the estrous stage of each female was determined on the day of scheduled sacrifice.

The pups were sexed and examined for macroscopically evident changes on PND 0. They were weighed on PND 1, 4, 7 and 13. Their viability was recorded. At necropsy on PND 13, all pups were sacrificed with CO2, under isoflurane anesthesia, and examined macroscopically for external and visceral findings. Anogenital distance (defined as the distance from the anus [center of the anal opening] to the base of the genital tubercle) measurements were conducted in a blind randomized fashion, using a measuring ocular on all live male and female pups on PND 1. All surviving pups were examined for the presence or absence of nipple/areola anlagen on PND 13. The number of nipple/areola anlagen were counted. Blood samples were taken from all surplus pups at PND 4 as well as one male and one female pup per litter at PND 13 by decapitation under isoflurane anesthesia for hormone measurement. Blood samples for choline determination were withdrawn from all parental males and females (with litter). Clinico-chemical and hematological examinations were performed in 5 animals per sex and group towards the end of the administration period. Blood samples from all dams at PND 14 and all males at termination were taken by puncturing the retrobulbar venous plexus under isoflurane anesthesia for hormone measurement. At the end of the administration period a functional observational battery was performed and motor activity was measured in 5 parental males and females per group. All F0 parental animals were sacrificed by decapitation, under isoflurane anesthesia, and were assessed by gross pathology. Weights of selected organs were recorded and a histopathological examination was performed.

Under the conditions of the present OECD 422 combined repeated dose toxicity study with the reproductive/developmental screening test in Wistar rats N,N-Dimethylaminoethanol caused signs of systemic toxicity in females consisting of decreased food consumption and body weight, clinically apparent signs (mortality, tremors and convulsions) together with axonal degeneration in cerebellum, medulla oblongata, cervical and thoracic spinal cord at a concentration of 5000 ppm in drinking water. Males of the same concentration group showed no adverse findings. Thus, the no observed adverse effect level (NOAEL) for general systemic toxicity was 5000 ppm (approx. 257 mg/kg bw/d) for males and 1000 ppm (approx. 89 mg/kg bw/d) for female Wistar rats. The NOAEL for fertility and reproductive performance was 5000 ppm (approx. 257 and 355 mg/kg bw/d, respectively) for male and female rats, the highest concentration tested. The NOAEL for developmental toxicity in the offspring was 1000 ppm based on adverse effects on pup number, status at delivery, viability/mortality and pup body weight at 5000 ppm.

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
27 MAY 2008 (Study plan) to Jun/July 2008 (end of experimental phase); 14 MAR 2019 (Report date of summary of results)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
Jul 1995
Deviations:
yes
Remarks:
Only females were dosed. No pairing/fertility part
Qualifier:
equivalent or similar to guideline
Guideline:
other: Postnatal part: EPA health Effects Test Guidelines, OPPTS 870.3550: Reproduction/Developmental Toxicity Screening Test
Version / remarks:
Jul 2000
Qualifier:
equivalent or similar to guideline
Guideline:
other: Prenatal part: Proposal for updating Guideline 414: Prenatal Developmental Toxicity Study (22 Jan 2001)
Principles of method if other than guideline:
The aim of this screening study was to obtain initial information on the effect of the test substance 2-dimethylaminoethanol after repeated oral administration (gavage) to pregnant female Wistar rats from gestation day (GD) 6 to GD 19 (prenatal study) and from GD 6 to postnatal day (PND) 3 (postnatal study). On GD 20 selected dams of each group (5 control animals, 5 low dose animals and 10 high dose animals) were sacrificed. For the postnatal study part, the remaining dams were allowed to litter and rear their pups until PND 4. On PND 4, all pups were sacrificed and examined grossly. This endpoint study record refers mainly to the postnatal study.
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
not specified
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Age at study initiation: about 10-15 weeks
- Housing: 1 animal / cage; from delivery to sacrifice (rearing) - 1 dam with its litter / cage, Makrolon cages type M III, Wooden gnawing blocks (Typ NGM E-022), Type Lignocel FS 14 fibres, dustfree bedding
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: During the acclimatization period, the animals were accustomed to the environmental conditions and to the diet.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24 °C
- Humidity (%): 30-70 %
- Air changes (per hr): 15 air changes per hour
- Photoperiod (hrs dark / hrs light):12 hours light (6:00 a.m. - 6:00 p.m.), 12 hours darkness (6:00 p.m. - 6:00 a.m.)


IN-LIFE DATES: From: 29 MAY TO: 23 JUN 2008 (PND 4/Sacrifice of the pups /Sacrifice of the female animals)
Route of administration:
oral: gavage
Vehicle:
olive oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
For the test substance preparation, the specific amount of test substance was weighed, topped up with olive oil in a volumetric flask and intensely shaken until it was completely dissolved

VEHICLE
- Amount of vehicle (if gavage): 4 mL/kg body weight
Details on mating procedure:
The animals paired by the breeder (time-mated animals) were supplied on the day of evidence of mating; this day is referred to as GD 0 and the following day as GD 1.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The stability of the test substance in olive oil over a period of up to 7 days at room temperature was verified analytically before the start of the study (Analytical report: 01Y0262/078001)
Duration of treatment / exposure:
From GD 6 through PND 3 (postnatal study part).
Frequency of treatment:
Once daily
Dose / conc.:
0 mg/kg bw/day
Remarks:
Group 0
Dose / conc.:
300 mg/kg bw/day
Remarks:
Group 1
Dose / conc.:
600 mg/kg bw/day
Remarks:
Group 2
No. of animals per sex per dose:
Control: 10 (5 in postnatal study)
300 mg/kg bw: 10 (5 in postnatal study)
600 mg/kg bw: 20 (10 in postnatal study)
Control animals:
yes
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- A check for moribund and dead animals was made twice daily from Monday to Friday and once daily on Saturday, Sunday and public holidays.
- A cageside examination was conducted at least once daily for any signs of morbidity, pertinent behavioral changes and signs of overt toxicity, further abnormalities, changes, littering and lactation behavior of the dams. Daily or twice a day check of littering and lactation behavior

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were recorded on GD 0 and on GD 1, 3, 6, 8,10, 13, 15, 17, 19, 20 and on PND 0 and PND 4.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption was recorded on GD 1, 3, 6, 8,10, 13, 15, 17, 19, 20 and on PND 4.
Oestrous cyclicity (parental animals):
no data
Sperm parameters (parental animals):
no data
Litter observations:
PARAMETERS EXAMINED
The following parameters were examined in [F1] offspring:
sex, liveborn or stillborn, number of all delivered pups, macroscopically evident changes, linical signs of toxicity, pup status/mortality, body weight.

GROSS EXAMINATION OF DEAD PUPS:
Stillborn pups, pups that die or are sacrificed in a moribund state, were eviscerated and examined for possible defects and/or the cause of death.

Postmortem examinations (parental animals):
SACRIFICE
- Maternal animals: All surviving animals on PND 4

GROSS NECROPSY
- Weight of liver
- Number of implantation sites
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring were sacrificed on PND 4.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

GROSS NECROPSY
- Pups were examined externally, eviscerated and their organs were assessed macroscopically.
Statistics:
Means and standard deviations will be calculated.
- DUNNETT test (two-sided): Food consumption, body weight and body weight change (parental females and pups); duration of gestation; number of delivered pups per litter
- FISHER'S exact test: Number of live and dead pups and different indices (e.g. viability index and lactation index) and number of litters with necropsy findings in pups
- WILCOXON test (one-sided): Proportion of pups with necropsy findings per litter
- KRUSKAL-WALLIS and WILCOXON test: Weight of liver
Reproductive indices:
Female fertility index, Live Birth index, Female Mating Index, Gestation Index
Offspring viability indices:
Viability Index
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
600 mg/kg bw/day:
- Respiratory sounds (7 out of 20 animals)
- Salivation after treatment (20 out of 20 animals)
- Postnatal study part (8 dams): Salivation after treatment (7 out of 7 animals)

300 mg/kg bw/day:
- Salivation after treatment (10 out of 10 animals)
- Postnatal study part (5 dams): Salivation after treatment (5 out of 5 animals)
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
600 mg/kg bw/day:
- One animal sacrificed moribund on GD 14 (gross pathological examination revealed stomach erosions and no feces in intestine).
- One animal found dead on GD 20 (gross pathological examination revealed stomach ulcerations).

300 mg/kg bw/day: no effects observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
600 mg/kg bw/day:
- Statistically significantly reduced mean body weight compared to the control group (set to 100 %), i.e. on GD 13 (93 %).
- Statistically significantly reduced mean body weight change compared to the control group (set to 100 %), i.e. between GD 8-10 (52 %).

300 mg/kg bw/day: no effects observed
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
600 mg/kg bw/day: Transient statistically significantly reduced mean food consumption compared to the control group (set to 100%), i.e. between GD 6-8 (81 %) and GD 8-10 (78 %).
- 600 mg/kg bw/day - Postnatal study part (8 dams): Statistically significantly reduced mean food consumption (81 % between PND 0-4) compared to the control group (set to 100 %).

300 mg/kg bw/day: no effects observed
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
Endocrine findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not specified
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
600 mg/kg body weight/day:
- One out of 8 animals did not deliver (7 animals left for further assessment)
- Live birth index of 91 % (control: 100 %)

300 mg/kg bw/day: no effects observed
Dose descriptor:
LOAEL
Remarks:
Local effects
Effect level:
300 mg/kg bw/day
Based on:
test mat.
Sex:
female
Basis for effect level:
gross pathology
Dose descriptor:
NOAEL
Remarks:
general toxicity
Effect level:
300 mg/kg bw/day
Based on:
test mat.
Sex:
female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
Dose descriptor:
LOAEL
Remarks:
Reproductive performance
Effect level:
600 mg/kg bw/day
Based on:
test mat.
Sex:
female
Basis for effect level:
reproductive performance
Clinical signs:
not specified
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
600 mg/kg body weight/day Postnatal study part (8 dams):
- 1/8 dams did not deliver (further assessment fo 7 litters)
- Six stillborn pups in 7 litters (64 pups in toto, 58 liveborn)
- Twenty-four pups out of 58 died ahead of schedule
- Nine pups out of 58 were cannibalized
- No more pups alive in 4 out of 7 litters (2 litters on PND 1, 1 on PND 2, 1 on PND 3)
- Viability index of 43 % (control: 100 %)

300 mg/kg body weight/day: Postnatal study part (5 dams): Pups: No test substance-related findings
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
600 mg/kg body weight/day - Postnatal study part (8 dams):
- Statistically significantly reduced mean body weight compared to the control group (set to 100 %), i.e. on PND 1 (76 %) & on PND 4 (71 %).
- Statistically significantly reduced mean body weight change (57 % between PND 1-4) compared to the control group (set to 100 %).

300 mg/kg body weight/day: Postnatal study part (5 dams): Pups: No test substance-related findings until
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Anogenital distance (AGD):
not examined
Nipple retention in male pups:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
600 mg/kg body weight/day - Postnatal study part (8 dams): Pups Post mortem autolysis (7/55), Empty stomach (4/55)
300 mg/kg body weight/day - Postnatal study part (5 dams): Pups: Hemorrhagic testis (1/48). Not considered as toxicologically relevant due to non-existent dose-response relationship and only single incidence.
Histopathological findings:
not examined
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Runts:
600 mg/kg body weight/day - Postnatal study part (8 dams):
- Twelve runts (no runt in the control)
300 mg/kg body weight/day: Postnatal study part (5 dams): Pups: No test substance-related findings

Post-implantation loss:
600 mg/kg bw/day - Postnatal study part (8 dams): Slightly increased post-implantation loss (9.9 % vs. 2.0% in control), not statistically significant.
300 mg/kg bw/day - Postnatal study part (8 dams): Slightly increased post-implantation loss (5.5 % vs. 2.0% in control), not statistically significant.
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
300 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
mortality
body weight and weight gain
Reproductive effects observed:
not specified

Results:

Gestation

Test group (mg/kg bw/d)

0 (0; control)

1 (300)

2 (300)

Mortality

-

-

1 animal sacrificed moribund on GD 14

1 animal found dead on GD 20

Clinical observation

NAD

Salivation after treatment (10/10)

Salivation after treatment (20/20)

Respiratory sounds (7/20)

Labored respiration (1/20)

Fur smeared with urine (1/20)

FC (day 6-8)

FC (day 8-10)

FC (day 0-6)

16.1 g

17.4 g

15.1 g

16.0 g (99 %)

16.9 g (97 %)

15.4 g (102 %)

13.1 g (81 %)**

13.5 g (78 %)

FC (day 6-20)

18.1 g

17.9 g (99 %)

16.7 g (92 %)

FC (day 0-20)

17.2 g

17.1 g (99 %)

16.4 g (95 %)

BW (day 0)

162.6 g

157.7 g (97 %)

160.5 g (99 %)

BW (day 6)

BW (day 13)

190.8 g

223.1 g

187.8 g (98 %)

215.3 g (96 %)

189.8 g (99 %)

208.5 g (93 %)*

BW (day 20)

281.4 g

268.5 g (95 %)

263.5 g (94 %)

BWC (day 8-10)

BWC (day 0-6)

11.8 g

28.2 g

8.7 g (74 %)

30.1 g (107 %)

6.2 g (52 %)**

29.3 g (104 %)

BWC (day 6-20)

90.5 g

80.7 g (89 %)

73.8 g (81 %)

BWC (day 0-20)

118.7 g

110.8 g (93 %)

103.3 g (87 %)

Duration of Gestation

21.8

22.0

22.3

Cesarean section

Test group

(mg/kg bw/d)

0 (0; control)

1 (300)

2 (600)

Uterus weight

52.8 g

43.5 g (82 %)

49.7 g (94 %)

Carcass weight

228.8 g

221.0 g (97 %)

216.1 g (94 %)

Corrected body weight gain

40.3 g

36.6 g (91 %)

28.7 g (71 %)

Implantation sites (mean/litter)

10.4

9.4

10.2

Postimplantation loss

5.2

15.2

11.8

Resorptions (mean/litter)

0.6

1.4

0.8

Live fetuses/dam

9.8

8.0

10.4

Placental weights

0.43 g

0.41 g (95 %)

0.39 g (91 %)

Fetal weights

3.5 g

3.7 g (104 %)

3.6 g (101 %)

Total external malformations

Fetuses:

Litter:

Affected fetuses/litter:

 

 

0/49 (0.0%)

0/5 (0.0%)

0.0%

 

 

0/40 (0.0 %)

 0/5 (0.0 %)

0.0 %

 

 

0/94 (0.0 %)

0/9 (0.0 %)

0.0 %

Total visceral malformations

Fetuses:

Litter:

Affected fetuses/litter:

 

 

0/24 (0.0%)

0/5 (0.0%)

0.0%

 

 

0/19 (0.0 %)

0/5 (0.0 %)

0.0 %

 

 

0/44 (0.0 %)

0/9 (0.0 %)

0.0 %

Total skeletal malformations

Fetuses:

Litter:

Affected fetuses/litter:

 

 

0/25 (0.0%)

0/5 (0.0%)

0.0 %

 

 

0/21 (0.0 %)

0/5 (0.0 %)

0.0 %

 

 

0/50 (0.0 %)

0/9 (0.0 %)

0.0 %

Lactation

Test group (mg/kg bw/d)

0 (0; control)

1 (300)

2 (600)

Clinical observation

NAD

Salivation after treatment (5/5)

Salivation after treatment (7/7)

Respiratory sounds (1/7)

No more pups alive (4/7)

FC (day 0-4)

24.8 g

25.4 g (103 %)

20.0 g (81 %)*

BW (day 0)

222.7 g

213.1 g (96 %)

218.3 g (98 %)

BW (day 4)

230.8 g

227.4 g (99 %)

219.4 g (95 %)

BWC (day 0-4)

8.2 g

14.3 g

1.1 g

Female fertility index

100%

100%

100

Implantation sites (mean/litter)

10.2

10.2

10.1

Postimplantation loss (mean/litter)

0.2

0.6

1.0

% Postimplantation loss (mean)

2.0

5.5

9.9

Females with live born

5

5

7

Females with stillborn

0

0

3

Pups delivered (mean/litter)

10.0

9.6

9.1

Live Birth Index

100 %

100 %

91 %*

Pups stillborn

0 (in 5 litters)

0 (in 5 litters)

6* (in 7 litters)

Pups died

0

0

24**

Pups cannibalized

0

0

9**

Viability Index

100 %

100 %

43 %**

Pups BW (day 1) (male+female)

6.8 g

6.7 g (99 %)

5.1 g (76 %)**

Pups BW (day 4) (male+ female)

10.4 g

10.4 g (100 %)

7.4 g (71 %)**

Pups BWC (day 1-4) (male+female)

3.6 g

3.7 g (101 %)

2.1 g (57 %)**

Runts

0

1

12

Pup Necropsy

NAD

Hemorrhagic testis (1/48)

Post mortem autolysis (7/55)

Empty stomach (4/55)

Test group (mg/kg bw/d)

0 (0; control)

1 (300)

2 (600)

Pathology (Dams died + sacrificed in moribund condition) Incidence of gross lesions

 

 

Stomach: Ulcerations (1/2)

Stomach: Erosions/filled with fluid and Intestines bloated/no feces (1/2)

Pathology (Dams sacrificed at schedule)

Incidence of gross lesions

NAD

Forestomach: Erosion/Ulcer (5/5)

Forestomach: Erosion/Ulcer (8/8)

Liver weights (absolute)

9.834 g

10.502 g (106 %)

10.259 g (104 %)

Liver weights (relative)

4.263 g

4.635 g (108 %)

4.703 g (110 %)

Conclusions:
In conclusion, signs of maternal toxicity occurred at 300 mg/kg bw/day, comprising local effects (strong ulcera in the stomach) and at 600 mg/kg bw/day, as evident by systemic effects. In the postnatal part of the study, in the dose group of 600 mg/kg bw/day the live birth index was reduced (91 %  vs. control 100 %). Pups of the high dose group were less viable compared to the control group (viability index 43 % and 100 %, respectively); and reduced body weights were recorded.
No substance related findings were observed in foetuses.

Based on these observations the following effect levels were determined:
Dams (F0):
- LOAEL for local effects: 300 mg/kg bw/day
- NOAEL for systemic effects: 300 mg/kg bw/day
- LOAEL for reproductive performance: 600 mg/kg bw/day
Offspring (F1)
- NOAEL for foetuses: not determinable; no effects observed
- NOAEL for pups: 300 mg/kg bw/day
Executive summary:

In the Pre- and Postnatal Developmental Toxicity Screening test, 2-Dimethylaminoethanol was administered via oral gavage to time-mated Wistar rats from GD 6 through GD 19 (prenatal study part) and from GD 6 through PND 3 (postnatal study part). The following concentrations were administered: i.e. 0 mg/kg bw/day (test group 0, 10 animals), 300 mg/kg bw/day (test group 1, 10 animals), 600 mg/kg bw/day (test group 2, 20 animals). The duration of treatment covered a 2-weeks in gestation and up to the 3rd day after parturition up to the day of scheduled sacrifice of the animals.

Animals dosed with the high dose of 600 mg/kg bw/day showed after treatment the following symptoms: salivation (20/20); respiratory sounds (7/20), statistically significantly reduced mean food consumption (i.e. between GD 6-8 (81 %) and GD 8-10 (78 %) compared to control group (100 %)), statistically significantly reduced mean body weight (i.e. on GD 13 (93 %) compared to the control group (100 %), statistically significantly reduced mean body weight change (i.e. between GD 8-10 (52 %) compared to the control group (100 %). Moreover, one animal was found dead on GD20 (gross pathological examination revealed stomach ulcerations) and one animal was sacrificed moribund on GD14 (gross pathological examination revealed stomach erosions and no feces in intestine).

For the prenatal study part, 10 dams were sacrificed on GD 20 and subsequently examined. In the dams the following observation were made: stomach erosions/ulcera (8 /10) and statistically significantly increased mean liver weight (118 %) compared to the control group (100 %). The following effects on reproduction performance were observed, but assessed statistically not significant and without a dose-response relationship: Slightly increased post-implantation loss (11.8 % vs. 5.2 % in control (compared to the control group and the historical control data)).

For the postnatal part, the remaining 8 animals were sacrificed on PND 4 and subsequently examined. In these dams the following observations were made: stomach erosions/ulcera (8/8), 1/8 dams did not deliver (7 animals left for further assessment), salivation after treatment (7/7), statistically significantly reduced mean food consumption (81 % between PND 0 – 4) compared to the control group (100 %) and a live birth index of 91 % compared to the control group (100 %). A slightly increased post-implantation loss (9.9 % vs. 2.0 % in control) was noted, but assessed as statistically not significant.

Animals dosed with the low dose of 300 mg/kg bw/day showed after treatment the following symptoms: salivation (10/10).

For the prenatal study part, 5 dams were sacrificed on GD 20 and subsequently examined, in 4/5 dams stomach erosions/ulcera was recorded. The following effects on reproduction performance were observed, but assessed statistically not significant and without a dose-response relationship: Slightly increased post-implantation loss (15.2 % vs. 5.2 % in control (compared to the control croup and the historical control data)) and slightly increased resorptions (mean/litter) (1.4 % vs. 0.6 % in control (compared to the control group and the historical control data)).

For the postnatal part, the remaining 5 animals were sacrificed on PND 4 and subsequently examined. In these dams the following local effects were observed: stomach erosions/ulcera (5/5). A slightly increased post-implantation loss (5.5 % vs. 2.0 % in control) was noted, but assessed as statistically not significant.

Concerning the effects and observations on foetuses of dams treated with 600 mg/kg bw/day, no test-substances-related findings were reported (prenatal part).

For the postnatal study part, pups derived of dams dosed with 600 mg/kg bw/day, the following is reported (as 1/8 dams did not deliver, only 7 litters left for further assessment): six stillborn pups in 7 litters (64 pups in toto, 58 liveborn), 24/58 pups died ahead of schedule, 9/58 pups were cannibalized, no more pups alive in 4 out of 7 litters (2 litters on PND 1, 1 on PND 2, 1 on PND 3), viability index of 43 % (control: 100 %), statistically significantly reduced mean body weight (i.e. on PND 1 (76 %) and on PND 4 (71 %) compared to the control group (100 %)), statistically significantly reduced mean body weight change ((57 % between PND 1-4) compared to the control group (100 %)) and 12 runts (no runts in the control).

In conclusion, signs of maternal toxicity occurred at 300 mg/kg bw/day, comprising local effects (strong ulcera in the stomach) and at 600 mg/kg bw/day, as evident by systemic effects. In the postnatal part of the study, in the dose group of 600 mg/kg bw/day the live birth index was reduced (91 %  vs. control 100 %). Pups of the high dose group were less viable compared to the control group (viability index 43 % and 100 %, respectively); and reduced body weights were recorded.

No substance related findings were observed in foetuses.

Based on these observations the following effect levels were determined:

Dams (F0):

- LOAEL for local effects: 300 mg/kg bw/day

- NOAEL for systemic effects: 300 mg/kg bw/day

- LOAEL for reproductive performance: 600 mg/kg bw/day

Offspring (F1)

- NOAEL for foetuses: not determinable; no effects observed

- NOAEL for pups: 300 mg/kg bw/day

Endpoint:
one-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
not reported, published 1996
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: no data on GLP or OECD compliance. Acceptable, well ducumented publication which meets basic scientific principles.
Reason / purpose for cross-reference:
reference to same study
Qualifier:
equivalent or similar to guideline
Guideline:
other: OECD 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
no
Limit test:
no
Species:
rat
Strain:
Fischer 344
Sex:
female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan Sprague-Dawley, Inc., Indianapolis, IN.
- Age at study initiation: 67-79 days old on arrival
- Weight at study initiation: not reported
- Fasting period before study: no
- Housing: in stainless-steel wire-meshcages
- Diet (e.g. ad libitum): ad libitum except during exposure
- Water (e.g. ad libitum): ad libitum except during exposure
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): not reported
- Humidity (%): not reported
- Air changes (per hr): not reported
- Photoperiod (hrs dark / hrs light):12/12
Route of administration:
inhalation: vapour
Type of inhalation exposure (if applicable):
whole body
Vehicle:
unchanged (no vehicle)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Liquid DMEA was metered from a piston pump into a heated glass maintained at the lowest temperature to vaporize the liquid. The resultant vapor was carried into the exposure chamber by a countercurrent flow of conditioned air through the evaporator. Exposure was conducted in 4320-litre stainless-steel and glass chambers at an airflow of 1000 L/min.
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: no data
- Proof of pregnancy: [vaginal plug ] referred to as [day 0] of pregnancy
- After successful mating each pregnant female was caged (how): singly
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Chamber atmosphere was analyzed for DMEA concentrations once every 32 min during each 6-h exposure, using a Perkin-Elmer 3920B gas chromatograph equipped with a flame ionization detector. Nominal concentrations weere calculated daily based on the amount of DMAE used and the chamber tube air flow during the exposure period.
Duration of treatment / exposure:
6 hours
Frequency of treatment:
on gestational days 6-15
Dose / conc.:
0 ppm (nominal)
Dose / conc.:
10 ppm (nominal)
Dose / conc.:
30 ppm (nominal)
Dose / conc.:
100 ppm (nominal)
No. of animals per sex per dose:
In a range-finding study, eight plug-positive females each were assigned to four DMEA-exposed groups (target DMEA concentrations 8, 25, 75 and 100 ppm) and an air-exposed control group.
In the definitive study, 25 plug-positive females each were assigned to three DMEA-exposed groups (10, 30 and 100 ppm) and a control group.
Control animals:
yes, sham-exposed
Details on study design:
- Dose selection rationale: Based on the results of the range-finding study.
The highest exposure concentration in the range-finding study, 100 ppm, was retained in the definitive study since it produced maternal toxicity (reduced body weights and weight gain, and clinical signs) and possible embryotoxicity (increased preimplantation loss) but no apparent fetotoxicity. The middle exposure concentration of 30 ppm chosen for the definitive study was slightly above the 25 ppm in the range-finding study which produced maternal toxicity (transient weight gain depression and clinical signs limited to the eyes) and possible embryotoxicity (reduced implantations, increased preimplantation loss and reduced number of viable fetuses per litter). The lowest exposure concentration, 10 ppm, was chosen as essentially the same as the 8 ppm in the range-finding study which produced no effects on maternal weights and only transient ocular changes and no evidence of embryofetal toxicity.
- Rationale for animal assignment (if not random): randomized
Positive control:
no
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: Maternal body weights were measured on gd 0, 6, 12, 15, 18 and 21.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 21
- Organs examined: The gravid uterus, ovaries (including corpora lutea), cervix, vagina and peritoneal and thoracic cavities were examined grossly. Ovarian corpora lutea of pregnancy were counted. Maternal liver and uterine weights were measured.
Oestrous cyclicity (parental animals):
no
Sperm parameters (parental animals):
no
Litter observations:
STANDARDISATION OF LITTERS
- Performed ongestation day 21
- If yes, maximum of [..all ] pups/litter

PARAMETERS EXAMINED
The following parameters were examined in [F1 ] offspring: [number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, other:]

GROSS EXAMINATION OF DEAD PUPS:
[no / yes, for external and internal abnormalities; possible cause of death was/was not determined for pups born or found dead.]
Postmortem examinations (parental animals):
SACRIFICE
- Maternal animals: All surviving animals [on gd 21]

GROSS NECROPSY
- Gross necropsy consisted of [external and internal examinations including the cervical, thoracic, and abdominal viscera.]

HISTOPATHOLOGY /
The tissues indicated in Table [#] were prepared for microscopic examination and weighed, respectively.

OTHER:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: Uteri were examined externally for signs of hemorrhage. All live and dead fetuses were recorded
Postmortem examinations (offspring):
- External examinations: Yes: [all per litter] including cleft palate
- Soft tissue examinations: Yes: [half per litter]
- Skeletal examinations: Yes: [half per litter]
- Head examinations: Yes: [half per litter]
Statistics:
The unit of comparison was the pregnant female or the litter. Continuous quantitative data were compared between the DMEA-exposed groups and air-exposed control group by the use of Levene's test for equal variances analysis of variance (ANOVA) and t-tests with Bonferroni probabilities. The t-tests were used when the F value from the ANOVA was significant. When Levene's test indicated homogeneous variances, and the ANOVA was significant, the pooled t-test was used. When Levene's test indicated heterogeneous variances, all groups were compared by an ANOVA for unequal variances," followed by the separate variance t-test when necessary. Non-parametric data obtained following laparohysterectomy were statistically treated using the Kruskal- Wallis test followed by the Mann-Whitney U test when appropriate. Incidence data were compared using Fisher's exact test. For all statistical tests, the fiducial limit of 0.05 (two-tailed) was used as the criterion for significance.
Reproductive indices:
listed in the tables 1 and 3 in "Remarks on results"
Offspring viability indices:
listed in the table 3 in "Remarks on results"
Clinical signs:
effects observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
not examined
Other effects:
not examined
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, treatment-related
Table 1 (in "Remarks on results") shows the pregnancy and litter data of all plug-positive females on study. There were no maternal deaths or abortions. Pregnancy rate ranged from 88 to 96% and all pregnant females had one or more live fetuses at scheduled sacrifice, except one dam at 100 ppm, which had a totally resorbed litter. Reduced body weight and reduced body weight gain were observed at 100 ppm (Table 2). Body weight reduced on gd 12 and 15 (during the exposure period) and on gd 15 and 21 (postexposure period). Body weight gain was reduced for all intervals except gd 6- 9 (pre-exposure) and gd 15-21 (post-exposure). There were no effects on body weight or body weight gain for the 10 or 30 ppm groups. Clinical examination showed that dams at 100 ppm only exhibited perinasal fur discoloration, presumably from chromodacryorrhea. At 30 and 100 ppm there were darkened (maroon), cloudy and hazy eyes, slight corneal vascularization and pupils dilated and fixed. Cloudy and hazy eyes were observed only during the exposure period. Darkened eyes were also observed at 10 ppm during the exposure period. There were no statistically significant differences in gravid uterine weight and absolute or relative liver weights between the DMEA-exposed groups and the controls.
Dose descriptor:
NOAEC
Effect level:
10 ppm (nominal)
Sex:
female
Basis for effect level:
other: ocular effects, reduced body weight. In dams, no effects related to treatment.
Clinical signs:
effects observed, treatment-related
Mortality / viability:
mortality observed, treatment-related
Body weight and weight changes:
no effects observed
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, treatment-related
Histopathological findings:
no effects observed
The present developmental toxicity evaluation revealed no treatment-related embryotoxicity at any exposure concentration employed. No consistent pattern of fetotoxicity was observed. Fetal body weights were elevated at 100 ppm relative to those in controls (Table 3), and only one skeletal district, the cervical centra, exhibited evidence of reduced ossification at 100 ppm (Table 4), an exposure concentration which also produced maternal toxicity. This finding is the only one which could be consistent with indicating possible minimal fetotoxicity; however, there were no other indications of fetotoxicity, such as reduced fetal body weight. No other skeletal districts, of the number identified as sensitive indicators of delayed development in rat fetuses,” exhibited a delay in ossification. No increases in malformations were observed at any concentration of DMEA employed, including those which produced maternal toxicity.
Reproductive effects observed:
not specified

Table 1. Pregnancy and litter data for Fischer 344 rats exposed whole body to N,N-dimethylethanolamine vapor

Exposure concentration (ppm)

0

10

30

100

Number in study

25

25

25

25

Number of early delivery

1

0

0

0

Number aborted

0

0

0

0

Number (%) pregnant at scheduled

22

23

22

24

sacrifice

(91.7)

(92.0)

(88.0)

(96.0)

Number of litters examined

22

23

22

23a

aOne dam carried a totally resorbed litter.

Table 2. Maternal body weights and weight gains in Fischer 344 rats exposed whole body to N,N-dimethyIethanoIamine vapor

Exposure conc. (ppm)

0

10

30

100

Number of dams

22

23

22

24

Maternal body weight3

 

 

 

 

gd 0

166.0 ± 8.1

165.0 ± 6.3

166.3 ± 7.6

166.6 ± 6.7

gd 6

178.1 ± 9.0

177.5 ± 5.9

178.8 ± 6.9

179.0 ± 7.4

gd 9

183.3 ± 9.9

181.8 ± 5.8

184.0 ± 6.7

179.8 ± 7.7

gd 12

193.1 ± 10.2

191.2 ± 6.0

193.4 ± 7.8

183.0 ± 8.2**

gd 15

203.3 ± 10.8

200.0 ± 7.2

203.6 ± 8.9

187.4 ± 8.7***

gd 18

223.7 ± 12.5

218.1 ± 9.2

221.9 ± 11.4

207.9 ± 12.5***

gd 21

246.6 ± 15.6

238.2 ± 15.2

243.5 ± 17.6

233.1 ± 18.1*

Body weight change3

 

 

 

 

gd 0-6 (pre-exposure)

12.1 ± 4.6

12.5 ± 3.2

12.5 ± 3.5

12.4 ± 3.1

gd 6-9

5.2 ± 3.3

4.3 ± 1.9

5.2 ± 2.8

0.7 ± 2.5***

gd 6-12

15.0 ± 3.4

13.8 ± 2.4

14.6 ± 3.4

4.0 ± 5.1***

gd 6-15 (exposure)

25.2 ± 5.1

22.6 ± 3.2

24.8 ± 5.3

8.4 ± 5.7***

gd 6-18

45.6 ± 7.4

40.6 ± 5.7

43.1 ± 9.3

28.9 ± 10.3***

gd 6-21

68.5 ± 9.9

60.7 ± 12.2

64.7 ± 16.2

54.1 ± 16.6**

gd 15-21 (postexposure)

43.3 ± 6.7

38.2 ± 9.9

39.9 ±11.9

45.7 ± 13.0

aln grams, mean ± SD.
*p < 0.05; **p < 0.01 and ***p < 0.001 versus control.

Table 3. Gestational parameters and fetal body weights in Fischer 344 rats exposed whole body toN,N-dimethylethanolamine vapor*

Exposure conc. (ppm)

0

10

30

100

Number of dams

22

23

22

24

Corpora lutea/dam

11.4 ± 1.2

11.1 ± 1.2b

11.3 ± 1.1

11.8 ± 1.2

Total implants/litter

9.6 ± 1.7

7.9 ± 3.0

8.6 ± 3.3

8.5 ± 3.0

Preimplantation loss (%)

15.5 ± 14.6

26.9 ± 25.4b

25.9 ± 26.8

28.1 ± 24.3

Viable implants/litter

9.5 ± 1.7

7.6 ± 3.1*

8.5 ± 3.4

8.0 ± 3.3

Non-viable implants/litter

0.0 ± 0.2

0.3 ± 0.6

0.1 ± 0.4

0.5 ± 1.5

Early resorptions

0.0 ± 0.0

0.2 ± 0.5

0.0 ± 0.2

0.4 ± 1.4

Late resorptions

0.0 ± 0.2

0.1 ± 0.3

0.1 ± 0.3

0.0 ± 0.0

Dead fetuses

0.0 ± 0.0

0.0 ± 0.2

0.0 ± 0.0

0.0 ± 0.2

Live fetuses/litter (%/litter)

99.5 ± 2.4

95.6 ± 9.0*

97.9 ± 6.1

94.4 ± 20.6

Sex ratio (% males)

56.0 ± 16.0

60.0 ± 21.0

41.6 ± 18.4*

49.0 ± 16.9c

Live litter size

9.5 ± 1.7

7.6 ± 3.1*

8.5 ± 3.4

8.4 ± 2.9c

Fetal body weight/litter (g)

 

 

 

 

All fetuses

4.47 ± 0.15

4.56 ± 0.26

4.53 ± 0.24

4.66 ± 0.27c

Male fetuses

4.63 ± 0.16

4.67 ± 0.24

4.61 ± 0.26d

4.82 ± 0.26*c

Female fetuses

4.28 ± 0.13

4.38 ± 0.26e

4.43 ± 0.26

4.52 ± 0.30**c

aValues presented as mean ± standard deviations; *p < 0.05 and **p < 0.01 versus control.
bn = 22 because the corpora lutea count from one dam was inadvertently not recorded.
cn = 23 because one dam carried a totally resorbed litter.
dn = 20 because two litters consisted of only female fetuses.
en = 22 because one litter consisted of only male fetuses.

Table 4. Skeletal variations in the fetuses of Fischer 344 rats exposed whole body toN,N-dimethylethanolamine vapor

 

Fetuses

Litters

Exposure concentration (ppm)

0

10

30

100

0

10

30

100

Number examined skeletally

102

82

89

91

22

23

20

23

Cervical centrum 6 poorly ossified

43

46

48

33

22

21

18

15**

Cervical centra 1, 2, 3 and/or 4 split

3

2

6

13

3

2

5

12*

Thoracic centrum 1 bilobed

8

14

15

11

6

14*

12

10

Thoracic centrum 9 bilobed

14

5

6

6

12

5*

6

6

Some proximal phalanges (forelimb) unossified

7

0

5

5

6

0*

5

5

Sternebra 5 bilobed

16

10

6

14

12

5*

5

11

*p < 0.05 and **p < 0.01 versus control.

Conclusions:
In summary, whole-body exposure to DMEA vapor of timed-pregnant Fischer 344 rats during organogenesis at 0, 10, 30 or 100 ppm resulted in maternal toxicity at 30 and 100 ppm (with transient minor ocular changes at 10 ppm). There was no evidence of embryonic or fetal toxicity, including teratogenicity, at any exposure concentration employed. Therefore, the no-observed-adverse-effect level is around 10 ppm for maternal toxicity and ≥ 100 ppm for embryofetal toxicity and teratogenicity in this study.
Executive summary:

Timed-pregnant Fischer 344 rats were exposed whole body to N,N-dimethylethanolamine vapor for 6 h per day on gestational days 6-15 at mean (±SD) analytically measured concentrations of 10.4 ± 0.86, 29.8 ± 2.14 and 100 ± 4.9 ppm. Dams were sacrificed on gestational day 21. There was no maternal mortality in any exposed groups. Maternal toxicity observed in the 100 ppm group included reduced body weight during and after exposures, reduced weight gain during exposure and ocular changes (darkened, cloudy and hazy eyes, slight corneal vascularization and fixed, dilated pupils). Ocular effects were also noted in the other two exposure groups; the effects were quite marked at 30ppm but only minimal and transient at 10 ppm. There were no effects of treatment on any gestational parameters, including pre- and postimplantation loss or sex ratio. Fetal body weights per litter were statistically significantly increased at 100 ppm relative to controls. There were no increases in the incidences of total malformations by category (external, visceral or skeletal) or individually. The incidence of six skeletal variations out of 120 noted differed in exposed groups relative to that of control. Four of these variations were decreases in incidence; only one fetal variation, the split (bipartite) cervical centrum, was elevated at 100 ppm relative to controls. In the absence of any other indications of delayed ossification or fetal body weights, the observed fetal variation does not suggest a consistent pattern of fetal toxicity. Hence, the no-observed-adverse-effect level is around 10 ppm for maternal toxicity and at or above 100 ppm for embryofetal toxicity and teratogenicity.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
257 mg/kg bw/day
Study duration:
subacute
Species:
rat
Effect on fertility: via inhalation route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEC
100 ppm
Study duration:
subacute
Species:
rat
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

There is - at the moment - not sufficient information on effects of DMAE on reproductive performance and fertility from repeated dose toxicity and developmental studies available. According to the decision with the decision number CCH-D-2114340872-49-01/F following a compliance check decision under Articles 50 and 51 of the REACH Regulation (Regulation (EC) No 1907/2006) issued in August 2016, among others an Extended one-generation reproductive toxicity study in rats, oral route with the registered substance 2-dimethylaminoethanol (according to OECD 443; 0 weeks premating exposure, Cohort A, Cohort B without extension as well as Cohorts 2A and 2B) has been requested.

Multiple laboratory delays occurred in relation to the OECD 443 study for the substance intended to be used for a read-across (CAS 111-42-2), triggering further delays for the planned screening tests for the registered substance, as well as a change in the intended read-across strategy.

The OECD 443 study is currently being conducted at Charles River laboratories (Ashland). The registrant will submit the testing results as soon as the study results with the registered substance are available.

Momentarily available data:

In the OECD 422 study (BASF, 2019, Key study), the test compound N,N-Dimethylaminoethanol was administered to groups of 10 male and 10 female healthy young Wistar rats (F0 animals) as a solution to the drinking water in different concentrations, i.e. 0 ppm (test group 0), 200 ppm (test group 1), 1000 ppm (test group 2) and 5000 ppm (test group 3). The duration of treatment covered a 2-weeks premating period and mating in both sexes (mating pairs were from the same test group), 3 days postmating in males and approximately 4-weeks postmating in two females (for sperm negative females) as well as the entire gestation and approximately 3 weeks of lactation period in females up to the day of scheduled sacrifice of the animals.

After 2 weeks of premating treatment the F0 animals were mated to produce F1 generation pups. Mating pairs were from the same test group. Mating was discontinued as soon as sperm were detected in the vaginal smear. F0 animals were examined for their reproductive performance including determination of the number of implantation sites and the calculation of postimplantation loss for all F0 females. A detailed clinical observation (DCO) was performed in all animals before the start of the administration period and, as a rule, thereafter at weekly intervals. Water consumption of the F0 parents was determined twice a week. However, during gestation and lactation water consumption of the F0 females were determined on gestation days (GD) 0-1, 4-5, 7-8, 10-11, 14-15, 17-18 and 19-20 and on postnatal days (PND) 1-2, 4-5, 7-8, 10-11 and 12-13. Food consumption of the F0 parents was determined once a week during premating. In dams food consumption was determined for GD 0-7, 7-14, 14-20 and PND 1-4, 4-7, 7-10 and 10-13. Body weights of F0 parents were determined once a week, in males throughout the study and in females during premating. During gestation and lactation period, F0 females were weighed on GD 0, 4, 7, 10, 14, 17 and 20, on the day after parturition (PND 1) and on PND 4, 7, 10 and 13. Estrous cycle data were evaluated for F0 generation females over a two weeks period prior to mating until evidence of mating occurred. Moreover, the estrous stage of each female was determined on the day of scheduled sacrifice.

The pups were sexed and examined for macroscopically evident changes on PND 0. They were weighed on PND 1, 4, 7 and 13. Their viability was recorded. At necropsy on PND 13, all pups were sacrificed with CO2, under isoflurane anesthesia, and examined macroscopically for external and visceral findings. Anogenital distance (defined as the distance from the anus [center of the anal opening] to the base of the genital tubercle) measurements were conducted in a blind randomized fashion, using a measuring ocular on all live male and female pups on PND 1. All surviving pups were examined for the presence or absence of nipple/areola anlagen on PND 13. The number of nipple/areola anlagen were counted. Blood samples were taken from all surplus pups at PND 4 as well as one male and one female pup per litter at PND 13 by decapitation under isoflurane anesthesia for hormone measurement. Blood samples for choline determination were withdrawn from all parental males and females (with litter). Clinico-chemical and hematological examinations were performed in 5 animals per sex and group towards the end of the administration period. Blood samples from all dams at PND 14 and all males at termination were taken by puncturing the retrobulbar venous plexus under isoflurane anesthesia for hormone measurement. At the end of the administration period a functional observational battery was performed and motor activity was measured in 5 parental males and females per group. All F0 parental animals were sacrificed by decapitation, under isoflurane anesthesia, and were assessed by gross pathology. Weights of selected organs were recorded and a histopathological examination was performed.

Under the conditions of the present OECD 422 combined repeated dose toxicity study with the reproductive/developmental screening test in Wistar rats N,N-Dimethylaminoethanol caused signs of systemic toxicity in females consisting of decreased food consumption and body weight, clinically apparent signs (mortality, tremors and convulsions) together with axonal degeneration in cerebellum, medulla oblongata, cervical and thoracic spinal cord at a concentration of 5000 ppm in drinking water. Males of the same concentration group showed no adverse findings. Thus, the no observed adverse effect level (NOAEL) for general systemic toxicity was 5000 ppm (approx. 257 mg/kg bw/d) for males and 1000 ppm (approx. 89 mg/kg bw/d) for female Wistar rats. The NOAEL for fertility and reproductive performance was 5000 ppm (approx. 257 and 355 mg/kg bw/d, respectively) for male and female rats, the highest concentration tested. The NOAEL for developmental toxicity in the offspring was 1000 ppm based on adverse effects on pup number, status at delivery, viability/mortality and pup body weight at 5000 ppm.

In the Pre- and Postnatal Developmental Toxicity Screening test, 2-Dimethylaminoethanol was administered via oral gavage to time-mated Wistar rats from GD 6 through GD 19 (prenatal study part) and from GD 6 through PND 3 (postnatal study part). The following concentrations were administered: i.e. 0 mg/kg bw/day (test group 0, 10 animals), 300 mg/kg bw/day (test group 1, 10 animals), 600 mg/kg bw/day (test group 2, 20 animals). The duration of treatment covered a 2-weeks in gestation and up to the 3rd day after parturition up to the day of scheduled sacrifice of the animals.

Animals dosed with the high dose of 600 mg/kg bw/day showed after treatment the following symptoms: salivation (20/20); respiratory sounds (7/20), statistically significantly reduced mean food consumption (i.e. between GD 6-8 (81 %) and GD 8-10 (78 %) compared to control group (100 %)), statistically significantly reduced mean body weight (i.e. on GD 13 (93 %) compared to the control group (100 %), statistically significantly reduced mean body weight change (i.e. between GD 8-10 (52 %) compared to the control group (100 %). Moreover, one animal was found dead on GD20 (gross pathological examination revealed stomach ulcerations) and one animal was sacrificed moribund on GD14 (gross pathological examination revealed stomach erosions and no feces in intestine).

For the prenatal study part, 10 dams were sacrificed on GD 20 and subsequently examined. In the dams the following observation were made: stomach erosions/ulcera (8 /10) and statistically significantly increased mean liver weight (118 %) compared to the control group (100 %). The following effects on reproduction performance were observed, but assessed statistically not significant and without a dose-response relationship: Slightly increased post-implantation loss (11.8 % vs. 5.2 % in control (compared to the control group and the historical control data)).

For the postnatal part, the remaining 8 animals were sacrificed on PND 4 and subsequently examined. In these dams the following observations were made: stomach erosions/ulcera (8/8), 1/8 dams did not deliver (7 animals left for further assessment), salivation after treatment (7/7), statistically significantly reduced mean food consumption (81 % between PND 0 – 4) compared to the control group (100 %) and a live birth index of 91 % compared to the control group (100 %). A slightly increased post-implantation loss (9.9 % vs. 2.0 % in control) was noted, but assessed as statistically not significant.

Animals dosed with the low dose of 300 mg/kg bw/day showed after treatment the following symptoms: salivation (10/10).

For the prenatal study part, 5 dams were sacrificed on GD 20 and subsequently examined, in 4/5 dams stomach erosions/ulcera was recorded. The following effects on reproduction performance were observed, but assessed statistically not significant and without a dose-response relationship: Slightly increased post-implantation loss (15.2 % vs. 5.2 % in control (compared to the control croup and the historical control data)) and slightly increased resorptions (mean/litter) (1.4 % vs. 0.6 % in control (compared to the control group and the historical control data)).

For the postnatal part, the remaining 5 animals were sacrificed on PND 4 and subsequently examined. In these dams the following local effects were observed: stomach erosions/ulcera (5/5). A slightly increased post-implantation loss (5.5 % vs. 2.0 % in control) was noted, but assessed as statistically not significant.

Concerning the effects and observations on foetuses of dams treated with 600 mg/kg bw/day, no test-substances-related findings were reported (prenatal part).

For the postnatal study part,pups derived of dams dosed with 600 mg/kg bw/day, the following is reported (as1/8 dams did not deliver, only 7 litters left for further assessment):six stillborn pups in 7 litters (64 pups in toto, 58 liveborn), 24/58 pups died ahead of schedule, 9/58 pups were cannibalized, no more pups alive in 4 out of 7 litters (2 litters on PND 1, 1 on PND 2, 1 on PND 3), viability index of 43 % (control: 100%), statistically significantly reduced mean body weight (i.e. on PND 1 (76 %) and on PND 4 (71 %) compared to the control group (100 %)), statistically significantly reduced mean body weight change ((57 % between PND 1-4) compared to the control group (100 %)) and 12 runts (no runts in the control).

In conclusion, signs of maternal toxicity occurred at 300 mg/kg bw/day, comprising local effects (strong ulcera in the stomach) and at 600 mg/kg bw/day, as evident by systemic effects. In the postnatal part of the study, in the dose group of 600 mg/kg bw/day the live birth index was reduced (91 %  vs. control 100 %). Pups of the high dose group were less viable compared to the control group (viability index 43 % and 100 %, respectively); and reduced body weights were recorded.

No substance related findings were observed in foetuses.

Based on these observations the following effect levels were determined:

Dams (F0):

- LOAEL for local effects: 300 mg/kg bw/day

- NOAEL for systemic effects: 300 mg/kg bw/day

- LOAEL for reproductive performance: 600 mg/kg bw/day

Offspring (F1)

- NOAEL for foetuses: not determinable; no effects observed

- NOAEL for pups: 300 mg/kg bw/day

In the developmental OECD 414 study (Leung et al., 1996, Key study) rats were exposed by whole body to DMAE vapor for 6 h per day on gestational days 6 -15 at mean concentrations of 10, 30 and 100 ppm. Inhaled DMAE during organogenesis resulted in maternal toxicity, expressed as reduced body weights and reduced body weight gain at 100 ppm and clinical signs of toxicity, involving the eyes at 30 and 100 ppm with slight transient ocular changes at 10 ppm. There were no maternal deaths or abortions. Pregnancy rate ranged from 88 to 96 % and all pregnant females had one or more live fetuses at scheduled sacrifice, except one dam at 100 ppm, which had a totally resorbed litter. Reduced body weight and reduced body weight gain were observed at 100 ppm. Body weight reduced on gestation day (gd) 12 and 15 (during the exposure period) and on gd 15 and 21 (postexposure period). Body weight gain was reduced for all intervals except gd 6- 9 (pre-exposure) and gd 15-21 (post-exposure). There were no effects on body weight or body weight gain for the 10 or 30 ppm groups. Clinical examination showed that dams at 100 ppm only exhibited perinasal fur discoloration, presumably from chromodacryorrhea. At 30 and 100 ppm there were darkened (maroon), cloudy and hazy eyes, slight corneal vascularization and pupils dilated and fixed. Cloudy and hazy eyes were observed only during the exposure period. Darkened eyes were also observed at 10 ppm during the exposure period. There were no statistically significant differences in gravid uterine weight and absolute or relative liver weights between the DMEA-exposed groups and the controls. There were no effects of treatment on gonads or on any gestational parameters, including pre- and post implantation loss or sex ratio. There were a few sporadic changes in some implantation data, but no consistent pattern of concentration-related effect was evident. For instance, viable implants per litter, percentage live fetuses/litter and live litter size were statistically significantly reduced at 10 ppm only, and the sex ratio (% males) was statistically significantly reduced at 30 ppm only. Fetal body weight per litter at 100 ppm was higher than controls for males and females separately, but not for combined sexes. The maternal NOAEC is considered to be 10 ppm.

In the sub-chronic inhalation repeated dose toxicity study there were no effects of treatment on gonads (Klonne et al., 1987). Pregnancies progressed to term in rats fed with a choline deficient diet supplemented with 1 % DMAE (Katyal and Lombardi, 1978, Zahniser et al, 1978). Litter size was similar with controls.

Effects on developmental toxicity

Description of key information

Key study, Millard 2021, GLP, OECD 414, New Zealand White rabbits, gavage, 0, 30, 100, 250 mg/kg bw of 2 -Dimethylaminoethanol.

Key. study, Leung et al., 1996. Developmental Toxicity Study in Fischer 344 Rats by Whole-body Exposure to N,N-Dimethylethanolamine Vapor. Journal of Applied Toxicology. Vol. 16(6), 533-538; Fischer 344 Rats, Inhalation, 0, 10, 30 and 100 ppm nominal

Supp. study, BASF SE, 2008. Modified Developmental Toxicity Screening Test (OECD 414, amended; OECD 421-postnatal part)., Wistar rats, gavage, 0, 300 and 600 mg/kg bw.  

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Remarks:
Prenatal Developmental Toxicity Study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
30 JAN 2020 to 23 MAR 2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
June 2018
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Version / remarks:
August 1998
Deviations:
no
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
- Physical Description: Clear, colorless liquid
- Storage Conditions: 18 °C to 24 °C, under nitrogen
- Density: 0.89 g/mL
Species:
rabbit
Strain:
New Zealand White
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Envigo, Inc., Denver, PA
- Age at study initiation: approximately 6 to 7 months old
- Weight at study initiation: 2964 and 3924 g
- Housing: Single/Individual in stainless steel cages with perforated flooring elevated above ground corncob bedding
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: yes

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 16 °C to 22 °C
- Humidity (%): 30 % to 70 %
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours dark.

IN-LIFE DATES: From: 30 Jan 2020 To: 11 Mar 2021
Route of administration:
oral: gavage
Vehicle:
water
Remarks:
Deionized (DI) water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Dose formulations were divided into aliquots where required to allow them to be dispensed on each dosing occasion. Dosing formulations were prepared at appropriate concentrations to meet dose level requirements. The dosing formulations were stirred continuously during dosing.
Frequency of Preparation: At least weekly

VEHICLE
- Concentration in vehicle: 0, 6, 20, 50 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg
- Purity: Not corrected for salt, purity, and water content.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses described below were performed by a gas chromatography method using flame ionization detection using a validated analytical procedure.
Concentration Analysis:
Sample Allocation: 2 x 1 mL (primary samples), 2 x 1 mL (backup samples)
Storage Conditions: Temperature set to maintain a target of 5 °C
Acceptance Criteria: Mean sample concentration within 100 % ± 10 % of theoretical concentration.

Stability Analysis: Test substance formulations have been previously shown to be stable over the range of concentrations used on this study for at least 24 hours of room temperature storage or 10 days of refrigerated (target of 5 °C) storage.
Details on mating procedure:
- Impregnation procedure: time-mated
Duration of treatment / exposure:
during Gestation Days 7 through 28
Frequency of treatment:
single daily oral gavage
Duration of test:
Gestation Days 5 through 29
Dose / conc.:
0 mg/kg bw/day
Remarks:
Control
Dose / conc.:
30 mg/kg bw/day
Dose / conc.:
100 mg/kg bw/day
Dose / conc.:
250 mg/kg bw/day
No. of animals per sex per dose:
25 females/control and 24 females/dose group (to obtain a sample size of 20 females/group at termination)
Control animals:
yes, concurrent vehicle
Details on study design:
Dose selection rationale: Based on previous studies (7-day tolerability study and dose range-finding study).
- In a previous 7-day tolerability study, 2-Dimethylaminoethanol was administered once daily by oral gavage to 4 groups of nonpregnant female rabbits at dosage levels of 35, 75, 150, and 300 mg/kg/day. Lower food consumption was noted at the high-dose group (300 mg/kg/day) generally throughout the study, but absolute body weights were unaffected across groups (approximately 2.5 %). At necropsy, no remarkable macroscopic findings were observed at any dosage level. Following microscopic examination of the stomachs from animals in the 150 and 300 mg/kg/day groups, gastric mucosal hemorrhage associated with increased mitoses and sometimes with minimal erosion and epithelial regeneration was detected at 300 mg/kg/day; no microscopic findings were noted in the stomach at 150 mg/kg/day.

- In a previous dose range-finding prenatal developmental toxicity study, 2-Dimethylaminoethanol was administered once daily by oral gavage to 3 groups of time-mated female rabbits at dosage levels
of 50, 150, and 450 mg/kg/day during Gestation Days 7–28. Severe body weight loss and reduced food consumption, with corresponding clinical observations, resulted in moribundity and abortion at 450 mg/kg/day. In addition, macroscopic (dark red areas, depressed areas, and thickening of the stomach) and microscopic (necrosis and/or heterophilic infiltration with or without congestion) findings were noted in the stomach at 150 and 450 mg/kg/day. Based on these results (body weight loss, reduced food consumption, moribundity and abortion at 450 mg/kg/day and microscopic findings noted at 300 and 450 mg/kg/day), dosage levels of 30, 100, and 250 mg/kg/day were selected for the current study.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least twice daily
- Cage side observations checked in table [No.?] were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once daily, beginning with the day of animal arrival and continuing through (and including) the day of euthanasia.

BODY WEIGHT: Yes
- Time schedule for examinations: Gestation Days 0 (by supplier), 5, 7–29 (daily)

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Gestation Days 5–29 (daily)
- Reported as g/animal/day for each corresponding body weight interval during gestation.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 29
- Organs examined:
Histopathology: Stomach, gross lesions;
Macroscopic Examination: The thoracic, abdominal, and pelvic cavities
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other:
- Number of viable and nonviable fetuses
- The placentae were examined
Fetal examinations:
- External examinations: Yes [all]
- Soft tissue examinations: Yes [all]
- Skeletal examinations: Yes [each eviscerated fetus]
- Head examinations: Yes: [all ]
Statistics:
Numerical data and clinical and necropsy observations data were summarized by sex and occasion or by litter.
All statistical tests were conducted at the 5 % significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1 % and 5 % levels, unless otherwise noted.
The pairwise comparisons of interest are listed below:
Group 2 vs. Group 1
Group 3 vs. Group 1
Group 4 vs. Group 1

Parametric/Non-parametric:
Variables: Ovarian and Uterine Content, Litter % of Fetuses with Gross/External/Visceral/Skeletal Abnormalities
Levene’s test was used to assess the homogeneity of group variances.
The groups were compared using an overall one-way ANOVA F-test if Levene’s test was not significant or the Kruskal-Wallis test if it was significant. If the overall F-test or Kruskal-Wallis test was found to be significant, then pairwise comparisons were conducted using Dunnett’s or Dunn’s test, respectively.

Non-Parametric:
Variables: Body Weight, Body Weight Gains, Food Consumption, Gravid Uterine Weight and Corrected Maternal Body Weights, Litter Means
The groups were compared using an overall Kruskal-Wallis test. If the overall Kruskal-Wallis test was found to be significant, then the above pairwise comparisons were conducted using Dunn’s test.

Incidence:
A Fisher’s exact test was used to conduct pairwise group comparisons of interest
Historical control data:
yes
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
In the 250 mg/kg/day group, 6 females (Nos. 4504, 4507, 4512, 4601, 4513, 4517) were noted with clinical observations (a thin body and/or excreta-related findings) noted at the daily examinations prior to death, abortion, or delivery.
Test substance-related increased incidences of decreased fecal output, which corresponded to reduced mean food consumption, were noted for surviving females in the 250 mg/kg/day group at the daily examinations.
No test substance-related clinical observations were noted at the daily examinations at 30 and 100 mg/kg/day or 1–2 hours postdosing at any dosage level.
Other observations noted in the test substance-treated groups, including scabbing and brown, yellow, or red staining on various body surfaces, occurred infrequently, at similar frequencies in the control group, and/or in a manner that was not dose-related.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, treatment-related
Description (incidence):
In the 250 mg/kg/day group, 1 female (No. 4513) was found dead on Gestation Day 23. The unscheduled death was considered secondary to the test substance-related effects on body weight and food consumption in this group.
In the 30 mg/kg/day group, Female No. 2501 was found dead on Gestation Day 25; at necropsy this female was noted with a perforated esophagus and fluid accumulation in the thoracic cavity, which were indicative of a dosing error. Therefore, this death was not attributed to the test substance.
In the control group, Female No. 1501 was euthanized on Gestation Day 8 due limited usage of a hindlimb, which was confirmed to be a tibial fracture at necropsy. All other females in the control, 30, 100, and 250 mg/kg/day groups survived to the scheduled necropsy on Gestation Day 29.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
In the 250 mg/kg/day group, 6 females (Nos. 4504, 4507, 4512, 4601, 4513, 4517) were noted with severe body weight losses (11.4 % to 21.7 % of Gestation Day 7 body weight).
Test substance-related mean body weight losses and lower mean body weight gains, with corresponding lower mean food consumption, were noted in the 250 mg/kg/day group throughout the treatment period (Gestation Days 7–29) when compared to the control group. As a result, absolute mean body weights in this group were 5.0 % to 11.2 % lower than the control group during Gestation Days 12–29.
In addition, lower mean corrected body weight, corrected mean body weight gain, and mean gravid uterine weight were noted in the 250 mg/kg/day group compared to the control group. Mean maternal body weights, body weight gains, corrected body weights, corrected body weight changes, gravid uterine weights, and food consumption in the 30 and 100 mg/kg/day groups were unaffected by test substance administration.

Mean body weights, body weight gains, corrected body weights, corrected body weight changes, and gravid uterine weights in the 30 and 100 mg/kg/day groups were unaffected by test substance administration. Differences from the control group were slight and not statistically significant.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
In the 250 mg/kg/day group, 6 females (Nos. 4504, 4507, 4512, 4601, 4513, 4517) were noted with reduced food consumption (0 to 86 g/day for 6 to 21 days).
In the 250 mg/kg/day group, lower mean food consumption, evaluated as g/animal/day, was noted throughout the treatment period (Gestation Days 7–10 [-50.7 %], 10–13 [54.5 %], 13–20 [54.9 %], and 20–29 [34.8 %]) and when the entire treatment period (Gestation Days 7–29; 42.5 %) was evaluated; differences from the control group were statistically significant.
The decrements in mean food consumption in this group corresponded to the mean body weight losses/lower mean body weight gains, and were considered test substance-related and adverse.
Mean maternal food consumption in the 30 and 100 mg/kg/day groups was unaffected by test substance administration. Any statistically significant differences from the control group did not impact mean body weights or body weights gains, and therefore were considered unrelated to the test substance.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not specified
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Endocrine findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not specified
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Test substance-related gross pathology findings were limited to the stomach of the 250 mg/kg/day group females and are summarized 'Any other information on results'.
A single dark red focus of the pyloric region was observed for 1 female in the 30 mg/kg/day group. This finding did not have a clear dose-response, lacked a histologic correlate, and was considered unlikely to be test substance-related.
Other gross findings observed were considered incidental, of the nature commonly observed in this strain and age of rabbits, and/or were of similar incidence in control and treated animals and, therefore, were considered unrelated to the test item.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Microscopic findings in the stomach were observed in all test substance-administered groups and included hemorrhage, necrosis, edema, fibrosis, and heterophilic infiltrates. Based on the absence of other signs of toxicity, the microscopic findings in the 30 and 100 mg/kg/day groups were considered nonadverse. Fore more details please refer to 'Details on results'
Histopathological findings: neoplastic:
not examined
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Test substance-related lower mean fetal body weights noted in 250 mg/kg bw/day resulted in a lower mean gravid uterine weight in this group.
Details on results:
Histopathology: Test substance-related changes in the stomach noted at terminal euthanasia in all dose groups were similar in character to those observed in the 250 mg/kg/day group females that did not survive to terminal euthanasia, aborted, or delivered, although a clear dose-response was not apparent. Hemorrhage ranged from minimal, which was observed primarily as small foci in the glandular mucosa, to moderate, extending from the mucosa and dissecting through the muscularis. Minimal to mild necrosis was noted in all dose groups and was characterized by foci of necrotic epithelium along the mucosa of the glandular stomach and rarely in the cardia. Necrosis and hemorrhage were variably accompanied by a minimal to moderate infiltrate of heterophils and edema. Fibrosis along the superficial muscularis was rarely observed but was considered likely secondary to hemorrhage and necrosis. Based on the absence of other signs of toxicity, the microscopic findings in the 30 and 100 mg/kg/day groups were considered nonadverse.
Number of abortions:
effects observed, treatment-related
Description (incidence and severity):
In the 250 mg/kg/day group, 4 females (Nos. 4504, 4507, 4512, and 4601) aborted during Gestation Days 20–28
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
Mean litter proportions of postimplantation loss, mean number of live fetuses, and mean fetal body weights were evaluated. Differences from the control group were slight, not statistically significant, and/or occurred in a manner that was not dose related.
Total litter losses by resorption:
not examined
Early or late resorptions:
not specified
Dead fetuses:
no effects observed
Description (incidence and severity):
Intrauterine survival in the 30, 100, and 250 mg/kg/day groups were unaffected by test substance administration.
Changes in pregnancy duration:
effects observed, treatment-related
Description (incidence and severity):
In the 250 mg/kg/day group, 1 female delivered on Gestation Day 29, prior to the scheduled necropsy.
Changes in number of pregnant:
not specified
Other effects:
no effects observed
Description (incidence and severity):
Mean numbers of corpora lutea and implantation sites and the mean litter proportions of pre-implantation loss were similar across all groups.
Details on maternal toxic effects:
Adverse effects on maternal survival, maternal body weight losses and/or lower body weight gain, reduced food consumption, and corresponding clinical observations at 250 mg/kg/day
Dose descriptor:
NOAEL
Remarks:
maternal toxicity
Effect level:
100 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
body weight and weight gain
food consumption and compound intake
mortality
other: clinical observations
Fetal body weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Test substance-related lower (12.16 % to 13.83 %) mean fetal body weights (male, female, and sexes combined) were noted in the 250 mg/kg/day group. However, the mean fetal weight values in this group were within the respective ranges of historical control data and the differences were considered secondary to the maternal toxicity noted at this dosage level, and nonadverse data.
Intrauterine growth in the 30 and 100 mg/kg/day groups was unaffected by test substance administration.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
Mean litter proportions of postimplantation loss, mean number of live fetuses, and mean fetal body weights were evaluated. Differences from the control group were slight, not statistically significant, and/or occurred in a manner that was not dose related.
Changes in sex ratio:
not specified
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
Mean litter proportions of postimplantation loss, mean number of live fetuses, and mean fetal body weights were evaluated. Differences from the control group were slight, not statistically significant, and/or occurred in a manner that was not dose related.
Anogenital distance of all rodent fetuses:
not examined
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Description (incidence and severity):
There were no test substance-related fetal malformations or developmental variations noted at any dosage level.
Skeletal malformations:
no effects observed
Description (incidence and severity):
There were no test substance-related fetal malformations or developmental variations noted at any dosage level.
Visceral malformations:
no effects observed
Description (incidence and severity):
There were no test substance-related fetal malformations or developmental variations noted at any dosage level.
Other effects:
not specified
Dose descriptor:
NOAEL
Remarks:
Prenatal development
Effect level:
250 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No substance-related effects observed
Abnormalities:
no effects observed
Developmental effects observed:
no

Results of Concentration Analyses

Mean Concentration, mg/mL (% of Target)

Date of Preparation

Group 2

(6 mg/mL)

Group 3

(20 mg/mL)

Group 4

50 mg/mL)

31 Jan 2020

6.30 (105)

22.0 (110)

55.2 (110)*

20 Feb 2020

6.56 (109)

22.2 (111)*

55.0 (110)

* = Back-up samples included in calculations.

Summary of Gross Pathology Findings – All Animals

Group

1

2

3

4

Dose (mg/kg/day)

0

30

100

250

No. Animals per Group

24

24

24

24

Stomach (No. Examined)

(24)

(24)

(24)

(24)

Focus, dark, red

0

1

0

7

Thick

0

0

0

3

Focus, raised

0

0

0

3

Discoloration, dark, red

0

0

0

2

Conclusions:
In conclusion, based on adverse effects on maternal survival, maternal body weight losses and/or lower body weight gain, reduced food consumption, and corresponding clinical observations at 250 mg/kg/day, considered secondary to local corrosion of the gastric mucosa, a dosage level of 100 mg/kg/day was considered to be the NOAEL for maternal toxicity. Lower mean fetal body weights observed at 250 mg/kg/day were considered secondary to the adverse maternal effects; therefore, a dosage level of 250 mg/kg/day was considered to be the NOAEL for prenatal development.
Executive summary:

The Prenatal Developmental Toxicity Study was conduced according to OECD 414 and in accordance with GLP.

2-Dimethylaminoethanol was administered orally by gavage to time-mated New Zealand White rabbits at doses of 0, 30, 100 and 250 mg/kg bw/day; animals were dosed once daily during Gestation Days 7–28. The following parameters and end points were evaluated in this study: clinical signs, body weights, body weight gains, gravid uterine weights, food consumption, gross necropsy findings, stomach histopathology, intrauterine growth and survival, and fetal morphology.

In the 250 mg/kg/day group, 1 female was found dead on Gestation Day 23, 4 females aborted during Gestation Days 20–28, and 1 female delivered on Gestation Day 29, prior to the scheduled necropsy. All 6 of these females were noted with severe body weight losses (11.4 % to 21.7 %), reduced food consumption (0 to 86 g/day), and corresponding clinical observations (a thin body and/or excreta-related findings) noted at the daily examinations prior to death, abortion, or delivery. At necropsy, macroscopic findings including dark red discoloration, dark red foci, thickened, and raised foci in the stomach with microscopic correlates (hemorrhage, necrosis, edema, fibrosis, and heterophilic infiltrates) were noted for the majority of these females. The effects on survival to the scheduled necropsy in the 250 mg/kg/day group were considered secondary to the test substance-related effects on body weight and food consumption in this group. One female in the 30 mg/kg/day group was found dead on Gestation Day 25; necropsy findings for this female were indicative of a dosing error, and therefore this death was not attributed to the test substance. In the control group, 1 female was euthanized in extremis on Gestation Day 8 due to limited usage of a hindlimb (tibial fracture). All other females survived to the scheduled necropsy on Gestation Day 29. Test substance-related increased incidences of decreased fecal output, which corresponded to reduced mean food consumption, were noted for surviving females in the 250 mg/kg/day group at the daily examinations. No test substance-related clinical observations were noted at the daily examinations at 30 and 100 mg/kg/day or 1–2 hours postdosing at any dosage level.

Test substance-related mean body weight losses and lower mean body weight gains, with corresponding lower mean food consumption, were noted in the 250 mg/kg/day group throughout the treatment period (Gestation Days 7–29) when compared to the control group. As a result, absolute mean body weights in this group were 5.0 % to 11.2 % lower than the control group during Gestation Days 12–29. In addition, lower mean corrected body weight, corrected mean body weight gain, and mean gravid uterine weight were noted in the 250 mg/kg/day group compared to the control group. Mean maternal body weights, body weight gains, corrected body weights, corrected body weight changes, gravid uterine weights, and food consumption in the 30 and 100 mg/kg/day groups were unaffected by test substance administration. Test substance-related gross observations were observed in the stomach of the 250 mg/kg/day group females at the scheduled necropsy, including dark red discoloration, dark red foci, thickened, and raised foci; comparable findings were also noted for females that did not survive to the scheduled necropsy. Microscopic findings in the stomach were observed in all test substance-administered groups and included hemorrhage, necrosis, edema, fibrosis, and heterophilic infiltrates. Based on the absence of other signs of toxicity, the microscopic findings in the 30 and 100 mg/kg/day groups were considered nonadverse.

Test substance-related lower (12.16 % to 13.83 %) mean fetal body weights (male, female, and sexes combined) were noted in the 250 mg/kg/day group, which resulted in a lower mean gravid uterine weight in this group. However, the mean fetal weight values in this group were within the respective ranges of values noted in historical control data, and the differences were considered secondary to the maternal toxicity noted at this dosage level, and nonadverse. Intrauterine growth in the 30 and 100 mg/kg/day groups and intrauterine survival in the 30, 100, and 250 mg/kg/day groups were unaffected by test substance administration. There were no test substance-related fetal malformations or developmental variations noted at any dosage level.

In conclusion, based on adverse effects on maternal survival, maternal body weight losses and/or lower body weight gain, reduced food consumption, and corresponding clinical observations at 250 mg/kg/day, considered secondary to local corrosion of the gastric mucosa, a dosage level of 100 mg/kg/day was considered to be the NOAEL for maternal toxicity. Lower mean fetal body weights observed at 250 mg/kg/day were considered secondary to the adverse maternal effects; therefore, a dosage level of 250 mg/kg/day was considered to be the NOAEL for prenatal development.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
not reported, published in 1996
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: no data on GLP or OECD compliance. Acceptable, well documented publication which meets basic scientific principles.
Reason / purpose for cross-reference:
reference to same study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
Fischer 344
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan Sprague-Dawley, Inc., Indianapolis, IN.
- Age at study initiation: 67-79 days old on arrival
- Weight at study initiation: not reported
- Fasting period before study: no
- Housing: in stainless-steel wire-meshcages
- Diet (e.g. ad libitum): ad libitum except during exposure
- Water (e.g. ad libitum): ad libitum except during exposure
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): not reported
- Humidity (%): not reported
- Air changes (per hr): not reported
- Photoperiod (hrs dark / hrs light):12/12
Route of administration:
inhalation: vapour
Type of inhalation exposure (if applicable):
whole body
Vehicle:
unchanged (no vehicle)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Liquid DMEA was metered from a piston pump into a heated glass maintained at the lowest temperature to vaporize the liquid. The resultant vapor was carried into the exposure chamber by a countercurrent flow of conditioned air through the evaporator. Exposure was conducted in 4320-litre stainless-steel and glass chambers at an airflow of 1000 L/min.

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Chamber atmosphere was analyzed for DMEA concentrations once every 32 min during each 6-h exposure, using a Perkin-Elmer 3920B gas chromatograph equipped with a flame ionization detector. Nominal concentrations were calculated daily based on the amount of DMEA used and the chamber tube air flow during the exposure period.
Details on mating procedure:
- Impregnation procedure:[cohoused]
- If cohoused:
- M/F ratio per cage: 1/1
- Length of cohabitation: not reported

- Further matings after two unsuccessful attempts: [no]
- Verification of same strain and source of both sexes: [yes / no (explain)]
- Proof of pregnancy: [vaginal plug] referred to as [day 0] of pregnancy
- Any other deviations from standard protocol:
Duration of treatment / exposure:
6 h per day
Frequency of treatment:
each day
Duration of test:
on gestational days 6-15
Dose / conc.:
0 ppm (nominal)
Dose / conc.:
10 ppm (nominal)
Dose / conc.:
30 ppm (nominal)
Dose / conc.:
100 ppm (nominal)
No. of animals per sex per dose:
In a range-finding study, eight plug-positive females each were assigned to four DMEA-exposed groups (target DMEA concentrations 8, 25, 75 and 100 ppm) and an air-exposed control group.
In the definitive study, 25 plug-positive females each were assigned to three DMEA-exposed groups (10, 30 and 100 ppm) and a control group.
Control animals:
yes, sham-exposed
Details on study design:
- Dose selection rationale: Based on the results of the range-finding study.
The highest exposure concentration in the range-finding study, 100 ppm, was retained in the definitive study since it produced maternal toxicity (reduced body weights and weight gain, and clinical signs) and possible embryotoxicity (increased preimplantation loss) but no apparent fetotoxicity. The middle exposure concentration of 30 ppm chosen for the definitive study was slightly above the 25 ppm in the range-finding study which produced maternal toxicity (transient weight gain depression and clinical signs limited to the eyes) and possible embryotoxicity (reduced implantations, increased preimplantation loss and reduced number of viable fetuses per litter). The lowest exposure concentration, 10 ppm, was chosen as essentially the same as the 8 ppm in the range-finding study which produced no effects on maternal weights and only transient ocular changes and no evidence of embryofetal toxicity.
- Rationale for animal assignment (if not random): randomized
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes - Time schedule: daily
DETAILED CLINICAL OBSERVATIONS: Yes - Time schedule: daily
BODY WEIGHT: Yes - Time schedule for examinations:Maternal body weights were measured on gd 0, 6, 12, 15, 18 and 21.
POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 21
- Organs examined: The gravid uterus, ovaries (including corpora lutea), cervix, vagina and peritoneal and thoracic cavities were examined grossly. Ovarian corpora lutea of pregnancy were counted. Maternal liver and uterine weights were measured.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: Uteri were examined externally for signs of hemorrhage. All live and dead fetuses were recorded-
Fetal examinations:
- External examinations: Yes: [all per litter] including cleft palate
- Soft tissue examinations: Yes: [half per litter]
- Skeletal examinations: Yes: [half per litter]
- Head examinations: Yes: [half per litter]
Statistics:
The unit of comparison was the pregnant female or the litter. Continuous quantitative data were compared between the DMEA-exposed groups and air-exposed control group by the use of Levene's test for equal variances analysis of variance (ANOVA) and t-tests with Bonferroni probabilities. The t-tests were used when the F value from the ANOVA was significant. When Levene's test indicated homogeneous variances, and the ANOVA was significant, the pooled t-test was used. When Levene's test indicated heterogeneous variances, all groups were compared by an ANOVA for unequal variances," followed by the separate variance t-test when necessary. Non-parametric data obtained following laparohysterectomy were statistically treated using the Kruskal- Wallis test followed by the Mann-Whitney U test when appropriate. Incidence data were compared using Fisher's exact test. For all statistical tests, the fiducial limit of 0.05 (two-tailed) was used as the criterion for significance.
Indices:
listed in the table 4 in "Remarks on results"
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Clinical examination showed that dams at 100 ppm only exhibited perinasal fur discoloration, presumably from chromodacryorrhea. At 30 and 100 ppm there were darkened (maroon), cloudy and hazy eyes, slight corneal vascularization and pupils dilated and fixed. Cloudy and hazy eyes were observed only during the exposure period. Darkened eyes were also observed at 10 ppm during the exposure period.
Mortality:
no mortality observed
Description (incidence):
There were no maternal deaths or abortions.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Reduced body weight and reduced body weight gain were observed at 100 ppm. Body weight reduced on gd 12 and 15 (during the exposure period) and on gd 15 and 21 (postexposure period). Body weight gain was reduced for all intervals except gd 6- 9 (pre-exposure) and gd 15-21 (post-exposure). There were no effects on body weight or body weight gain for the 10 or 30 ppm groups.
Ophthalmological findings:
effects observed, treatment-related
Description (incidence and severity):
At 30 and 100 ppm there were darkened (maroon), cloudy and hazy eyes, slight corneal vascularization and pupils dilated and fixed. Cloudy and hazy eyes were observed only during the exposure period. Darkened eyes were also observed at 10 ppm during the exposure period.
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
There were no statistically significant differences in gravid uterine weight and absolute or relative liver weights between the DMEA-exposed groups and the controls.
Description (incidence and severity):
There were no effects of treatment on gonads.
Number of abortions:
no effects observed
Description (incidence and severity):
There were no maternal deaths or abortions.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
There were no effects of treatment on pre- and post implantation loss.
Description (incidence and severity):
Pregnancy rate ranged from 88 to 96% and all pregnant females had one or more live fetuses at scheduled sacrifice, except one dam at 100 ppm, which had a totally resorbed litter.
Description (incidence and severity):
Pregnancy rate ranged from 88 to 96% and all pregnant females had one or more live fetuses at scheduled sacrifice, except one dam at 100 ppm, which had a totally resorbed litter.
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
There were no effects of treatment on any gestational parameters.
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
Pregnancy rate ranged from 88 to 96% and all pregnant females had one or more live fetuses at scheduled sacrifice, except one dam at 100 ppm, which had a totally resorbed litter.
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
Table 2 (in "Remarks on results") shows the pregnancy and litter data of all plug-positive females on study. There were no maternal deaths or abortions. Pregnancy rate ranged from 88 to 96% and all pregnant females had one or more live fetuses at scheduled sacrifice, except one dam at 100 ppm, which had a totally resorbed litter. Reduced body weight and reduced body weight gain were observed at 100 ppm. Body weight reduced on gd 12 and 15 (during the exposure period) and on gd 15 and 21 (postexposure period). Body weight gain was reduced for all intervals except gd 6- 9 (pre-exposure) and gd 15-2 1 (post-exposure). There were no effects on body weight or body weight gain for the 10 or 30 ppm groups. Clinical examination showed that dams at 100 ppm only exhibited perinasal fur discoloration, presumably from chromodacryorrhea. At 30 and 100 ppm there were darkened (maroon), cloudy and hazy eyes, slight corneal vascularization and pupils dilated and fixed. Cloudy and hazy eyes were observed only during the exposure period. Darkened eyes were also observed at 10ppm during the exposure period. There were no statistically significant differences in gravid uterine weight and absolute or relative liver weights between the DMEA-exposed groups and the controls.
Dose descriptor:
NOAEL
Effect level:
10 ppm (nominal)
Basis for effect level:
body weight and weight gain
clinical signs
ophthalmological examination
other: maternal toxicity
Fetal body weight changes:
no effects observed
Description (incidence and severity):
Fetal body weights were elevated at 100 ppm relative to those in controls.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
The present developmental toxicity evaluation revealed no treatment-related embryotoxicity at any exposure concentration employed. No consistent pattern of fetotoxicity was observed.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
The present developmental toxicity evaluation revealed no treatment-related embryotoxicity at any exposure concentration employed. No consistent pattern of fetotoxicity was observed. There were no effects of treatment on sex ratio.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
The present developmental toxicity evaluation revealed no treatment-related embryotoxicity at any exposure concentration employed. No consistent pattern of fetotoxicity was observed.
Changes in postnatal survival:
no effects observed
Description (incidence and severity):
The present developmental toxicity evaluation revealed no treatment-related embryotoxicity at any exposure concentration employed. No consistent pattern of fetotoxicity was observed.
External malformations:
no effects observed
Description (incidence and severity):
No increases in malformations were observed at any concentration of DMEA employed, including those which produced maternal toxicity.
Skeletal malformations:
no effects observed
Description (incidence and severity):
One skeletal district, the cervical centra, exhibited evidence of reduced ossification at 100 ppm, an exposure concentration which also produced maternal toxicity. This finding is the only one which could be consistent with indicating possible minimal fetotoxicity; however, there were no other indications of fetotoxicity, such as reduced fetal body weight. No other skeletal districts, of the number identified as sensitive indicators of delayed development in rat fetuses,” exhibited a delay in ossification. No increases in malformations were observed at any concentration of DMEA employed, including those which produced maternal toxicity.
Visceral malformations:
no effects observed
Description (incidence and severity):
No increases in malformations were observed at any concentration of DMEA employed, including those which produced maternal toxicity.
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
The present developmental toxicity evaluation revealed no treatment-related embryotoxicity at any exposure concentration employed. No consistent pattern of fetotoxicity was observed. Fetal body weights were elevated at 100 ppm relative to those in controls (Table 3), and only one skeletal district, the cervical centra, exhibited evidence of reduced ossification at 100 ppm (Table 4), an exposure concentration which also produced maternal toxicity. This finding is the only one which could be consistent with indicating possible minimal fetotoxicity; however, there were no other indications of fetotoxicity, such as reduced fetal body weight. No other skeletal districts, of the number identified as sensitive indicators of delayed development in rat fetuses,” exhibited a delay in ossification. No increases in malformations were observed at any concentration of DMEA employed, including those which produced maternal toxicity.
Dose descriptor:
NOAEL
Effect level:
>= 100 ppm (nominal)
Basis for effect level:
other: see "Remarks"
Abnormalities:
not specified
Developmental effects observed:
not specified

Table 2. Pregnancy and litter data for Fischer 344 rats exposed whole body to N,N-dimethylethanolamine vapor

Exposure concentration (ppm)

0

10

30

100

Number in study

25

25

25

25

Number of early delivery

1

0

0

0

Number aborted

0

0

0

0

Number (%) pregnant at scheduled

22

23

22

24

sacrifice

(91.7)

(92.0)

(88.0)

(96.0)

Number of litters examined

22

23

22

23a

aOne dam carried a totally resorbed litter.

Table 3. Gestational parameters and fetal body weights in Fischer 344 rats exposed whole body to N,N-dimethylethanolamine vapor*

Exposure conc. (ppm)

0

10

30

100

Number of dams

22

23

22

24

Corpora lutea/dam

11.4 ± 1.2

11.1 ± 1.2b

11.3 ± 1.1

11.8 ± 1.2

Total implants/litter

9.6 ± 1.7

7.9 ± 3.0

8.6 ± 3.3

8.5 ± 3.0

Preimplantation loss (%)

15.5 ± 14.6

26.9 ± 25.4b

25.9 ± 26.8

28.1 ± 24.3

Viable implants/litter

9.5 ± 1.7

7.6 ± 3.1*

8.5 ± 3.4

8.0 ± 3.3

Non-viable implants/litter

0.0 ± 0.2

0.3 ± 0.6

0.1 ± 0.4

0.5 ± 1.5

Early resorptions

0.0 ± 0.0

0.2 ± 0.5

0.0 ± 0.2

0.4 ± 1.4

Late resorptions

0.0 ± 0.2

0.1 ± 0.3

0.1 ± 0.3

0.0 ± 0.0

Dead fetuses

0.0 ± 0.0

0.0 ± 0.2

0.0 ± 0.0

0.0 ± 0.2

Live fetuses/litter (%/litter)

99.5 ± 2.4

95.6 ± 9.0*

97.9 ± 6.1

94.4 ± 20.6

Sex ratio (% males)

56.0 ± 16.0

60.0 ± 21.0

41.6 ± 18.4*

49.0 ± 16.9c

Live litter size

9.5 ± 1.7

7.6 ± 3.1*

8.5 ± 3.4

8.4 ± 2.9c

Fetal body weight/litter (g)

 

 

 

 

All fetuses

4.47 ± 0.15

4.56 ± 0.26

4.53 ± 0.24

4.66 ± 0.27c

Male fetuses

4.63 ± 0.16

4.67 ± 0.24

4.61 ± 0.26d

4.82 ± 0.26*c

Female fetuses

4.28 ± 0.13

4.38 ± 0.26e

4.43 ± 0.26

4.52 ± 0.30**c

aValues presented as mean ± standard deviations; *p < 0.05 and **p < 0.01 versus control.
bn = 22 because the corpora lutea count from one dam was inadvertently not recorded.
cn = 23 because one dam carried a totally resorbed litter.
dn = 20 because two litters consisted of only female fetuses.
en = 22 because one litter consisted of only male fetuses.

Table 4. Skeletal variations in the fetuses of Fischer 344 rats exposed whole body to N,N-dimethylethanolamine vapor

 

Fetuses

Litters

Exposure concentration (ppm)

0

10

30

100

0

10

30

100

Number examined skeletally

102

82

89

91

22

23

20

23

Cervical centrum 6 poorly ossified

43

46

48

33

22

21

18

15**

Cervical centra 1, 2, 3 and/or 4 split

3

2

6

13

3

2

5

12*

Thoracic centrum 1 bilobed

8

14

15

11

6

14*

12

10

Thoracic centrum 9 bilobed

14

5

6

6

12

5*

6

6

Some proximal phalanges (forelimb) unossified

7

0

5

5

6

0*

5

5

Sternebra 5 bilobed

16

10

6

14

12

5*

5

11

*p < 0.05 and **p < 0.01 versus control.

Conclusions:
In summary, whole-body exposure to DMEA vapor of timed-pregnant Fischer 344 rats during organogenesis at 0, 10, 30 or 100 ppm resulted in maternal toxicity at 30 and 100 ppm (with transient minor ocular changes at 10 ppm). There was no evidence of embryonic or fetal toxicity, including teratogenicity, at any exposure concentration employed. Therefore, the no-observed-adverse-effect level (NOAEL) is around 10 ppm for maternal toxicity and ≥ 100 ppm for embryofetal toxicity and teratogenicity in this study.
Executive summary:

Timed-pregnant Fischer 344 rats were exposed whole body to N,N-dimethylethanolamine vapor for 6 h per day on gestational days 6-15 at mean (± SD) analytically measured concentrations of 10.4 ± 0.86, 29.8 ± 2.14 and 100 ± 4.9 ppm. Dams were sacrificed on gestational day 21. There was no maternal mortality in any exposed groups. Maternal toxicity observed in the 100 ppm group included reduced body weight during and after exposures, reduced weight gain during exposure and ocular changes (darkened, cloudy and hazy eyes, slight corneal vascularization and fixed, dilated pupils). Ocular effects were also noted in the other two exposure groups; the effects were quite marked at 30 ppm but only minimal and transient at 10 ppm. There were no effects of treatment on any gestational parameters, including pre- and postimplantation loss or sex ratio. Fetal body weights per litter were statistically significantly increased at 100 ppm relative to controls. There were no increases in the incidences of total malformations by category (external, visceral or skeletal) or individually. The incidence of six skeletal variations out of 120 noted differed in exposed groups relative to that of control. Four of these variations were decreases in incidence; only one fetal variation, the split (bipartite) cervical centrum, was elevated at 100 ppm relative to controls. In the absence of any other indications of delayed ossification or fetal body weights, the observed fetal variation does not suggest a consistent pattern of fetal toxicity. Hence, the no-observed-adverse-effect level (NOAEL) is around 10 ppm for maternal toxicity and at or above 100 ppm for embryofetal toxicity and teratogenicity.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
27 MAY 2008 (Study plan) to Jun/July 2008 (end of experimental phase); 14 MAR 2019 (Report date of summary of results)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
equivalent or similar to guideline
Guideline:
other: Prenatal part: Proposal for updating Guideline 414: Prenatal Developmental Toxicity Study
Version / remarks:
22 Jan 2001
Qualifier:
equivalent or similar to guideline
Guideline:
other: Postnatal part: OECD guideline for the Testing of Chemicals; No.421 (SIDS): reproduction/Developmental Toxicity Screening Test
Version / remarks:
July 1995
Deviations:
yes
Remarks:
no pairing/fertility part
Qualifier:
equivalent or similar to guideline
Guideline:
other: EPA Health Effects Test Guideline, OPPTS 870.3550: Reproduction/Developmental Toxicity Screening Test (July 2000)
Version / remarks:
Jul 2000
Principles of method if other than guideline:
The aim of this screening study was to obtain initial information on the effect of the test substance after repeated oral administration (gavage) to pregnant female Wistar rats from gestation day (GD) 6 to GD 19 (prenatal study) and from GD 6 to postnatal day (PND) 3 (postnatal study). For the prenatal study part, selected dams of each group (5 animals of the control group, 5 animals of dose group 1 (300 mg/kg bw/day) and 10 animals of dose group 2 (600 mg/kg bw/day)) were sacrificed on GD 20; dams and fetuses were examined.
This endpoint study record refers mainly to the prenatal study.
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Age at study initiation: about 10-15 weeks
- Housing: 1 animal / cage; from delivery to sacrifice (rearing) - 1 dam with its litter / cage, Makrolon cages type M III, Wooden gnawing blocks (Typ NGM E-022), Type Lignocel FS 14 fibres, dustfree bedding
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: During the acclimatization period, the animals were accustomed to the environmental conditions and to the diet.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 30-70%
- Air changes (per hr): 15 air changes per hour
- Photoperiod (hrs dark / hrs light):12 hours light (6:00 a.m. - 6:00 p.m.), 12 hours darkness (6:00 p.m. - 6:00 a.m.)


IN-LIFE DATES: From: 29 MAY TO: 18 JUN 2008 (GD 20/Sacrifice of selected animals)
Route of administration:
oral: gavage
Vehicle:
olive oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
For the test substance preparation, the specific amount of test substance was weighed, topped up with olive oil in a volumetric flask and intensely shaken until it was completely dissolved

VEHICLE
- Amount of vehicle (if gavage): 4 mL/kg body weight
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The stability of the test substance in olive oil over a period of up to 7 days at room temperature was verified analytically before the start of the study (Analytical report: 01Y0262/078001)
Details on mating procedure:
The animals paired by the breeder (time-mated animals) were supplied on the day of evidence of mating; this day is referred to as GD 0 and the following day as GD 1.
Duration of treatment / exposure:
from GD 6 through GD 19
Frequency of treatment:
Once daily
Duration of test:
25 days
Dose / conc.:
0 mg/kg bw/day
Remarks:
Group 0
Dose / conc.:
300 mg/kg bw/day
Remarks:
Group 1
Dose / conc.:
600 mg/kg bw/day
Remarks:
Group 2
No. of animals per sex per dose:
Control: 10 (5 in prenatal study)
300 mg/kg bw: 10 (5 in prenatal study)
600 mg/kg bw: 20 (10 in prenatal study)
Control animals:
yes
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- A check for moribund and dead animals was made twice daily from Monday to Friday and once daily on Saturday, Sunday and public holidays.
- A cageside examination was conducted at least once daily for any signs of morbidity, pertinent behavioral changes and signs of overt toxicity, further abnormalities, changes, littering and lactation behavior of the dams.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were recorded on GD 0 and on GD 1, 3, 6, 8,10, 13, 15, 17, 19, 20

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption was recorded on GD 1, 3, 6, 8,10, 13, 15, 17, 19, 20

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Gross-pathological examination
- Weight of the unopened uterus
- Weight of liver (livers will be retained in 4% buffered formaldehyde solution and transferred to Pathology Laboratory for potential histopathological processing and evaluation)


Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Weight of the unopened uterus
- Number of corpora lutea
- Number of implantations (differentiated according to live and dead fetuses and early or late resorptions)
- Early resorptions in animals that do not appear to be pregnant and animals with single-horn pregnancy
- Site of implantations in the uterus
Fetal examinations:
After the fetuses have been removed from the uterus, the following examinations or weight determinations were carried out:
- Weight of each fetus
- Sex
- Weight of the placentas
- Gross-pathological examination of the fetuses after dissection from the uterus (including abnormalities of the fetal membranes, placentas, amniotic fluid and umbilical cord); all fetuses are sacrificed by subcutaneous injection of pentobarbital.
- About half of the fetuses of each dam: Skeletons/cartilage examination
- About half of the fetuses of each dam: Soft tissue examination
Statistics:
Means and standard deviations will be calculated.
- DUNNETT test (two-sided): Food consumption, body weight and body weight change; duration of gestation
- KRUSKAL-WALLIS and WILCOXON test: Weight of liver
Indices:
Female fertility index
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
600 mg/kg bw/day: Salivation (20 out of 20 animals), respiratory sounds (7 out of 20 animals) were observed.
300 mg/kg bw/day: Salivation after treatment (10 out of 10 animals).
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, treatment-related
Description (incidence):
600 mg/kg bw/day:
- One animal sacrificed moribund on GD 14 (gross pathological examination revealed stomach erosions and no feces in intestine).
- One animal found dead on GD 20 (gross pathological examination revealed stomach ulcerations).

300 mg/kg bw/day: no effects observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
600 mg/kg bw/day:
- Transient statistically significantly reduced mean body weight compared to the control group (set to 100 %), i.e. on GD 13 (93%). No statistical relevant difference on GD 20.
- Transient statistically significantly reduced mean body weight change compared to the control group (set to 100 %), i.e. between GD 8-10 (52 %). No statistically significantly difference between GD 0-20.

300 mg/kg bw/day: no effects observed
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
600 mg/kg bw/day: Transient statistically significantly reduced mean food consumption compared to the control group (set to 100 %), i.e. between GD 6-8 (81 %) and GD 8-10 (78 %). No statistically significantly difference between GD 0-20.

300 mg/kg bw/day: no effects observed.
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
Endocrine findings:
not examined
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):
600 mg/kg bw/day - Prenatal study part (10 dams sacrificed on GD 20): Statistically significantly increased mean liver weight (118 %) compared to the control group (set to 100 %). Since no macroscopic or microscopic examinations were made, no conclusion on adversity is possible.

300 mg/kg bw/day: no effects observed.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
600 mg/kg bw/day - Prenatal study part (10 dams sacrificed on GD 20): Stomach erosions/ulcera (8 out of 10 animals)
300 mg/kg bw/day - Prenatal study part (5 dams sacrificed on GD 20): Stomach erosions/ulcera (4 out of 5 animals)
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not specified
Number of abortions:
not specified
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
600 mg/kg bw/day - Prenatal study part (10 dams sacrificed on GD 20): Slightly increased post-implantation loss compared to the control group and the historical control data (11.8 % vs. 5.2 % in control), not statistically significant.
300 mg/kg bw/day - Prenatal study part (5 dams sacrificed on GD 20): Slightly increased post-implantation loss compared to the control group and the historical control data (15.2 % vs. 5.2 % in control), not statistically significant.
Total litter losses by resorption:
not specified
Early or late resorptions:
no effects observed
Description (incidence and severity):
600 mg/kg bw/day - Prenatal study part (5 dams sacrificed on GD 20): Slightly increased resorptions (mean/litter) compared to the control group (0.8 % vs. 0.6 % in control), not statistically significant and without dose-response relation.
300 mg/kg bw/day - Prenatal study part (5 dams sacrificed on GD 20): Slightly ncreased resorptions (mean/litter) compared to the control group and the historical control data (1.4 % vs. 0.6 % in control),not statistically significant and without dose-response relationship
Dead fetuses:
no effects observed
Description (incidence and severity):
600 and 300 mg/kg bw/day: no effects observed
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
600 and 300 mg/kg bw/day: no effects observed
Changes in number of pregnant:
effects observed, treatment-related
Description (incidence and severity):
600 mg/kg bw/day - Postnatal study part (8 dams): One out of 8 animals did not deliver (7 animals left for further assessment)

300 mg/kg bw/day: no effects observed.
Other effects:
not specified
Details on maternal toxic effects:
Maternal toxicity: yes
Dose descriptor:
LOAEL
Remarks:
local effects
Effect level:
300 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
gross pathology
Dose descriptor:
NOAEL
Remarks:
general toxicity
Effect level:
300 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
body weight and weight gain
clinical signs
food consumption and compound intake
mortality
Fetal body weight changes:
no effects observed
Description (incidence and severity):
600 mg/kg body weight/day - Prenatal study part (10 dams sacrificed on GD 20): Fetuses: No test substance-related findings
300 mg/kg body weight/day - Prenatal study part (5 dams): Fetuses: No test substance-related findings
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
600 mg/kg body weight/day - Prenatal study part (10 dams sacrificed on GD 20): Fetuses: No test substance-related findings
300 mg/kg body weight/day - Prenatal study part (5 dams): Fetuses: No test substance-related findings
Changes in sex ratio:
no effects observed
Description (incidence and severity):
600 mg/kg body weight/day - Prenatal study part (10 dams sacrificed on GD 20): Fetuses: No test substance-related findings
300 mg/kg body weight/day - Prenatal study part (5 dams): Fetuses: No test substance-related findings
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
600 mg/kg body weight/day - Prenatal study part (10 dams sacrificed on GD 20): Fetuses: No test substance-related findings
300 mg/kg body weight/day - Prenatal study part (5 dams): Fetuses: No test substance-related findings
Anogenital distance of all rodent fetuses:
not examined
Changes in postnatal survival:
no effects observed
Description (incidence and severity):
600 mg/kg body weight/day - Prenatal study part (10 dams sacrificed on GD 20): Fetuses: No test substance-related findings
300 mg/kg body weight/day - Prenatal study part (5 dams): Fetuses: No test substance-related findings
External malformations:
no effects observed
Description (incidence and severity):
600 mg/kg body weight/day - Prenatal study part (10 dams sacrificed on GD 20): Fetuses: No test substance-related findings
300 mg/kg body weight/day - Prenatal study part (5 dams): Fetuses: No test substance-related findings
Skeletal malformations:
no effects observed
Description (incidence and severity):
600 mg/kg body weight/day - Prenatal study part (10 dams sacrificed on GD 20): Fetuses: No test substance-related findings
300 mg/kg body weight/day - Prenatal study part (5 dams): Fetuses: No test substance-related findings
Visceral malformations:
no effects observed
Description (incidence and severity):
600 mg/kg body weight/day - Prenatal study part (10 dams sacrificed on GD 20): Fetuses: No test substance-related findings
300 mg/kg body weight/day - Prenatal study part (5 dams): Fetuses: No test substance-related findings
Other effects:
not specified
Dose descriptor:
NOAEL
Basis for effect level:
other: No test item-related findings
Remarks on result:
not determinable
Remarks:
No NOAEL identified
Abnormalities:
not specified
Developmental effects observed:
no

Gestation

Test group (mg/kg bw/d)

0 (0; control)

1 (300)

2 (600)

Mortality

-

-

1 animal sacrificed moribund on GD 14

1 animal found dead on GD 20

Clinical observation

NAD

Salivation after treatment (10/10)

Salivation after treatment (20/20)

Respiratory sounds (7/20)

Labored respiration (1/20)

Fur smeared with urine (1/20)

FC (day 6-8)

FC (day 8-10)

FC (day 0-6)

16.1 g

17.4 g

15.1 g

16.0 g (99 %)

16.9 g (97 %)

15.4 g (102 %)

13.1 g (81 %)**

13.5 g (78 %)**

15.6 g (103 %)

FC (day 6-20)

18.1 g

17.9 g (99 %)

16.7 g (92 %)

FC (day 0-20)

17.2 g

17.1 g (99 %)

16.4 g (95 %)

BW (day 0)

162.6 g

157.7 g (97 %)

160.5 g (99 %)

BW (day 6)

BW (day 13)

190.8 g

223.1 g

187.8 g (98%) 215.3 g (96 %)

189.8 g (99 %)

208.5 g (93 %)*

BW (day 20)

281.4 g

268.5 g (95 %)

263.5 g (94 %)

BWC (day 8-10)

BWC (day 0-6)

11.8 g

28.2 g

8.7 g (74 %)

30.1 g (107 %)

6.2 g (52 %)**

29.3 g (104 %)

BWC (day 6-20)

90.5 g

80.7 g (89 %)

73.8 g (81 %)

BWC (day 0-20)

118.7 g

110.8 g (93 %)

103.3 g (87 %)

Duration of Gestation

21.8

22.0

22.3

Cesarean section

Test group

(mg/kg bw/d)

0 (0; control)

1 (300)

2 (600)

Uterus weight

52.8 g

43.5 g (82 %)

49.7 g (94 %)

Carcass weight

228.8 g

221.0 g (97 %)

216.1 g (94 %)

Corrected body weight gain

40.3 g

36.6 g (91 %)

28.7 g (71 %)

Implantation sites (mean/litter)

10.4

9.4

10.2

Postimplantation loss

5.2

15.2

11.8

Resorptions (mean/litter)

0.6

1.4

0.8

Live fetuses/dam

9.8

8.0

10.4

Placental weights

0.43 g

0.41 g (95 %)

0.39 g (91 %)

Fetal weights

3.5 g

3.7 g (104 %)

3.6 g (101 %)

Total external malformations

Fetuses:

Litter:

Affected fetuses/litter:

 

 

0/49 (0.0 %)

0/5 (0.0 %)

0.0 %

 

 

0/40 (0.0 %)

 0/5 (0.0 %)

0.0 %

 

 

0/94 (0.0 %)

0/9 (0.0 %)

0.0 %

Total visceral malformations

Fetuses:

Litter:

Affected fetuses/litter:

 

 

0/24 (0.0 %)

0/5 (0.0 %)

0.0 %

 

 

0/19 (0.0 %)

0/5 (0.0 %)

0.0 %

 

 

0/44 (0.0 %)

0/9 (0.0 %)

0.0 %

Total skeletal malformations

Fetuses:

Litter:

Affected fetuses/litter:

 

 

0/25 (0.0 %)

0/5 (0.0 %)

0.0 %

 

 

0/21(0.0 %)

0/5 (0.0 %)

0.0 %

 

 

0/50(0.0 %)

0/9 (0.0 %)

0.0 %

Cesarean section: Pathology (Dams)

Test group

(mg/kg bw/d)

0 (0; control)

1 (300)

2 (600)

Incidence of gross lesions

NAD

Forestomach: Erosion/Ulcer (4/5)

Forestomach: Erosion/Ulcer (8/10)

Liver weights (absolute)

10.886 g

11.106 g (102 %)

12.203 g (112 %)

Liver weights (relative)

4.761 g

5.018 g (105 %)

5.632 g (118 %)*

Conclusions:
In conclusion, signs of maternal toxicity occurred at 300 mg/kg bw/day, comprising local effects (strong ulcera in the stomach) and at 600 mg/kg bw/day, as evident by systemic effects. In the postnatal part of the study, in the dose group of 600 mg/kg bw/day the live birth index was reduced (91 %  vs. control 100 %). Pups of the high dose group were less viable compared to the control group (viability index 43 % and 100 %, respectively); and reduced body weights were recorded.
No substance related findings were observed in foetuses.

Based on these observations the following effect levels were determined:
Dams (F0):
- LOAEL for local effects: 300 mg/kg bw/day
- NOAEL for systemic effects: 300 mg/kg bw/day
- LOAEL for reproductive performance: 600 mg/kg bw/day
Offspring (F1)
- NOAEL for foetuses: not determinable; no effects observed
- NOAEL for pups: 300 mg/kg bw/day
Executive summary:

In the Pre- and Postnatal Developmental Toxicity Screening test the test compound 2-Dimethylaminoethanol was administered via oral gavage to time-mated Wistar rats from GD 6 through GD 19 (prenatal study part) and from GD 6 through PND 3 (postnatal study part). The following concentrations were administered: i.e. 0 mg/kg bw/day (test group 0, 10 animals), 300 mg/kg bw/day (test group 1, 10 animals), 600 mg/kg bw/day (test group 2, 20 animals). The duration of treatment covered a 2-weeks in gestation and up to the 3rd day after parturition up to the day of scheduled sacrifice of the animals.

Animals dosed with the high dose of 600 mg/kg bw/day showed after treatment the following symptoms: salivation (20/20); respiratory sounds (7/20), statistically significantly reduced mean food consumption (i.e. between GD 6-8 (81 %) and GD 8-10 (78 %) compared to control group (100 %)), statistically significantly reduced mean body weight (i.e. on GD 13 (93 %) compared to the control group (100 %), statistically significantly reduced mean body weight change (i.e. between GD 8-10 (52 %) compared to the control group (100 %). Moreover, one animal was found dead on GD20 (gross pathological examination revealed stomach ulcerations) and one animal was sacrificed moribund on GD14 (gross pathological examination revealed stomach erosions and no feces in intestine).

For the prenatal study part, 10 dams were sacrificed on GD 20 and subsequently examined. In the dams the following observation were made: stomach erosions/ulcera (8 /10) and statistically significantly increased mean liver weight (118 %) compared to the control group (100 %). The following effects on reproduction performance were observed, but assessed statistically not significant and without a dose-response relationship: Slightly increased post-implantation loss (11.8 % vs. 5.2 % in control (compared to the control group and the historical control data)).

For the postnatal part, the remaining 8 animals were sacrificed on PND 4 and subsequently examined. In these dams the following observations were made: stomach erosions/ulcera (8/8), 1/8 dams did not deliver (7 animals left for further assessment), salivation after treatment (7/7), statistically significantly reduced mean food consumption (81 % between PND 0 – 4) compared to the control group (100 %) and a live birth index of 91 % compared to the control group (100 %). A slightly increased post-implantation loss (9.9 % vs. 2.0% in control) was noted, but assessed as statistically not significant.

Animals dosed with the low dose of 300 mg/kg bw/day showed after treatment the following symptoms: salivation (10/10).

For the prenatal study part, 5 dams were sacrificed on GD 20 and subsequently examined, in 4/5 dams stomach erosions/ulcera was recorded. The following effects on reproduction performance were observed, but assessed statistically not significant and without a dose-response relationship: Slightly increased post-implantation loss(15.2 % vs. 5.2 % in control (compared to the control croup and the historical control data)) and slightly increased resorptions (mean/litter) (1.4% vs. 0.6% in control (compared to the control group and the historical control data)).

For the postnatal part, the remaining 5 animals were sacrificed on PND 4 and subsequently examined. In these dams the following local effects were observed: stomach erosions/ulcera (5/5).A slightly increased post-implantation loss (5.5 % vs. 2.0% in control) was noted, but assessed as statistically not significant.

Concerning the effects and observations on foetuses of dams treated with 600 mg/kg bw/day, no test-substances-related findings were reported (prenatal part).

For thepostnatal study part,pups derived of dams dosed with 600 mg/kg bw/day, thefollowing is reported (as1/8 dams did not deliver, only 7 litters left for further assessment):six stillborn pups in 7 litters (64 pups in toto, 58 liveborn), 24/58 pups died ahead of schedule, 9/58 pups were cannibalized, no more pups alive in 4 out of 7 litters (2 litters on PND 1, 1 on PND 2, 1 on PND 3), viability index of 43 % (control: 100%), statistically significantly reduced mean body weight (i.e. on PND 1 (76 %) and on PND 4 (71 %) compared to the control group (100 %)), statistically significantly reduced mean body weight change ((57 % between PND 1-4) compared to the control group (100 %)) and 12 runts (no runts in the control).

In conclusion, signs of maternal toxicity occurred at 300 mg/kg bw/day, comprising local effects (strong ulcera in the stomach) and at 600 mg/kg bw/day, as evident by systemic effects. In the postnatal part of the study, in the dose group of 600 mg/kg bw/day the live birth index was reduced (91 %  vs. control 100 %). Pups of the high dose group were less viable compared to the control group (viability index 43 % and 100 %, respectively); and reduced body weights were recorded.

No substance related findings were observed in foetuses.

Based on these observations the following effect levels were determined:

Dams (F0):

- LOAEL for local effects: 300 mg/kg bw/day

- NOAEL for systemic effects: 300 mg/kg bw/day

- LOAEL for reproductive performance: 600 mg/kg bw/day

Offspring (F1)

- NOAEL for foetuses: not determinable; no effects observed

- NOAEL for pups: 300 mg/kg bw/day

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
250 mg/kg bw/day
Study duration:
subacute
Species:
rabbit
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEC
100 ppm
Study duration:
subacute
Species:
rat
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

There is - at the moment - not sufficient information on effects of DMAE on reproductive performance and fertility from repeated dose toxicity and developmental studies available. According to the decision with the decision number CCH-D-2114340872-49-01/F following a compliance check decision under Articles 50 and 51 of the REACH Regulation (Regulation (EC) No 1907/2006) issued in August 2016, among others an Extended one-generation reproductive toxicity study in rats, oral route with the registered substance 2-dimethylaminoethanol (according to OECD 443; 0 weeks premating exposure, Cohort A, Cohort B without extension as well as Cohorts 2A and 2B) has been requested.

Multiple laboratory delays occurred in relation to the OECD 443 study for the substance intended to be used for a read-across (CAS 111-42-2), triggering further delays for the planned screening tests for the registered substance, as well as a change in the intended read-across strategy.

The OECD 443 study is currently being conducted at Charles River laboratories (Ashland). The registrant will submit the testing results as soon as the study results with the registered substance are available.

Available Studies

The Prenatal Developmental Toxicity Study in rabbits was conduced as requested according to OECD 414 and in accordance with GLP (Millard, 2021). 2-Dimethylaminoethanol was administered orally by gavage to time-mated New Zealand White rabbits at doses of 0, 30, 100 and 250 mg/kg bw/day; animals were dosed once daily during Gestation Days 7–28. Based on adverse effects on maternal survival, maternal body weight losses and/or lower body weight gain, reduced food consumption, and corresponding clinical observations at 250 mg/kg/day, considered secondary to local corrosion of the gastric mucosa, a dosage level of 100 mg/kg/day was considered to be the NOAEL for maternal toxicity. Lower mean fetal body weights observed at 250 mg/kg/day were considered secondary to the adverse maternal effects; therefore, a dosage level of 250 mg/kg/day was considered to be the NOAEL for prenatal development.

Leung et al. exposed pregnant rats to vapours of DMAE at 1, 10, 30 and 100 ppm for 6 h per day on gestational days 6-15 (analytically measured mean concentrations (±

SD) of 10.4 ± 0.86, 29.8 ± 2.14 and 100 ± 4.9 ppm . There was no evidence of embryonic or fetal toxicity, including teratogenicity, at any exposure concentration employed (including that which produced maternal toxicity). There were no statistically significant increases in the incidences of individual malformations, malformations by category (external, visceral including craniofacial and skeletal) or total malformations at any exposure concentration relative to controls. The incidence of litters with one or more fetuses having variations did not differ for any individual external or visceral variations, or for total external, visceral or skeletal variations, or for total variations. The incidences of six skeletal variations out of 120 evaluated were statistically significantly different in DMEA-exposed groups relative to that of controls.

Four of these variations were decreased in incidence; only one fetal variation, the split (bipartite) cervical centrum, was elevated at 100 ppm relative to controls (an exposure concentration which also produced maternal toxicity). In the absence of any other indications of delayed ossification or fetal body weights, the observed fetal variation does not suggest a consistent pattern of fetal toxicity. Inconsistent pattern of skeletal variations reported were poorly ossified cervical centrum, bilobed thoracic centrum, bilobed sternebrae, unossified proximal phalanges of the forelimb, and increased incidences of split cervical centra, and bilobed thoracic centrum.

Fetal body weights per litter were statistically significantly increased at 100 ppm relative to controls. There were no other indications of fetotoxicity such as reduced fetal body weight. The NOAEL is at or above 100 ppm for embryofetal toxicity and teratogenicity.

In the Modified Developmental Toxicity Screening Study in Wistar rats with DMAE (BASF, 2008), 2 -dimethylaminoethanol was administered to time-pregnant female rats orally by gavage from gestation day (GD) 6 through GD 19 (prenatal study part) or GD 6 through postnatal day (PND) 3 (postnatal study part) at dose levels 300 and 600 mg/kg bw/day.

Signs of maternal toxicity occurred at 300 mg/kg bw/day, comprising local effects (strong ulcera in the stomach) and at 600 mg/kg bw/day, as evident by systemic effects. In the prenatal part of study, no test substance-related findings were observed in fetuses at the both dose levels. In the high dose group (600 mg/kg bw), viability of pups was severely affected (viability index 43 % compared to 100% in control). Six out of 64 pups were stillborn. From the 58 liveborn pups, 24 died ahead of schedule. Nine pups were cannibalized. No pups were alive in 4 out of 7 litters. Body weight was significantly reduced compared to the control group (set to100%), i.e, on PND 1 (76 %) and on PND 4 (71 %) and body weight change (57 % between PND 1 -4). Twelve runts were born (no runts in the control). Pups in the 300 mg/kg bw dose group were not affected.

In conclusion, effects on pups occurred only at doses of maternal toxicity.

Toxicity to reproduction: other studies

Additional information

DMAE and Choline

There is evidence that DMAE can act as toxicant for developing conceptus under certain conditions. Pups delivered by rats fed during pregnancy a choline-deficient diet containing 1 % DMAE died within 36 h of birth (Katyal and Lombardi, 1978, Zahniser et al., 1978). The concentration of sphingomyelins, phophatidyl cholines, and disaturated phosphatidyl cholines in the lungs of these pups were lower than those in the lungs of pups delivered by dams fed a choline-supplemented diet (CS) (Katyal and Lombardi, 1978). The amount of surfactant isolated from the lungs of the pups was also reduced. These changes were accompanied by alterations in the activity of enzymes (choline kinase and phosphotransferase) involved in the synthesis of lung lecithins. The authors strongly suggest that pups delivered by dams fed a choline deficient diet containing 1 % DMAE died of respiratory distress syndrome due to altered metabolism of lung surfactant. But since these results were only obtained, when the rats were fed a choline deficient diet, and since it is not clear which influence this diet had on the development of the fetal body, one has to jugde this carefully.

 

In another oral feeding study, pregnant rats were either maintained on a choline deficient diet or a choline deficient diet supplemented with 0.8 % choline chloride, 1 % DMAE, or 1 % monomethylethanolamine (MMEA) from gestation-day six through two weeks postpartum (Zahniser et al., 1978). Pregnancies progressed to term equally well for all treatment groups and litters of similar sizes were delivered. However, only 18/253 offspring of rats exposed to DMAE, survived for more than 36 hours after birth, whereas all offspring of control rats survived to 15 days (end of observation period). It confirms findings of Katyal and Lombardi showing also high mortality of new born rats.

Pups born to dams fed the DMAE-supplemented diet demonstrated moderate degrees of glycogen and fatty infiltrations in their livers. Measurable amounts of DMAE (72.2 ± 12.7 nmol/g) were observed in the brains of pups from DMAE-supplemented dams. No DMAE was found in the brains of pups from choline-deficient diets or choline-supplemented diets. Levels of choline and acetylcholine in the brains were elevated 53 % and 36 % in pups from the DMAE supplemented group, relative to the choline-deficient pup brains. Distributions of phosphatidyl choline (38.9 %) and phosphatidyl aminoethanol (16.2 %) from the brains of DMAE supplemented pups were markedly lower than from pups derived from the choline-deficient 52.4 % and 25.3 %, respectively) or choline-supplement groups (52.4 % and 22.7 %, respectively) (Zahniser et al., 1978).

 

DMAE is a potent inhibitor of choline uptake and choline kinase in vitro

Fisher et al studied choline metabolism in gastrulation/neurulation stage mouse embryos in vitro (Fisher et al., 2002). The embryo culture was treated with 375 µM DMAE and free choline, betaine, incorporation of choline into phosphocholine , phosphatidylcholine (PtdCho) and sphingomyelin were measured. After 10 h of embryo culture with DMAE, 14C-choline in the embryo was reduced by 60 %; in both embryos and yolk sacs, incorporation of 14C-choline into phosphocholine, PtdCho, and sphingomyelin was decreased to 25 %, 35 % and 50 % of control values, respectively. In DMAE-treated embryos, labeled betaine was threefold higher than in controls. In embryos and yolk sacs, treatment with DMAE resulted in reduced phosphatidylethanolamine (PtdEtn) synthesized from 3H-ethanolamine. Treatment with either DMAE resulted in a 15 % increase in embryonic ceramide. Analysis of [14C] choline uptake by the embryos and yolk sacs indicated that DMAE-treatment inhibited choline uptake and choline kinase. The inhibition of choline uptake and metabolism resulted in increased cell death and craniofacial and neural tube defects in neurulation stage mouse embryos grown in culture. The authors suggest that reduced PtdCho availability caused by decreased choline transport and/or inhibition of PtdCho synthesis through the CDP-choline pathway could be responsible for the defects. DMAE caused a reduction in PtdEtn synthesis, reflecting that besides being a choline analogue, it is structurally related to ethanolamine. Further investigations into the effect of DMAE on ethanolamine metabolism is necessary to understand the full impact of DMAE on embryonic development.

Justification for classification or non-classification

Fertility and developmental findings in the available studies on DMAE and MMEA

There is no two generation study/ extended one generation reproductive toxicity study available for N,N-Dimethylethanolamine (CAS No. 108-01-1). However, according to the decision with the decision number CCH-D-2114340872-49-01/F following a compliance check decision under Articles 50 and 51 of the REACH Regulation (Regulation (EC) No 1907/2006) issued in August 2016, among others an Extended one-generation reproductive toxicity study in rats, oral route with the registered substance 2-dimethylaminoethanol (according to OECD 443; 0 weeks premating exposure, Cohort A, Cohort B without extension as well as Cohorts 2A and 2B) and a Pre-natal developmental toxicity study in rabbits (OECD 414), oral route with the registered substance 2-dimethylaminoethanol has been requested.

Multiple laboratory delays occurred in relation to the OECD 443 study for the substance intended to be used in a read-across (CAS 111-42-2), triggering further delays for the planned screening tests for the registered substance, as well as a change in the intended read-across strategy.

The OECD 443 study is currently being conducted at Charles River laboratories (Ashland). The registrant will submit the testing results as soon as the study results with the registered substance are available. The results of the OECD 414 are available and integrated in the data set.

The Prenatal Developmental Toxicity Study was conduced according to OECD 414 and in accordance with GLP (Millard, 2021). 2-Dimethylaminoethanol was administered orally by gavage to time-mated New Zealand White rabbits at doses of 0, 30, 100 and 250 mg/kg bw/day; animals were dosed once daily during Gestation Days 7–28. The following parameters and end points were evaluated in this study: clinical signs, body weights, body weight gains, gravid uterine weights, food consumption, gross necropsy findings, stomach histopathology, intrauterine growth and survival, and fetal morphology.

In the 250 mg/kg/day group, 1 female was found dead on Gestation Day 23, 4 females aborted during Gestation Days 20–28, and 1 female delivered on Gestation Day 29, prior to the scheduled necropsy. All 6 of these females were noted with severe body weight losses (11.4 % to 21.7 %), reduced food consumption (0 to 86 g/day), and corresponding clinical observations (a thin body and/or excreta-related findings) noted at the daily examinations prior to death, abortion, or delivery. At necropsy, macroscopic findings including dark red discoloration, dark red foci, thickened, and raised foci in the stomach with microscopic correlates (hemorrhage, necrosis, edema, fibrosis, and heterophilic infiltrates) were noted for the majority of these females. The effects on survival to the scheduled necropsy in the 250 mg/kg/day group were considered secondary to the test substance-related effects on body weight and food consumption in this group. One female in the 30 mg/kg/day group was found dead on Gestation Day 25; necropsy findings for this female were indicative of a dosing error, and therefore this death was not attributed to the test substance. In the control group, 1 female was euthanized in extremis on Gestation Day 8 due to limited usage of a hindlimb (tibial fracture). All other females survived to the scheduled necropsy on Gestation Day 29. Test substance-related increased incidences of decreased fecal output, which corresponded to reduced mean food consumption, were noted for surviving females in the 250 mg/kg/day group at the daily examinations. No test substance-related clinical observations were noted at the daily examinations at 30 and 100 mg/kg/day or 1–2 hours postdosing at any dosage level.

Test substance-related mean body weight losses and lower mean body weight gains, with corresponding lower mean food consumption, were noted in the 250 mg/kg/day group throughout the treatment period (Gestation Days 7–29) when compared to the control group. As a result, absolute mean body weights in this group were 5.0 % to 11.2 % lower than the control group during Gestation Days 12–29. In addition, lower mean corrected body weight, corrected mean body weight gain, and mean gravid uterine weight were noted in the 250 mg/kg/day group compared to the control group. Mean maternal body weights, body weight gains, corrected body weights, corrected body weight changes, gravid uterine weights, and food consumption in the 30 and 100 mg/kg/day groups were unaffected by test substance administration. Test substance-related gross observations were observed in the stomach of the 250 mg/kg/day group females at the scheduled necropsy, including dark red discoloration, dark red foci, thickened, and raised foci; comparable findings were also noted for females that did not survive to the scheduled necropsy. Microscopic findings in the stomach were observed in all test substance-administered groups and included hemorrhage, necrosis, edema, fibrosis, and heterophilic infiltrates. Based on the absence of other signs of toxicity, the microscopic findings in the 30 and 100 mg/kg/day groups were considered nonadverse.

Test substance-related lower (12.16 % to 13.83 %) mean fetal body weights (male, female, and sexes combined) were noted in the 250 mg/kg/day group, which resulted in a lower mean gravid uterine weight in this group. However, the mean fetal weight values in this group were within the respective ranges of values noted in historical control data, and the differences were considered secondary to the maternal toxicity noted at this dosage level, and nonadverse. Intrauterine growth in the 30 and 100 mg/kg/day groups and intrauterine survival in the 30, 100, and 250 mg/kg/day groups were unaffected by test substance administration. There were no test substance-related fetal malformations or developmental variations noted at any dosage level.

In conclusion, based on adverse effects on maternal survival, maternal body weight losses and/or lower body weight gain, reduced food consumption, and corresponding clinical observations at 250 mg/kg/day, considered secondary to local corrosion of the gastric mucosa, a dosage level of 100 mg/kg/day was considered to be the NOAEL for maternal toxicity. Lower mean fetal body weights observed at 250 mg/kg/day were considered secondary to the adverse maternal effects; therefore, a dosage level of 250 mg/kg/day was considered to be the NOAEL for prenatal development.

The available data on effects of DMAE on reproductive performance and fertility and fetal and neonatal development show DMAE not to be a reproductive or developmental toxicant. In the repeated dose toxicity studies, no histopathological changes in the gonads were observed after repeated exposure to DMAE in a 90-day inhalation study in rats (Klonne et al., 1987). In the OECD 414 study (Leung and Ballantyne, 1996) there were also no treatment-related effects on gonads or on any gestational parameters, including pre- and post implantation loss or sex ratio. There were a few sporadic changes in some implantation data, but no consistent pattern of concentration-related effect was evident. A NOAEL of 100 ppm (4.10 mmol/m³) or greater was established for embryofetal toxicity and teratogenicity. A NOAEL for maternal toxicity was estimated at 10 ppm (0.41 mmol/m³). In an older publication, gestation and parturition of pregnant rats fed with a choline deficient diet supplemented with 1 % DMAE proceeded normally (Katyal and Lombardi, 1978, Zahniser et al., 1978). Litters of equal size were delivered compared to the control group. Furthermore, no signs of maternal toxicity were noted (Zahniser et al., 1978). However, only 18/253 offspring derived from pregnant rats maintained on a choline-deficient diet supplemented with 1 % DMAE survived for more than 36 hours postpartum. Opposite to this fact, pups delivered by dams fed with choline deficient diet but without DMAE survived. The pups demonstrated moderate degrees of glycogen and fatty infiltrations in their livers. Measurable amounts of DMAE (72.2 ± 12.7 nmol/g) were observed in their brains. In addition, levels of choline and acetylcholine in the brains were elevated 53 % and 36 % respectively. No histological or cytological alterations were detected in the brain of the pups. Authors supposed that death of pups was probably due to alteration of choline uptake resulted in respiratory distress syndrome because of altered metabolism of lung surfactant.

 

In the Pre- and Postnatal Developmental Toxicity Screening test the test compound 2-Dimethylaminoethanol was administered via oral gavage to time-mated Wistar rats from GD 6 through GD 19 (prenatal study part) and from GD 6 through PND 3 (postnatal study part). The following concentrations were administered: i.e. 0 mg/kg bw/day (test group 0, 10 animals), 300 mg/kg bw/day (test group 1, 10 animals), 600 mg/kg bw/day (test group 2, 20 animals). The duration of treatment covered a 2-weeks in gestation and up to the 3rd day after parturition up to the day of scheduled sacrifice of the animals.

Animals dosed with the high dose of 600 mg/kg bw/day showed after treatment the following symptoms: salivation (20/20); respiratory sounds (7/20), statistically significantly reduced mean food consumption (i.e. between GD 6-8 (81 %) and GD 8-10 (78 %) compared to control group (100 %)), statistically significantly reduced mean body weight (i.e. on GD 13 (93 %) compared to the control group (100 %), statistically significantly reduced mean body weight change (i.e. between GD 8-10 (52 %) compared to the control group (100 %). Moreover, one animal was found dead on GD20 (gross pathological examination revealed stomach ulcerations) and one animal was sacrificed moribund on GD14 (gross pathological examination revealed stomach erosions and no feces in intestine).

For the prenatal study part, 10 dams were sacrificed on GD 20 and subsequently examined. In the dams the following observation were made: stomach erosions/ulcera (8 /10) and statistically significantly increased mean liver weight (118 %) compared to the control group (100 %). The following effects on reproduction performance were observed, but assessed statistically not significant and without a dose-response relationship: Slightly increased post-implantation loss (11.8 % vs. 5.2 % in control (compared to the control group and the historical control data)).

For the postnatal part, the remaining 8 animals were sacrificed on PND 4 and subsequently examined. In these dams the following observations were made: stomach erosions/ulcera (8/8), 1/8 dams did not deliver (7 animals left for further assessment), salivation after treatment (7/7), statistically significantly reduced mean food consumption (81 % between PND 0 – 4) compared to the control group (100 %) and a live birth index of 91 % compared to the control group (100 %). A slightly increased post-implantation loss (9.9 % vs. 2.0 % in control) was noted, but assessed as statistically not significant.

Animals dosed with the low dose of 300 mg/kg bw/day showed after treatment the following symptoms: salivation (10/10).

For the prenatal study part, 5 dams were sacrificed on GD 20 and subsequently examined, in 4/5 dams stomach erosions/ulcera was recorded. The following effects on reproduction performance were observed, but assessed statistically not significant and without a dose-response relationship: Slightly increased post-implantation loss(15.2 % vs. 5.2 % in control (compared to the control croup and the historical control data)) and slightly increased resorptions (mean/litter) (1.4 % vs. 0.6 % in control (compared to the control group and the historical control data)).

For the postnatal part, the remaining 5 animals were sacrificed on PND 4 and subsequently examined. In these dams the following local effects were observed: stomach erosions/ulcera (5/5). A slightly increased post-implantation loss (5.5 % vs. 2.0 % in control) was noted, but assessed as statistically not significant.

Concerning the effects and observations on foetuses of dams treated with 600 mg/kg bw/day, no test-substances-related findings were reported (prenatal part).

For the postnatal study part,pups derived of dams dosed with 600 mg/kg bw/day, thefollowing is reported (as1/8 dams did not deliver, only 7 litters left for further assessment):six stillborn pups in 7 litters (64 pups in toto, 58 liveborn), 24/58 pups died ahead of schedule, 9/58 pups were cannibalized, no more pups alive in 4 out of 7 litters (2 litters on PND 1, 1 on PND 2, 1 on PND 3), viability index of 43 % (control: 100%), statistically significantly reduced mean body weight (i.e. on PND 1 (76 %) and on PND 4 (71 %) compared to the control group (100 %)), statistically significantly reduced mean body weight change ((57 % between PND 1-4) compared to the control group (100 %)) and 12 runts (no runts in the control).

In conclusion, signs of maternal toxicity occurred at 300 mg/kg bw/day, comprising local effects (strong ulcera in the stomach) and at 600 mg/kg bw/day, as evident by systemic effects. In the postnatal part of the study, in the dose group of 600 mg/kg bw/day the live birth index was reduced (91 %  vs. control 100 %). Pups of the high dose group were less viable compared to the control group (viability index 43 % and 100 %, respectively); and reduced body weights were recorded.

No substance related findings were observed in foetuses.

Based on these observations the following effect levels were determined:

Dams (F0):

- LOAEL for local effects: 300 mg/kg bw/day

- NOAEL for systemic effects: 300 mg/kg bw/day

- LOAEL for reproductive performance: 600 mg/kg bw/day

Offspring (F1)

- NOAEL for foetuses: not determinable; no effects observed

- NOAEL for pups: 300 mg/kg bw/day

 

Evaluation of reprotoxic findings

Taken together, it is clear that DMAE (CAS No. 108-01-1) if administered per oral to rats show its effects on reproductive performance and developmental parameters only in the presence of systemic parental toxicity (Zahniser et al., 1978, Modified Developmental Toxicity Screening Study with DMAE, BASF, 2008, OECD 422 study performed with DMAE, 2019). Parental effects seen in the oral studies with DMAE (Katyal and Lombardi, 1977, Zahniser et al., 1978, BASF, 2008) reduced body weight, strong ulcera in the stomach) might be also the reason of post implantation losses and then to be a reason of a low viability of pups in neonatal stage. The effects observable by dams in the OECD 422 study performed with DMEA in 2019 were signs of systemic toxicity in females consisting of decreased food consumption and body weight, clinically apparent signs (mortality, tremors and convulsions) together with axonal degeneration in cerebellum, medulla oblongata, cervical and thoracic spinal cord at a concentration of 5000 ppm in drinking water. Males of the same concentration group showed no adverse findings.

 

DMAE as choline uptake inhibitor

Fisher et al. (2001, 2002) reported that the DMAE-induced perturbations of choline uptake and metabolism caused neural tube defects and craniofacial hypoplasia in neurulating mouse embryos in vitro. Incubation of mouse embryos (harvested at gestation-day nine) for 26 hours in DMAE containing medium (0, 250, 375, 500, or 750 μM; 0, 22.5, 33.7, 45.0, or 67.0 μg/mL) resulted in a statistically significant, dose-dependent increase in malformation rate and severity. Malformations included neural tube defects, caudal dysgenesis, craniofacial hypoplasia, and abnormal circulation. Average embryonic protein content was also reduced, significantly so in the 375 μM (33.7 μg/mL) and above DMAE treatment groups. Analysis of [14C]choline uptake by the embryos and yolk sacs indicated that DMAE treatment reduced choline uptake by 70 % in the 375 μM group (33.7 μg/mL), relative to untreated control embryos. Follow-on studies to investigate the impact of choline metabolism and to identify mechanisms associated with growth and developmental abnormalities caused by DMAE were conducted in gastrulation/ neurulation stage mouse embryos. DMAE (375 μM; 33.7 μg/mL) decreased the incorporation of [14C]choline into phosphocholine, phosphatidylcholine, and sphingomyelin to 25 %, 35 %, and 50 % of control values, respectively. Labeled betaine was threefold higher in the DMAE-treated embryos than in the control embryos. Reduced phosphatidylethanolamine synthesis from [3H]ethanolamine was noted in both treated embryos and yolk sac. DMAE treatment produced a 15 % increase in embryonic ceramide, an important cell-signaling molecule (Fisher et al., 2002).

 

Possible metabolic pathways of DMAE

Two decades ago there was an extensive search for the interrelationship between choline uptake, synthesis of phospholipids, sphyngomyelins and alkanolamines administration. Regarding DMAE, there are contradictive finding on this issue. On the one hand, it is suggested that DMAE is methylated to choline and incorporated into phospholipids (Dormard et al., 1975a) or crossed blood-brain barrier, is methylated to choline and then to acetylcholine. On the other hand, there are assumptions that DMAE is demethylated to ethanolamine and then routed toward normal metabolic pathway (Jope and Jenden, 1979). In humans, 33 % of an injected 1 g (10 mmol) dose of DMAE was excreted unchanged. It was suggested that the remaining dose might have been demethylated to ethanolamine directed toward normal metabolic pathways (“Toxicological Summary for Dimethylamine and Selected Salts and Esters”, 2002).

 

It is unclear to what extent DMAE is methylated and substituted into acetylcholine. There are totally different findings on this issue. In fetal rat brain aggregating cell cultures exposed to varying concentrations of [3H]DMAE for 72 hours, 95 %, and 5 % of the radioactivity was associated with phosphatidyl-DMAE and phosphatidylcholine, respectively. The rate of formation of radioactive products was concentration dependant up to 4 mM [3H]DMAE, the highest concentration tested. The authors calculated an apparent half-life of 24 hours (Dainous and Kanfer, 1988, cited in “Toxicological Summary for Dimethylamine and Selected Salts and Esters”, 2002).

Jope and Jenden found both in vivo and in vitro studies, that DMAE was not methylated to form choline in brain tissues. Neither acute (in vitro) nor chronic (in vivo) treatments with [2H6]DMAE had the capacity to alter levels of acetylcholine in the brain tissues. Treatment with HC-3, effectively reducing the concentration of acetylcholine in the brain, did not promote acetylcholine synthesis from DMAE in the rat brain. Although DMAE uptake, which is diffusion-dependant, was unaffected by HC-3, acetylcholine levels dropped dramatically, failing to support the contention that DMAE can supplement the synthesis of acetylcholine. Zahniser et al., (1977, 1978) obtained similar results. Zahniser et al. (1978) found also that in brains of pups those mothers were fed with choline deficient but DMAE or MMEA supplemented diet the phosphatidyl-Ch and phosphatidylaminoethanol (PAE) contents were markedly reduced. At the same time, significant amounts of DMAE of phosphatidyl-N,N dimethylaminoethanol( PDME) was present in the same brain areas.

For DEA (Diethanolamine), not methylated but containing two ethanol groups the incorporation into phosphoglycerides and sphingomyelin was described in an animal study (Mathews et al., 1994). DEA was metabolized by biosynthetic routes common to ethanolamine and was conserved, O-phosphorylated, N-methylated, and incorporated into phosphoglyceride and sphingomyelin analogues as the parent compound and as its N-methyl and N,N-dimethyl derivatives.

 

Discussion and conclusion

It appears possible that the effects of DMAE and MMEA on pre- and post-implantation losses as well as on the viability of pups may be mediated by effects on the cholin homeostasis (as described above) rather than through direct embryo toxicity. Some ethanolamines cause inhibition of cholin-uptake in the liver, subsequent perturbation of cholin-homeostasis, perturbation of C1-metabolism, perturbation of DNA-methylation and perturbation of the lipid metabolism as a consequence. Cholin metabolism is connected to phosphatidylcholine and betaine. The latter is reported to be central for the synthesis of SAM (S-Adenosyl-Methionine), a principle methylating agent for biosynthetic pathways and maintenance of critical gene methylation patterns (Stott et al. 2004; Zeisel and Blusztajn, 1994). The role of DNA methylation for normal embryonic development and cellular differentiation in high organisms is crucial (Jaenisch and Bird, 2003, Ehrlisch et al., 1982). Therefore, it is suggested that affected fertility by MMEA and DMEA as well as the low viability of pups caused by these two alkanolamines might be related to perturbation of intracellular methylation processes. Alterations of DNA methylation have also been recognized as an important component of cancer development (Daura-Oller et al., 2009).

Final evaluation of the reproductive and developmental toxicity potential of DMEA will be conducted as soon as the results of the ongoing OECD 443 and OECD 414 are available.

 

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