2-dimethylaminoethanol

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

- Key study, Millard, 2021. EOGRTS (OECD 443). Crl:CD(SD) rat, oral:gavage, 0, 15, 50, 150 mg/kg bw/day 2-Dimethylaminoethanol, NOAEL (F0 and F1 systemic toxicity) 50 mg/kg bw/day, NOAEL (F0 reproductive-, F1 neonatal-, F1 developmental toxicity and F1 immunotoxicity) 150 mg/kg/bw day.

- Supp study, Millard 2021, Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test (OECD 422), Crl:CD(SD) rat, oral: gavage, 0, 15, 50, 150 mg/kg bw/day 2-Dimethylaminoethanol, NOEAL (systemic-, reproductive-, neonatal/developmental toxicity) 150 mg/kg bw/day.

- Supp 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, NOAEL (systemic-, reproductive toxicity and fertility) 5000 ppm, NOAEL (developmental toxicity) 1000 ppm

- 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.  NOAEL (F0 systemic toxicity) 300 mg/kg bw/day, LOAEL (F0 reproductive toxicity) 600 mg/kg bw/day, NOAEL (F1 pups) 300 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. NOAEL (maternal toxicity) 10 ppm, NOAEL (embryonal toxicity and teratogenicity) ≥ 100 ppm

Link to relevant study records

Reference

Reference 1

Endpoint:

extended one-generation reproductive toxicity - with developmental neurotoxicity (Cohorts 1A, 1B without extension, 2A and 2B)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22 Apr 2020 (study Initiation Date); 05 May 2020 (Experimental Start Date & Initiation of Dosing); 24 Nov 2020 (Completion of In-life); 15 Sept 2021 (Experimental termination Date), 07 Oct 2021 (issue of final report)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to other study

Remarks:

DRF of OECD 443

Qualifier:

according to guideline

Guideline:

OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)

Version / remarks:

Jun 2018

Deviations:

no

GLP compliance:

yes

Limit test:

no

Justification for study design:

SPECIFICATION OF STUDY DESIGN FOR EXTENDED ONE-GENERATION REPRODUCTION TOXICITY STUDY WITH JUSTIFICATIONS [please address all points below]:

- Premating exposure duration for parental (P0) animals : 10 weeks as specified in Decision CCH-D-2114340872-49-01/F
- Basis for dose level selection : as specified in Decision CCH-D-2114340872-49-01/F: Dose level setting shall aim to induce some toxicity at the highest dose level. This will be determined in a Dose-range-finder (according to OECD 422).
- Inclusion/exclusion of extension of Cohort 1B: Exclusion of extention of Chort 1B as specified in Decision CCH-D-2114340872-49-01/F
- Termination time for F2 : not applicable as specified in Decision CCH-D-2114340872-49-01/F
- Inclusion/exclusion of developmental neurotoxicity Cohorts 2A and 2B: Inclusion of developmental neurotoxicity Cohorts 2A and 2B as specified in Decision CCH-D-2114340872-49-01/F
- Inclusion/exclusion of developmental immunotoxicity Cohort 3 : Exclusion of developmental immunotoxicity Cohort 3 as specified in Decision CCH-D-2114340872-49-01/F
- Route of administration : oral as specified in Decision CCH-D-2114340872-49-01/F
- Other considerations, e.g. on choice of species, strain, vehicle and number of animals [if applicable]: not applicable

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) of test material: 2-Dimethylaminoethanol (DMAE, CAS 108-01-0) from Taminco US LLC
- Receipt date: 06 Aug 2019
- Retest Date: 20 Aug 2021

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Kept in a controlled temperature area set to maintain 18 °C to 24 °C, under nitrogen
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: Test substance formulations have been previously shown to be stable over the range of concentrations used on this study for 24 hours and 10 days at room temperature and 10 days refrigerated (target of 5 °C; Engda, 2020, 01300001).
- Stability in the medium, i.e. sensitivity of the test material to hydrolysis and/or photolysis: see above
- Solubility and stability of the test material in the solvent/vehicle and the exposure medium: see above
- Reactivity of the test material with the incubation material used (e.g. plastic ware): not expected

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing (e.g. warming, grinding): none
- Preliminary purification step (if any): none
- Final concentration of a dissolved solid, stock liquid or gel: o mg/mL; 3 mg/mL for the low dose group of 15 mg/kg/day); 10 mg/mL for the mid dose group of 50 mg/kg/day and 30 mg/mL for the high dose group of 150 mg/kg/day
- Final preparation of a solid (e.g. stock crystals ground to fine powder using a mortar and pestle): not applicable

FORM AS APPLIED IN THE TEST (if different from that of starting material)
- Specify the relevant form characteristics if different from those in the starting material, such as state of aggregation, shape of particles or particle size distribution: physical decription: Colorless, clear liquid

OTHER SPECIFICS
- Other relevant information needed for characterising the tested material, e.g. if radiolabelled, adjustment of pH, osmolality and precipitate in the culture medium to which the test chemical is added: not available

Species:

rat

Strain:

other: Crl:CD(SD)

Details on species / strain selection:

The Crl:CD(SD) rat is recognized as appropriate for reproduction studies. Charles River Ashland has reproductive historical data for the Crl:CD(SD) rat. This animal model has been proven to be susceptible to the effects of reproductive toxicants.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Ashland
- Animal identification: Each animal was identified using a subcutaneously implanted electronic identification chip (BMDS system). Offspring were identified by tattoo markings applied to the digits after parturition and by microchip after weaning. Pups selected for the F1 generation retained the dam number, followed by a hyphen "-" and the digit tattoo marking (i.e., 9999-01).
- Females (if applicable) nulliparous and non-pregnant: [yes]
- Age at study initiation: (P) ca. 6 x wks old at receipt (study initiation) (P) ca. 8 x wks at experimental starting date
- Weight at study initiation: (P) Males: 178-262 g; Females: 145-210 g;
- Fasting period before study: no
- Housing: On arrival, the F0 animals were group housed (2 to 3 animals of the same sex). During cohabitation, the F0 animals were paired for mating in the home cage of the male. Following the breeding period, animals were individually housed. Following weaning (F1), animals were group housed (2 to 3 animals of the same sex) until euthanasia. Offspring selected to constitute Cohort 2B were single housed overnight prior to euthanasia. Animals were housed in solid-bottom cages containing appropriate bedding equipped with an automatic watering valve. Animals were separated during designated procedures/activities. Each cage was clearly labeled with a color-coded cage card indicating study, group, animal, cage number(s), dosage level, and sex. Cages were arranged on the racks in group order. Animals were maintained in accordance with the Guide for the Care and Use of Laboratory Animals (National Research Council, 2011). The animal facilities at Charles River Ashland are accredited by AAALAC International.
- Use of restrainers for preventing ingestion (if dermal): no - not applicable
- Diet (e.g. ad libitum): PMI Nutrition International, LLC Certified Rodent LabDiet® 5002 meal was provided ad libitum throughout the study, except during designated procedures. The feed was analyzed by the supplier for nutritional components and environmental contaminants. Results of the analysis are provided by the supplier and are on file at the Testing Facility. It is considered that there are no known contaminants in the feed that would interfere with the objectives of the study.
- Water (e.g. ad libitum): Municipal tap water after treatment by reverse osmosis and ultraviolet irradiation was freely available to each animal via an automatic watering system. Water bottles were provided, if required. Periodic analysis of the water is performed, and results of these analyses are on file at the Testing Facility. It is considered that there are no known contaminants in the water that could interfere with the outcome of the study.
- Acclimation period: yes (After receipt at the Testing Facility, the Crl:CD(SD) rats were acclimated prior to initiation of dosing.)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 68 °F to 78 °F (20 °C to 26 °C)
- Humidity (%): 30 % to 70 %
- Air changes (per hr): not specified
- Photoperiod (hrs dark / hrs light): 12-hour light/12-hour dark cycle

IN-LIFE DATES: 05 May 2020 (experimental starting date) From: To: 24 Nov 2020

Remarks:
The number of animals selected for this study was based on the OECD Guideline for the Testing of Chemicals, Guideline 443, Extended One-Generation Reproductive Toxicity Study, Jun 2018, which recommends including a sufficient number of mating pairs to yield at least 20 pregnant females per dose group. Given the possibility of nongravid animals, unexpected deaths, total litter losses, or test substance-related moribundity and/or mortality in each generation of the study, 25 F0 animals/sex/group was an appropriate number of animals to achieve the desired number of animals in each generation.

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

deionized water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Test substance dosing formulations were prepared based at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared approximately weekly and an adequate amount of each formulation was dispensed into daily aliquots, which were stored refrigerated (target of 5 °C) until use. The dosing formulations were stirred continuously during dosing.
The schedule of sample collection and analysis is given unter 'Any other information'.

VEHICLE
Preparation of Vehicle: The vehicle, deionized water, was dispensed approximately weekly for administration to Group 1 control animals and preparation of test substance formulations. Details of the dispensing of the vehicle have been retained in the Study Records.
- Justification for use and choice of vehicle (if other than water): Deionized water
- Concentration in vehicle: 0, 3, 10, 30 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: If evidence of mating was not apparent after 14 days, the animals were separated, with no further opportunity for mating.
- Proof of pregnancy: vaginal plug or sperm in vaginal smear
- After successful mating each pregnant female was caged (how): individually

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- Concentration Analysis
Duplicate sets of samples (1.0 mL) for each sampling time point were transferred to the analytical laboratory; the remaining samples were retained at the Testing Facility as backup samples. Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 10 % of theoretical concentration. After acceptance of the analytical results, backup samples were discarded.
- Homogeneity and Stability Analyses:
Test substance formulations have been previously shown to be stable over the range of concentrations used on this study for 24 hours and 10 days at room temperature and 10 days refrigerated (target of 5 °C; Engda, 2020, 01300001). Therefore, stability of test substance formulations was not assessed on this study.
The test substance is freely miscible in water. The Sponsor has documentation on file to demonstrate that the test substance formulations are solution at the range of concentrations evaluated during this study. The Sponsor provided this documentation for inclusion in the Study Records. Therefore, homogeneity assessments were not conducted on this study.

Analyses described above were performed by gas chromatography with flame ionization detection using a validated analytical procedure.
Schedule for collection of dose formulation samples for analysis are indicated in Text Table 2 under 'Any other information'.

Duration of treatment / exposure:

- F0 males: 70 consecutive days prior to mating and continuing through the day prior to euthanasia (for 19 weeks).
- F0 females: for 70 consecutive days prior to mating and continuing throughout mating, gestation, and lactation, through the day prior to euthanasia.
- Offspring selected for the F1 generation began dosing following weaning until the day prior to euthanasia (PND 52 [Cohort 1 Surplus], PND 78 [Cohort 2A], PND 91 [Cohort 1A], PND 22 [Cohort 2B], and PND 98 [Cohort 1B]).

Frequency of treatment:

once daily

Dose / conc.:

0 mg/kg bw/day

Remarks:

Control

Dose / conc.:

15 mg/kg bw/day

Dose / conc.:

50 mg/kg bw/day

Dose / conc.:

150 mg/kg bw/day

No. of animals per sex per dose:

25 / sex / dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The dosage levels for the current study were determined from the results of a previous combined 28-day toxicity study with the reproduction/developmental toxicity screening test (Millard, 2021, 01300002). In that study, male and female rats (10/group) were administered the test substance via oral gavage at dosage levels of 15, 50, and 150 mg/kg/day for 28 days (males) or for 14 days prior to mating, continuing through mating, gestation, and lactation until Lactation Day 21 (females) (weaning). F1 male and female pups (up to 10/group) were directly administered the test substance from PND 22 through 36. No effects on survival, body weights, or food consumption were noted during the F0 generation. At necropsy, test substance-related findings were noted in the glandular area of the stomach which included erosion, mixed cell infiltrate or inflammation, and edema in the submucosa in the 15, 50, and 150 mg/kg/day group females. Test substance-related gross observations of dark red area(s) in the stomach at necropsy correlated with microscopic findings of erosion and mixed cell inflammation or infiltration in the stomach of the 15 and 50 mg/kg/day group females. Test substance-related higher mean liver weights were noted in the 50 and 150 mg/kg/day group females but without any correlating gross observations or microscopic findings. No remarkable effects were noted in the F1 animals following direct dose administration. Based on these results, a high-dose level of 150 mg/kg/day was selected as it was determined that it would be tolerated for the duration of the current study, while a higher dose would not be tolerated. Dosage levels of 15 and 50 mg/kg/day were selected to determine a dose-response relationship.

- Fasting period before blood sampling for clinical biochemistry: Animals were fasted overnight prior to blood collection.

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations: general health/mortality and moribundity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily

BODY WEIGHT: Yes
- Time schedule for examinations: on a weekly basis, beginning 1 week prior to dosing, and continuing throughout the study until prior to the scheduled necropsy. Once evidence of mating was observed, female body weights were recorded on Gestation Days 0, 4, 7, 11, 14, 17, and 20 and on Lactation Days 0 (when possible), 1, 4, 7, 14, and 21. Any animals that were fasted had a body weight collected prior to and after fasting. Terminal body weights were not collected from animals found dead or euthanized moribund.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Time schedule: on a weekly basis, beginning 1 week prior to dosing, and continuing throughout the study, except during the mating period. Once evidence of mating was observed, female food consumption was recorded on Gestation Days 0, 4, 7, 11, 14, 17, and 20 and Lactation Days 1, 4, 7, 14, and 21. Food consumption was not recorded following the end of the breeding period (for females with no evidence of mating or that failed to deliver) or following weaning of offspring (for females that delivered, between weaning and euthanasia).
Food efficiency (body weight gained as a percentage of food consumed) was calculated and reported.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OTHER:
During the period of expected parturition, females were observed twice daily for initiation and completion of parturition and for dystocia (prolonged or difficult labor) or other difficulties. Beginning on the day parturition was initiated, the numbers of stillborn and live pups were recorded. Individual gestation length was calculated using the date delivery was first observed.

For more detailed information please refer to 'Any other information'

Oestrous cyclicity (parental animals):

Vaginal lavages were performed daily, and the slides were evaluated microscopically to determine the stage of the estrous cycle of each F0 female for 14 days prior to cohabitation and continuing until evidence of mating was observed or until the end of the mating period. The average cycle length was calculated for complete estrous cycles. Vaginal lavages were also performed on the day of necropsy to determine the stage of the estrous cycle.
At the end of the study, the overall pattern of each female was characterized as regularly cycling, irregularly cycling, not cycling, or insufficient data.
For more detailed information please refer to 'Any other information'

Sperm parameters (parental animals):

Parameters examined in all F0 male parental generations: Epididymis weight, sperm motility, sperm morphology, homogenization resistant spermatid count.
For more detailed information please refer to 'Any other information'

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes

PARAMETERS EXAMINED
The following parameters were examined in F1offspring: General health/mortality and moribundity, litter size, postnatal survival, total litter loss, detailed clinical observation, sex determination, weight gain, AGD, thoracic nipples/areola (males), thyroid hormone analyses (T4 and TSH),
Developmental Landmarks, Sensory Function, and Neurobehavioral Testing: Balanopreputial separation, vaginal patency, auditory startle response (Cohort 2A), FOB assessments (Cohort 2A), motor activity (Cohort 2A)
Additional observations F1 Generation: Food consumption, food efficiency (body weight gained as a percentage of food consumed), estrous cyclicity (Cohort 1A), clinical pathology (Cohort 1A)
For more details please refer to the corresponding paragraphs unter 'Any other information'

GROSS EXAMINATION OF DEAD PUPS:
yes, a necropsy was conducted for animals that died on study, and specified tissues were saved. Intact offspring that were found dead or euthanized for humane reasons during PND 0–4 were necropsied using a fresh dissection technique, which included examination of the heart and major vessels (Stuckhardt and Poppe, 1984). Findings were recorded as developmental variations or malformations, as appropriate. A gross necropsy was performed on any pup found dead or euthanized for humane reasons after PND 4.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals, including females that failed to deliver or with total litter loss, were euthanized by carbon dioxide inhalation following the selection of the F1 generation.
- Maternal animals: All surviving animals, were euthanized by carbon dioxide inhalation following the selection of the F1 generation.

GROSS NECROPSY
- Sperm Evaluations: The right cauda epididymis was excised and weighed, motility determinations were performed and microscopic examination to evaluate sperm morphology and abnormal forms of sperm (double heads, double tails, microcephalic, or megacephalic, etc.). The left testis and cauda epididymis from all males were weighed. The left cauda epididymis from all males was homogenized and analyzed for determination of homogenization resistant spermatid count.
- Complete necropsy examination: examination of the external surface, all orifices, the cranial cavity, the external surfaces of the brain and spinal cord, and the thoracic, abdominal, and pelvic cavities, including viscera. Special attention was paid to the organs of the reproductive system. The numbers of former implantation sites were recorded for females that delivered. The number of unaccounted-for sites was calculated for each female by subtracting the number of pups born from the number of former implantation sites observed. For females that failed to deliver, a pregnancy status was determined, and specific emphasis was placed on anatomic or pathologic findings that may have interfered with pregnancy.
For more details please refer to the corresponding paragraphs under 'Any other information'.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in corresponding tables under 'Any other information' were prepared for microscopic examination and weighed, respectively.

CLINICAL PATHOLOGY
- Hematology: Blood samples were analysed for parameters specified in Text Table 8 under 'Any other information'
- Coagulation: Plasma samples were analysed for the following coagulation parameters: Activated partial thromboplastin time (APTT), Prothrombin time (PT)
- Serum Chemistry: Serum was analyzed for the parameters specified in Text Table 10 under 'Any other information'
- Urinalysis: Urine samples were analysed for the parameters specified in Text Table 11 under 'Any other information'

Postmortem examinations (offspring):

SACRIFICE
- On PND 4, culled pups were euthanized by exsanguination (those pups used for blood/thyroid collection) or an intraperitoneal injection of sodium pentobarbital.
- On PND 21, nonselected pups were euthanized by exsanguination (those pups used for blood collection) or by carbon dioxide inhalation.
- Offspring selected for the F1 generation: PND 52 [Cohort 1 Surplus], PND 78 [Cohort 2A], PND 91 [Cohort 1A], PND 22 [Cohort 2B], and PND 98 [Cohort 1B]).

Terminal procedures are indicated in Text Table 26 (F1 Litters), Text Table 30 (F1 Generation, Cohort 1) and Text Table 34 (Cohort 2) under 'Any other information'

Animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

GROSS NECROPSY
- On PND 4, 1 culled pup/sex/litter was subjected to a complete necropsy examination. Pups were necropsied using a fresh dissection technique, which included examination of the heart and major vessels.
- On PND 21, nonselected pups were subjected to a complete necropsy examination, with emphasis on developmental morphology and organs of the reproductive system.

Cohort 1:
- All animals were subjected to a complete necropsy examination, which included examination of the external surface, all orifices, the cranial cavity, the external surfaces of the brain and spinal cord, and the thoracic, abdominal, and pelvic cavities, including viscera.
- Clinical Pathology (Cohort 1A)
- Splenic Lymphocyte Immunophenotyping (Cohort 1A)
- Spleen from 10 F1 animals/sex/group weighted. Determination of total spleen leukocytes for each subset (please refer to Text Table 33 in 'Any other information') and absolute lymphocyte count (cells/organ) of each subset.
- Sperm Evaluations (Cohort 1A)
- Right cauda epididymis was excised and weighed, motility, microscopic examination (abnormal forms of sperm (double heads, double tails, microcephalic, or megacephalic, etc.)), left testis and cauda epididymis from all males were weighed. The left cauda epididymis from all males was homogenized and analyzed for determination of homogenization resistant spermatid count. Sperm production rate was calculated.

Cohort 2:
- Neuropathology (Cohort 2)
- Neuropathological assessment: Central and peripheral nervous system tissues from all animals were dissected. Any abnormal coloration or lesions of the external brain and spinal cord were recorded.

HISTOPATHOLOGY / ORGAN WEIGTHS
Cohort 1
- Organ weights: Cohort 1A and/or 1B: All tissues indicated in Text Table 31 under 'Any other information' were weighed.
- Histopathology: Cohort 1A (all animals in the control and high-dose groups and from all animals found dead and euthanized in extremis):
- Tissues identified (see corresponding paragraph under 'Any other information')
- Examination of ovaries
- Examination of the testis included a qualitative assessment of the stages of spermatogenesis (Russell et al., 1990).
- Qualitative assessment of the relationships between spermatogonia, spermatocytes, spermatids, and spermatozoa seen in cross-sections of the seminiferous tubules (for males that survived to the scheduled necropsy).
- In both testes, presence of degenerative changes (e.g., vacuolation of the germinal epithelium, a preponderance of Sertoli cells, sperm stasis, inflammatory changes, mineralization, and fibrosis).
- When possible, examination of sections of the rete testis (in males)

Cohort 2
- Neuropathology (Cohort 2)
- Organ Weights and Measurements: The whole brains were removed (including olfactory bulbs), weighed, and the dimensions (length [excluding olfactory bulbs] and width) were recorded.
- Cohort 2B: (on PND 22) evaluation of sections from all major brain regions (including olfactory bulbs, cerebral cortex, hippocampus, basal ganglia, thalamus, hypothalamus, midbrain, pons, and cerebellum), all animals in C and high-dose group.
- Cohort 2A: (on PND 78) tissues identified in Text Table 35 for microscopic examination were evaluated from all animals in the control and high-exposure group.
- Cohort 2A: (on PND 78) Histopathological examination included a simple morphometric analysis (for details on the morphometric analysis, please refer to corresponding paragraph under 'Any other information'.

Statistics:

Please refer to 'Any other information'.

Reproductive indices:

Reproductive performance: male and female mating, fertility, copulation, and conception indices

Offspring viability indices:

- Mean Live Litter Size = (Total Viable Pups on PND 0)/(No. Litters with Viable Pups on PND 0)
- Postnatal Survival Between Birth and PND 0 or PND 4 (Pre-Selection) (% Per Litter) = ((Sum of (Viable Pups/Litter on PND 0 or PND 4 [Pre-Selection]/No. of Pups Born/Litter)/ (No. of Litters/Group)) x 100
- Postnatal Survival for All Other Intervals (% Per Litter) = ((Sum of (Viable Pups/Litter at End of Interval
- N/Viable Pups/Litter at Start of Interval N)) / (No. of Litters/Group)) x 100. Where N = PND 0–1, 1–4 (Pre-Selection), 4 (Post-Selection)–7, 7–14, 14–21, or 4 (Post-Selection)–21
- Total litter loss was determined when the last pup in the litter was found dead or euthanized in extremis prior to the scheduled euthanasia

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Test substance-related, adverse clinical observations of convulsions (clonic and tonic) and rales were noted at the daily examinations and postdosing observations for F0 males and females in the 150 mg/kg/day group sporadically throughout the study.
No other test substance-related clinical observations were noted.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

There were no test substance-related effects on survival for F0 animals at any dose level.
One male in the 150 mg/kg/day group was euthanized in extremis after swallowing the cannula during dose administration. In addition, 1 female in the 50 mg/kg/day group was found dead on Study Day 10. In the absence of clinical observations, necropsy findings, effects on body weight, and the lack of any evidence of mortality at 150 mg/kg/day (the highest dose tested), this death was not considered test substance-related.
In the control group, 1 female was euthanized in extremis on Gestation Day 25 due to dystocia.
All other F0 animals survived to the scheduled necropsy.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no test substance-related effects on mean absolute body weights, or body weight gains for F0 males throughout the generation and for F0 females during the premating, gestation, and lactation treatment periods at any dosage level.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There were no test substance-related effects on food consumption, or food efficiency for F0 males throughout the generation and for F0 females during the premating, gestation, and lactation treatment periods at any dosage level.

Food efficiency:

no effects observed

Description (incidence and severity):

There were no test substance-related effects on food consumption, or food efficiency for F0 males throughout the generation and for F0 females during the premating, gestation, and lactation treatment periods at any dosage level.

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

There were no test substance-related effects on hematology, coagulation, or serum chemistry for F0 males and females at any dosage level.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

There were no test substance-related effects on hematology, coagulation, or serum chemistry for F0 males and females at any dosage level.

Endocrine findings:

no effects observed

Description (incidence and severity):

No test substance-related effects on thyroid hormone concentrations (T4 or TSH) were noted for F0 males and females in the 15, 50, and 150 mg/kg/day groups.

Urinalysis findings:

no effects observed

Description (incidence and severity):

There were no test substance-related effects on urinalysis for F0 males and females at any dosage level.

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

In F0 animals, the test substance was associated microscopically with nonadverse, minimal to mild mixed cell inflammation in the stomach of males (50 and 150 mg/kg/day) and females (150 mg/kg/day), and nonadverse minimal to mild erosion of the glandular stomach of males and females in the 150 mg/kg/day group. However, the administration of the test substance was not associated with macroscopic findings or effects on organ weights or reproductive performance in F0 animals.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

There were no test substance-related effects on F0 estrous cyclicity, precoital intervals, reproductive performance (male and female mating and fertility, male copulation, and female conception indices), gestation lengths, and the process of parturition. Mean numbers of implantation sites and unaccounted for sites were comparable across groups.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

No test substance-related effects on F0 sperm parameters (mean epididymal sperm numbers, motility, progressive motility, and morphology) were noted at any dosage level.

Reproductive performance:

no effects observed

Description (incidence and severity):

There were no test substance-related effects on F0 estrous cyclicity, precoital intervals, reproductive performance (male and female mating and fertility, male copulation, and female conception indices), gestation lengths, and the process of parturition. Mean numbers of implantation sites and unaccounted for sites were comparable across groups.

Dose descriptor:

NOAEL

Remarks:

for F0 systemic toxicity

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

Dose descriptor:

NOAEL

Remarks:

for F0 reproductive toxicity

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: all tested parameters

Remarks on result:

other: based on the lack of adverse effects the highest dose tested was set was NOAEL

Critical effects observed:

no

Clinical signs:

no effects observed

Description (incidence and severity):

Prior to weaning, there were no clinical observations or effects on mean body weights or body weight changes, and absolute and relative anogenital distance on PND 1 were comparable for F1 pups in the 15, 50, and 150 mg/kg/day groups.
Following weaning, there were no test substance-related effects on survival for F1 males and females at any dose level.

Following weaning: Test substance-related, adverse clinical observations of convulsions (clonic) and rales were noted for F1 males and females in the 150 mg/kg/day group at the daily examinations and postdosing observations sporadically throughout the study in this group, and were not observed in the control group.
These observations were also noted for males and females in the F0 generation. No other test substance-related clinical observations were noted for F1 males and females at any dosage level.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

Pre-weaning:
In the 150 mg/kg/day group, lower mean postnatal survival was noted during birth to PND 4 (pre-selection) compared to the concurrent control group and the respective minimum mean values in the laboratories' historical control data (version 2020.01).
The differences noted at 150 mg/kg/day were primarily attributed to 2 females in this group with total litter loss on Lactation Day 2 or 3, and therefore not considered test substance-related.
Postnatal survival during birth and PND 4 (pre-selection) in the 15 and 50 mg/kg/day groups, and the mean number of pups born, live litter size, percentage of male per litter at birth, and postnatal survival between birth and PND 0, 0–1, 4 (post-selection)–7, 7–14, 14–21, and 4 (post-selection)–21 in the 15, 50, and 150 mg/kg/day groups were unaffected by test substance administration.

Following weaning:
Ten F1 animals in the 150 mg/kg/day group were found dead or euthanized in extremis during PND 25–89. Of these animals, 7 were noted with microscopic findings that were consistent with gavage accidents. The cause of death of the remaining 3 animals (1 male and 2 females) in this group was undetermined, but was considered unrelated to the test substance given the minimal incidence of mortality at this dose level, the lack of any test substance-related mortality in the F0 generation, and a comparable number of unscheduled deaths in the control group (2 males). Furthermore, 1 female in the 50 mg/kg/day group was euthanized in extremis on PND 86; the cause of death for this female was undetermined, but not considered test substance-related based on the lack of mortality/moribundity at 150 mg/kg/day. Two males in the control group were found dead on PND 43 or 84. All other F1 males and females survived to the scheduled necropsy.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no test substance-related effects on mean absolute body weights, body weight gains, food consumption, and food efficiency noted for F1 males and females in the 15, 50, and 150 mg/kg/day groups throughout the generation (PND 21–98).

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There were no test substance-related effects on mean absolute body weights, body weight gains, food consumption, and food efficiency noted for F1 males and females in the 15, 50, and 150 mg/kg/day groups throughout the generation (PND 21–98).

Food efficiency:

no effects observed

Description (incidence and severity):

There were no test substance-related effects on mean absolute body weights, body weight gains, food consumption, and food efficiency noted for F1 males and females in the 15, 50, and 150 mg/kg/day groups throughout the generation (PND 21–98).

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

not examined

Ophthalmological findings:

not specified

Haematological findings:

no effects observed

Description (incidence and severity):

Following weaning: There were no test substance-related effects on hematology, coagulation, serum chemistry, and urinalysis parameters for F1 males and females (Cohort 1A) in the 15, 50, and 150 mg/kg/day groups.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Following weaning: There were no test substance-related effects on hematology, coagulation, serum chemistry, and urinalysis parameters for F1 males and females (Cohort 1A) in the 15, 50, and 150 mg/kg/day groups.

Urinalysis findings:

no effects observed

Description (incidence and severity):

Following weaning: There were no test substance-related effects on hematology, coagulation, serum chemistry, and urinalysis parameters for F1 males and females (Cohort 1A) in the 15, 50, and 150 mg/kg/day groups.

Sexual maturation:

no effects observed

Description (incidence and severity):

The mean ages of attainment for balanopreputial separation and vaginal patency and the mean body weights at that the age of attainment for F1 males and females, respectively, in the 15, 50, and 150 mg/kg/day groups were unaffected by test substance administration.
In F1 generation females (Cohort 1A), there were no test substance-related effects on the mean age at first estrus or the mean duration from vaginal opening to first estrus, as well as mean estrous cycle length. Also, in F1 generation males (Cohort 1A), there were no test substance-related effects on sperm parameters.

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

Prior to weaning, there were no clinical observations or effects on mean body weights or body weight changes, and absolute and relative anogenital distance on PND 1 were comparable for F1 pups in the 15, 50, and 150 mg/kg/day groups.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

There were no retained nipples/areolae for F1 male pups at any dosage level on PND 13.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Pre-weaning:
There were no macroscopic findings and/or effects on organ weights noted for F1 pups that died or were euthanized in extremis or for PND 4 (culled) or PND 21 (nonselected or selected for hormone analysis) pups that could be attributed to F0 parental administration of the test substance.

Following weaning: Administration of the test substance was not associated with macroscopic findings or effects on organ/brain weights, ovarian follicle counts, gross brain measurements, or microscopic brain measurements in F1 animals.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Pre-weaning:
There were no macroscopic findings and/or effects on organ weights noted for F1 pups that died or were euthanized in extremis or for PND 4 (culled) or PND 21 (nonselected or selected for hormone analysis) pups that could be attributed to F0 parental administration of the test substance.

Following weaning: Administration of the test substance was not associated with macroscopic findings or effects on organ/brain weights, ovarian follicle counts, gross brain measurements, or microscopic brain measurements in F1 animals.

Histopathological findings:

effects observed, treatment-related

Description (incidence and severity):

In the F1 reproductive cohorts, the test substance was associated microscopically with nonadverse, minimal to moderate mixed cell inflammation in the stomach of males (50 and 150 mg/kg/day) and females (15, 50, and 150 mg/kg/day), and nonadverse erosion of the glandular stomach in one male in the 150 mg/kg/day group.

Other effects:

no effects observed

Description (incidence and severity):

Endocrinological system:
Pre-weaning: There were no effects on T4 or TSH concentrations for PND 4 (culled) or PND 21 (nonselected) pups.
Following weaning: There were no test substance-related effects on T4 or TSH concentrations for F1 males and females in the 15, 50, and 150 mg/kg/day groups on PND 91.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Following weaning: There were no test substance-related effects on F1 male and female (Cohort 2A) neurobehavior parameters (startle response, FOB, and motor activity) at any dosage level.

Developmental immunotoxicity:

no effects observed

Description (incidence and severity):

Following weaning: There were no test substance-related effects on splenic immunophenotyping parameters in F1 generation male and female animals following exposure to the test substance.

Dose descriptor:

NOAEL

Remarks:

for F1 systemic toxicity

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

Dose descriptor:

NOAEL

Remarks:

for F1 neonatal toxicity

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: all tested parameters

Remarks on result:

other: Based on the lack of adverse effects the highest concentration tested was set as NOAEL.

Dose descriptor:

NOAEL

Remarks:

F1 developmental neurotoxicity

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: all tested parameters

Remarks on result:

other: Based on the lack of adverse effects the highest concentration tested was set as NOAEL.

Dose descriptor:

NOAEL

Remarks:

for F1 immunotoxicity

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: all tested parameters

Remarks on result:

other: Based on the lack of adverse effects the highest concentration tested was set as NOAEL.

Critical effects observed:

no

Reproductive effects observed:

no

Conclusions:

In conclusion, based on the adverse clinical observations or rales and clonic and/or tonic convulsion noted for F0 and F1 animals, a dose level of 50 mg/kg/day was considered to be the no-observed-adverse-effect level (NOAEL) for F0 and F1 systemic toxicity of 2-Dimethylaminoethanol when administered orally by gavage to male and female Crl:CD(SD) rats. Based on the lack of adverse effects for all other tested parameters, a dose level of 150 mg/kg/day (the highest dose level tested) was considered to be the NOAEL for F0 reproductive toxicity, F1 neonatal toxicity, F1 developmental neurotoxicity, and F1 immunotoxicity.

Executive summary:

The extended one-generation reproductive toxicity was conduced according to OECD 443 and in accordance with GLP. The objective of this study was to evaluate the potential adverse effects of  2‑Dimethylaminoethanol on reproduction. This included evaluation of life stages not covered by other types of toxicity studies and tested for effects that may occur as a result of pre- and postnatal chemical exposure.

The study design was as follows:

Group Number	Test Substance	DosageLevel^a(mg/kg/day)	Dose Concentration (mg/mL)	DoseVolume (mL/kg)	Number of Animals
					Males	Females
1	Vehicle Control	0	0	5	25	25
2	2‑Dimethylaminoethanol	15	3	5	25	25
3	2‑Dimethylaminoethanol	50	10	5	25	25
4	2‑Dimethylaminoethanol	150	30	5	25	25
^a  Not corrected for salt, purity, and water content.

F0 animals were dosed via oral gavage once daily for 70 consecutive days prior to mating and continuing through the day prior to euthanasia. The offspring in the F1 generation were potentially exposed in utero during gestation and through nursing during lactation. The offspring selected to become the F1 generation were dosed beginning at weaning and continuing until euthanasia.

The following parameters and end points were evaluated in this study: clinical signs, body weights, body weight gains, food consumption, estrous cycles, reproductive performance, parturition, litter viability and survival, pre- and postweaning developmental landmarks, neurobehavior, thyroid hormones, clinical pathology, macroscopic findings, sperm parameters, immunophenotyping, organ weights, microscopic examinations, and neuropathologic and brain morphometric examinations.

F1 animals were further subdivided into cohorts following weaning and specifically evaluated for the following: Cohort 1 (A and B) for reproductive/developmental toxicity testing and Cohort 2 (A and B) for developmental neurotoxicity testing. Animals assigned to Cohort 1A were evaluated on PND 91 (including estrous cycles and sperm evaluations); Cohort 1B animals were maintained on study for possible assessment of reproductive performance and to generate an F2 generation; however, additional breeding was not ultimately required on this study. Animals assigned to Cohort 2B were evaluated on PND 22 (brain morphometry) and Cohort 2A animals were maintained on study until PND 78 for neurobehavioral testing and for evaluation of adult neuropathology.

The analyzed dosing formulations were within the protocol-specified range of concentrations for solutions, with the following exceptions. The analyzed dosing formulations on 22 Jun 2020 and 24 Aug 2020 exceeded the protocol specified maximum concentration. Backup samples were analyzed, and the results confirmed the original analysis. Fresh formulations were prepared on 24 Jun 2020 and 26 Aug 2020, and the results of these new formulations met the acceptance criteria. These excursions did not negatively impact the quality or integrity of the data or the outcome of the study because the assayed levels were only marginally higher (2 % to 4 %) than the acceptable range and animals were dosed with at least the nominal dosage levels.

F0 Generation: There were no test substance-related effects on survival for F0 animals at any dose level. One male in the 150 mg/kg/day group was euthanized in extremis after swallowing the cannula during dose administration. In addition, 1 female in the 50 mg/kg/day group was found dead on Study Day 10. In the absence of clinical observations, necropsy findings, effects on body weight, and the lack of any evidence of mortality at 150 mg/kg/day (the highest dose tested), this death was not considered test substance-related. In the control group, 1 female was euthanized in extremis on Gestation Day 25 due to dystocia.

All other F0 animals survived to the scheduled necropsy. Test substance-related, adverse clinical observations of convulsions (clonic and tonic) and rales were noted at the daily examinations and postdosing observations for F0 males and females in the 150 mg/kg/day group sporadically throughout the study. No other test substance-related clinical observations were noted.

There were no test substance-related effects on mean absolute body weights, body weight gains, food consumption, or food efficiency for F0 males throughout the generation and for F0 females during the premating, gestation, and lactation treatment periods at any dosage level.

There were no test substance-related effects on F0 estrous cyclicity, precoital intervals, reproductive performance (male and female mating and fertility, male copulation, and female conception indices), gestation lengths, and the process of parturition. Mean numbers of implantation sites and unaccounted-for sites were comparable across groups. No test substance-related effects on F0 sperm parameters (mean epididymal sperm numbers, motility, progressive motility, and morphology) were noted at any dosage level.

No test substance-related effects on thyroid hormone concentrations (T4 or TSH) were noted for F0 males and females in the 15, 50, and 150 mg/kg/day groups.

There were no test substance-related effects on hematology, coagulation, serum chemistry, or urinalysis for F0 males and females at any dosage level.

In F0 animals, the test substance was associated microscopically with nonadverse, minimal to mild mixed cell inflammation in the stomach of males (50 and 150 mg/kg/day) and females (150 mg/kg/day), and nonadverse minimal to mild erosion of the glandular stomach of males and females in the 150 mg/kg/day group. Administration of the test substance was not associated with macroscopic findings or effects on organ weights or reproductive performance in F0 animals.

F1 Generation: In the 150 mg/kg/day group, lower mean postnatal survival was noted during birth to PND 4 (pre-selection) compared to the concurrent control group and the respective minimum mean values in the Charles River Ashland historical control data (version 2020.01). The differences noted at 150 mg/kg/day were primarily attributed to 2 females in this group with total litter loss on Lactation Day 2 or 3, and therefore not considered test substance-related.

Postnatal survival during birth and PND 4 (pre-selection) in the 15 and 50 mg/kg/day groups, and the mean number of pups born, live litter size, percentage of male per litter at birth, and postnatal survival between birth and PND 0, 0–1, 4 (post-selection)–7, 7–14, 14–21, and 4 (post-selection)–21 in the 15, 50, and 150 mg/kg/day groups were unaffected by test substance administration.

Prior to weaning, there were no clinical observations or effects on mean body weights or body weight changes, and absolute and relative anogenital distance on PND 1 were comparable for F1 pups in the 15, 50, and 150 mg/kg/day groups. In addition, there were no retained nipples/areolae for F1 male pups at any dosage level on PND 13, and there were no effects on T4 or TSH concentrations for PND 4 (culled) or PND 21 (nonselected) pups, and there were no macroscopic findings and/or effects on organ weights noted for F1 pups that died or were euthanized in extremis or for PND 4 (culled) or PND 21 (nonselected or selected for hormone analysis) pups that could be attributed to F0 parental administration of the test substance.

Following weaning, there were no test substance-related effects on survival for F1 males and females at any dose level. Ten F1 animals in the 150 mg/kg/day group were found dead or euthanized in extremis during PND 25–89. Of these animals, 7 were noted with microscopic findings that were consistent with gavage accidents. The cause of death of the remaining 3 animals (1 male and 2 females) in this group was undetermined, but was considered unrelated to the test substance given the minimal incidence of mortality at this dose level, the lack of any test substance-related mortality in the F0 generation, and a comparable number of unscheduled deaths in the control group (2 males). Furthermore, 1 female in the 50 mg/kg/day group was euthanized in extremis on PND 86; the cause of death for this female was undetermined, but not considered test substance-related based on the lack of mortality/moribundity at 150 mg/kg/day.

Two males in the control group were found dead on PND 43 or 84. All other F1 males and females survived to the scheduled necropsy. Test substance-related, adverse clinical observations of convulsions (clonic) and rales were noted for F1 males and females in the 150 mg/kg/day group at the daily examinations and postdosing observations sporadically throughout the study in this group, and were not observed in the control group.

These observations were also noted for males and females in the F0 generation. No other test substance-related clinical observations were noted for F1 males and females at any dosage level.

There were no test substance-related effects on mean absolute body weights, body weight gains, food consumption, and food efficiency noted for F1 males and females in the 15, 50, and 150 mg/kg/day groups throughout the generation (PND 21–98).

The mean ages of attainment for balanopreputial separation and vaginal patency and the mean body weights at that the age of attainment for F1 males and females, respectively, in the 15, 50, and 150 mg/kg/day groups were unaffected by test substance administration.

In F1 generation females (Cohort 1A), there were no test substance-related effects on the mean age at first estrus or the mean duration from vaginal opening to first estrus, as well as mean estrous cycle length. Also, in F1 generation males (Cohort 1A), there were no test substance-related effects on sperm parameters.

There were no test substance-related effects on T4 or TSH concentrations for F1 males and females in the 15, 50, and 150 mg/kg/day groups on PND 91.

There were no test substance-related effects on hematology, coagulation, serum chemistry, and urinalysis parameters for F1 males and females (Cohort 1A) in the 15, 50, and 150 mg/kg/day groups.

There were no test substance-related effects on F1 male and female (Cohort 2A) neurobehavior parameters (startle response, FOB, and motor activity) at any dosage level.

In the F1 reproductive cohorts, the test substance was associated microscopically with nonadverse, minimal to moderate mixed cell inflammation in the stomach of males (50 and 150 mg/kg/day) and females (15, 50, and 150 mg/kg/day), and nonadverse erosion of the glandular stomach in one male in the 150 mg/kg/day group. Administration of the test substance was not associated with macroscopic findings or effects on organ/brain weights, ovarian follicle counts, gross brain measurements, or microscopic brain measurements in F1 animals.

There were no test substance-related effects on splenic immunophenotyping parameters in F1 generation male and female animals following exposure to the test substance.

In conclusion, based on the adverse clinical observations or rales and clonic and/or tonic convulsion noted for F0 and F1 animals, a dose level of 50 mg/kg/day was considered to be the no-observed-adverse-effect level (NOAEL) for F0 and F1 systemic toxicity of 2-Dimethylaminoethanol when administered orally by gavage to male and female Crl:CD(SD) rats. Based on the lack of adverse effects for all other tested parameters, a dose level of 150 mg/kg/day (the highest dose level tested) was considered to be the NOAEL for F0 reproductive toxicity, F1 neonatal toxicity, F1 developmental neurotoxicity, and F1 immunotoxicity.

Reference 2

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

22 NOV 2019 (audit of final protocol) to 21 JAN 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to other study

Remarks:

OECD 422 used as DRF for OECD 443

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

29 July 2016

Deviations:

no

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) of test material: 2-Dimethylaminoethanol (DMAE, CAS 108-01-0) from Taminco US LLC
- Receipt date: 06 Aug 2019
- Retest Date: 20 Aug 2021

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Kept in a controlled temperature area set to maintain 18 °C to 24 °C, under nitrogen

FORM AS APPLIED IN THE TEST (if different from that of starting material)
- Specify the relevant form characteristics if different from those in the starting material, such as state of aggregation, shape of particles or particle size distribution: physical decription: Colorless, clear liquid

Species:

rat

Strain:

other: Crl:CD(SD)

Details on species / strain selection:

The Crl:CD(SD) rat is recognized as appropriate for reproduction studies. The laboratory has reproductive historical control data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, NC.
- Animal Identification: Each animal was identified using a subcutaneously implanted electronic identification chip (BMDS system). Offspring were identified by tattoo markings applied to the digits after parturition and by microchip after weaning.
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: ~ 11 weeks old
- Weight at study initiation: (P) Males: 360 - 535 g; Females: 217 - 282 g at the initiation of dosing.
- Housing: On arrival, animals were group housed (up to 3 animals of the same sex) until cohabitation During cohabitation, animals were paired for mating in the home cage of the male. Following the breeding period, animals were individually housed. Animals were housed in solid-bottom cages containing appropriate bedding equipped with an automatic watering valve throughout the study. All offspring, not euthanized at weaning, were housed in groups of 2–3 by sex (by litter, if possible) in solid-bottom cages containing appropriate bedding equipped with an automatic watering valve until euthanasia. Each cage was clearly labeled with a color-coded cage card indicating study, group, animal, cage number(s), dosage level, and sex. Cages were arranged on the racks in group order. Animals were maintained in accordance with the Guide for the Care and Use of Laboratory Animals (National Research Council, 2011). The animal facilities at Charles River Ashland are accredited by AAALAC International.
- Use of restrainers for preventing ingestion (if dermal): no - not applicable
- Diet (e.g. ad libitum): PMI Nutrition International, LLC Certified Rodent LabDiet® 5002 (meal) was provided ad libitum throughout the study, except during periods of fasting for clinical pathology. The feed was analyzed by the supplier for nutritional components and environmental contaminants. Results of the analysis are provided by the supplier and are on file at the Testing Facility. It is considered that there are no known contaminants in the feed that would interfere with the objectives of the study.
- Water (e.g. ad libitum): Municipal tap water after treatment by reverse osmosis was freely available to each animal via an automatic watering system. Water bottles were provided, if required. Periodic analysis of the water is performed, and results of these analyses are on file at the Testing Facility. It is considered that there are no known contaminants in the water that could interfere with the outcome of the study.
- Acclimation period: After receipt at the Testing Facility, the Crl:CD(SD) rats were acclimated prior to initiation of dosing. The testes were palpated at least once for all males during acclimation.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Target temperatures of 68 °F to 78 °F (20 °C to 26 °C) was maintained
- Humidity (%): relative target humidity of 30 % to 70 % was maintained
- Air changes (per hr): Ten or greater air changes per hour with 100 % fresh air (no air recirculation) were maintained in the animal rooms
- Photoperiod (hrs dark / hrs light): 12-hour light/12-hour dark cycle was maintained

IN-LIFE DATES: From: 17 DEC 2019 (experimental starting date) To: 12 MAR 2020

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

Deionized water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Test substance dosing formulations were prepared at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared approximately weekly and an adequate amount of each formulation was dispensed into daily aliquots, which were stored refrigerated (target of 5 °C) until use. The dosing formulations and control vehicle were stirred continuously during dosing. Details of the preparation and dispensing of the test substance have been retained in the Study Records.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Deionized water
- Concentration in vehicle: 0, 3, 10, 30 mg/mL
- Amount of vehicle (if gavage): 5 mg/kg

Details on mating procedure:

- M/F ratio per cage: 1:1
- Proof of pregnancy: by presence of a vaginal copulatory plug or the presence of sperm in a vaginal lavage.
- If evidence of mating was not apparent after 14 days, the animals were separated, with no further opportunity for mating
- Animals cohabited over a 12-hour dark cycle were considered to have been paired for 1 day.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses were performed by a gas chromatography method using flame ionization detection using a validated analytical procedure (Engda, 2020, 01300001).
- Concentration Analysis: Duplicate sets of samples (1.0 mL) for each sampling time point were transferred to the analytical laboratory; the remaining samples were retained at the Testing Facility as backup samples. Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 10 % of theoretical concentration. After acceptance of the analytical results, backup samples were discarded.
- Homogeneity Analysis: The Sponsor has provided data that demonstrate that the test substance is soluble in the vehicle when prepared under the same mixing conditions at concentrations bracketing those used in the present study. Solubility data provided by the Sponsor have been retained in the Study Records.
- Stability Analysis: Stability analyses performed previously in conjunction with Study No. 01300001 demonstrated that the test substance is stable in the vehicle when prepared and stored under the same conditions at concentrations bracketing those used in the present study. Stability data have been retained in the Study Records for Study No. 01300001

Duration of treatment / exposure:

F0 males were dosed for 14 days prior to mating, throughout mating, and continuing until the day prior to euthanasia.
F0 females were dosed for 14 days prior to mating and continuing through Lactation Day 20.
The offspring of the F0 generation (F1 litters) in the present study were potentially exposed to the test substance in utero, as well as via the milk while nursing. Selected F1 pups (1 pup/sex/litter, if possible) were directly administered the test substance from PND 22 through 36, inclusively.

Frequency of treatment:

The test substance and vehicle were administered as a single daily oral gavage.

Dose / conc.:

0 mg/kg bw/day

Remarks:

Control

Dose / conc.:

15 mg/kg bw/day

Dose / conc.:

50 mg/kg bw/day

Dose / conc.:

150 mg/kg bw/day

No. of animals per sex per dose:

10 / sex / dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dosage levels for this study were determined from the results of a previous 14-day tolerability study (Millard, 2020, 01300008). In the previous study, male and female rats were administered the test substance via oral gavage for 14 days at dosage levels of 0, 100, 200, and 300 mg/kg/day. Mortality and/or moribundity were noted for 1 and 3 males in the 200 and 300 mg/kg/day groups, respectively, as well as 1 female in the 200 mg/kg/day group during Study Days 4–12. Respiratory-related clinical observations, including labored/shallow breathing and abnormal breathing sounds, were noted for the 2 surviving males and all 5 females in the 300 mg/kg/day group, as well as 2 females in the 200 mg/kg/day group during Study Days 5–14. Lower body weight gain and/or body weight loss, with corresponding reduced food consumption, were noted for males and females in the 300 mg/kg/day group when the entire treatment period (Study Days 0–14) was evaluated, resulting in absolute body weights in this group that were 23.9% and 6.9% lower than the control group on Study Day 14. In the 200 mg/kg/day group, lower body weight gains, in the absence of a remarkable effect on food consumption, were noted for males and females when the entire treatment period was evaluated; however, these were not of sufficient magnitude to affect absolute body weights. There were no significant effects on mean body weight gains or food consumption noted in the 100 mg/kg/day group throughout the study. As a result of the previous study, dosage levels of 15, 50, and 150 mg/kg/day were chosen for this study to assess male and female reproduction within the scope of this screening study.
- Fasting period before blood sampling for clinical biochemistry: Animals were fasted overnight prior to blood collection
- Other: Rationale for administration route: The route of administration was oral (gavage) because this is a potential route of exposure for humans. Historically, this route has been used extensively for studies of this nature.

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations: general health/mortality and moribundity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily

BODY WEIGHT: Yes
- Time schedule for examinations: weekly throughout the study and prior to the scheduled necropsy. Once evidence of mating was observed, female body weights were recorded on Gestation Days 0, 4, 7, 11, 14, 17, and 20 and on Lactation Days 0 (when possible), 1, 4, 7, 10, 13, 17, and 20. A fasted weight was recorded on the day of necropsy. Terminal body weights were not collected from animals found dead or euthanized moribund.

FOOD CONSUMPTION AND COMPOUND INTAKE:
Food consumption was quantitatively measured weekly until cohabitation. Once evidence of mating was observed, female food consumption was recorded on Gestation Days 0, 4, 7, 11, 14, 17, and 20 and Lactation Days 1, 4, 7, 10, 13, 17, and 20.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OTHER:
Neurobehavioral Testing:
- FOB assessments were recorded for 5 animals/sex/group during the last week of dosing (Study Day 24, males) or on Lactation Day 20 (females). Further details are given under 'Any other information'.
- Motor activity was assessed for 5 animals/sex/group during the last week of dosing (Study Day 24, males) or on Lactation Day 20 (females). Further details are given under 'Any other information'

Clinical Pathology: Animals were fasted overnight prior to blood collection.
- Hematology: Blood samples were analyzed for the parameters specified in 'Any other information'
- Coagulation: Blood samples were processed for plasma, and the plasma was analyzed for the parameters specified in 'Any other information'
- Serum Chemistry: Blood samples were processed for serum, and the serum was analyzed for the parameters specified in 'Any other information'

Thyroid Hormone Analysis: Blood samples were analyzed for Thyroxine (Total T4).

Oestrous cyclicity (parental animals):

For all females, vaginal lavages were performed daily for 14 days prior to randomization and continuing until evidence of mating was observed. Vaginal lavages were also performed on the day of necropsy to determine the stage of the estrous cycle.

Sperm parameters (parental animals):

not specified

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
- A detailed clinical observation was performed on PND 1, 4, 7, 10, 13, 17, and 21, and daily beginning on PND 22 until euthanasia
- Pups were individually sexed on PND 0, 4, 13, and 21
- Pups were weighed individually on PND 1, 4, 7, 10, 13, 17, and 21
- Pups selected for the F1 postweaning phase were weighed twice weekly following weaning
- Pup food consumption was recorded on a weekly basis, beginning on PND 21, and continuing until euthanasia.

Preweaning Developmental Landmarks:
- Anogenital distance of all pups was measured on PND 1
- On PND 13, all male pups were evaluated for the presence of nipples/areolae. The number of nipples was recorded.

Thyroid Hormone Analysis
- On PND 4 blood samples were collected of at least 2/litter
- OM PND 21 blood samples were collected of 1/sex/litter for T4

GROSS EXAMINATION OF DEAD PUPS:
A necropsy was conducted for animals that died on study, and specified tissues were saved.
Intact offspring that were found dead during PND 0–4 were necropsied using a fresh dissection technique, which included examination of the heart and major vessels (Stuckhardt and Poppe, 1984). Findings were recorded as developmental variations or malformations, as appropriate. A gross necropsy was performed on any pup found dead after PND 4.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals were sacrificed on Study Day 28
- Maternal animals: All surviving animals were sacrificed on Lactation Day 21

Terminal procedures are summarized in 'Any other information'

GROSS NECROPSY
- F0 animals were subjected to a complete necropsy examination, which included examination of the external surface, all orifices, the cranial cavity, the external surfaces of the brain and spinal cord, and the thoracic, abdominal, and pelvic cavities, including viscera. The numbers of former implantation sites were recorded for females that delivered. The number of unaccounted-for sites was calculated for each female by subtracting the number of pups born from the number of former implantation sites observed.

HISTOPATHOLOGY / ORGAN WEIGHTS
- Organ Weights: The tissues indicated in 'Any other information' were weighed at necropsy for all scheduled euthanasia animals. Paired organs were weighed together, unless otherwise indicated. Organ to body weight ratio (using the terminal body weight) and organ to brain weight ratios were calculated.
- Tissue Collection and Preservation: Representative samples of the tissues are given in 'Any other information'.
- Histology/Histopathology: Tissues given in 'Any other information from 5 animals/sex in the control and high-dose groups, as well as gross lesions from all groups.

Postmortem examinations (offspring):

SACRIFICE
Pups of F1 Litters were sacrificed on PND 21. Terminal procedures of F1 Litters are summarized in 'Any other information'
Pups of F1 Generation were sacrifieced on PND 37. Terminal procedures of F1 Generation are summarized in 'Any other information'

GROSS NECROPSY
- Pups of F1 Litters and F1 Generation were subjected to a complete necropsy examination, with emphasis on developmental morphology and organs of the reproductive system.

HISTOPATHOLOGY / ORGAN WEIGTHS
- Tissue Collection and Preservation: The thyroids of pups of F1 Litters (with parathyroids, if present) were collected from 1 pup/sex/litter at the scheduled euthanasia. The stomach and gross lesions of pups of F1 Generation were collected from 1 pup/sex/litter at the scheduled euthanasia.
- Histology/Histopathology: Microscopic examination of the stomach was conducted for all animals assigned to the F1 generation in all groups, and from all animals dying spontaneously. Gross lesions were also examined from all animals.

Statistics:

See 'Any other information'

Reproductive indices:

Male/Female Mating Index (%), Male/Female Fertility Index (%), Male Copulation Index (%), Female Conception Index (%), Estrous Cycle Length (days), Pre-Coital Interval (days)

Clinical signs:

no effects observed

Description (incidence and severity):

No test substance-related clinical observations were noted for F0 males and females at any dosage level. Clinical observations noted in the test substance-treated groups at the daily examinations and the 1–2 hour postdosing observations were noted 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:

no mortality observed

Description (incidence):

All F0 males and females in the control, 15, 50, and 150 mg/kg/day groups survived to the scheduled necropsies.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

- Males: Mean body weights and body weight gains in the 15, 50, and 150 mg/kg/day group males were unaffected by test substance administration throughout the study (Study Days 0-27). Differences from the control group were generally slight, not statistically significant, and/or did not occur in a clear dose-responsive manner.
- Females: Mean body weights and body weight gains in the 15, 50, and 150 mg/kg/day group females were unaffected by test substance administration during the premating period (Study Days 0–13), gestation and lactation. None of the differences from the control group were statistically significant.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

- Males: Mean food consumption, evaluated as g/animal/day, in the 15, 50, and 150 mg/kg/day group males was unaffected by test substance administration during the premating period (Study Days 0–13). Statistically significantly higher mean food consumption was noted in the 150 mg/kg/day group during the first week of dosing (Study Days 0–7). However, this difference did not result in noteworthy changes in mean body weight, and therefore was considered unrelated to the test substance. No other statistically significant differences were noted at any dosage level.
- Females: Mean food consumption, evaluated as g/animal/day, in the 15, 50, and 150 mg/kg/day group females was unaffected by test substance administration during the premating period (Study Days 0–13) and gestation. None of the differences from the control group were statistically significant. Slightly higher (statistically significant) consumption values were noted sporadically throughout lactation in the 50 and 150 mg/kg/day groups when compared to the control group; however, these differences were considered unrelated to the test substance.

Food efficiency:

not specified

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

There were no test substance-related effects on hematology and coagulation parameters. Any statistically significant differences showed no dose-response relationship, no similar occurrence in the opposite sex, and the changes were of minimal magnitude. These differences from the control group were considered to be the result of normal biological variation and were not considered to be of toxicological significance.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

There were no test substance-related effects on serum chemistry. Differences from the control group were not statistically significant and were considered to be the result of normal biological variation and not of toxicological significance.
There were no test substance-related effects on T4 concentrations in the F0 males at any dosage level. Differences from the control group were not statistically significant and were considered to be the result of normal biological variation and not of toxicological significance.

Endocrine findings:

no effects observed

Description (incidence and severity):

see info under clinical biochemistry findings and organ weights

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Functional Observational Battery
- Home Cage parameters, handling parameters, open field parameters, sensory parameters and physiological parameters were unaffected by test substance administration. There were no statistically significant differences for the test substance-treated groups when compared to the control group on Study Day 24 (males) or Lactation Day 20 (females).
- Neuromuscular parameters were unaffected by test substance administration. There were no statistically significant differences for the test substance-treated groups when compared to the control group on Study Day 24 (males) or Lactation Day 20 (females), with the following exception. A statistically significantly higher mean hindlimb grip strength was noted for F0 males in the 150 mg/kg/day group; however, higher grip strength is not considered to be test substance-related or adverse.
- Motor Activity: Motor activity patterns (total activity as well as ambulatory activity counts) in F0 animals were unaffected by test substance administration at all concentrations when evaluated on Study Day 24 (males) and Lactation Day 20 (females). Values obtained from the 6 subintervals evaluated (0–10, 11–20, 21–30, 31–40, 41–50 and 51–60 minutes) and the overall 60-minute test session values were comparable to the concurrent control values and the historical control data of the laboratory. Differences from the control group were slight and not statistically significant. No remarkable shifts in the pattern of habituation occurred in any of the test substance-treated groups when the F0 animals were evaluated.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

In the stomach, microscopic findings of erosion and mixed cell inflammation/infiltrate were noted in the stomach of the 15, 50, and 150 mg/kg/day group females. Additionally, an increased incidence and severity of edema in the submucosa of the stomach was noted in the 15, 50, and 150 mg/kg/day group females. However, there was no clear dose-dependent increase in incidence or severity of these findings. Therefore, the relationship of these findings in the stomachs of 15, 50, and 150 mg/kg/day group females to test substance administration was uncertain.

Edema was noted in the stomach in the 15 mg/kg/day group males (minimal, 1 out of 10), the 50 mg/kg/day group males (minimal, 1 out of 10), and the 150 mg/kg/day group males (2 out of 10, minimal to mild). The incidence was similar to the control group females and, in the absence of additional test substance-related findings in the stomach of treated males, was not considered test substance-related.

Other microscopic findings observed were considered incidental, of the nature commonly observed in this strain and age of rat, and/or were of similar incidence and severity in control and treated animals and, therefore, were considered unrelated to administration of 2-Dimethylaminoethanol.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

No test substance-related effects on Estrous Cycle Length (days) were observed at any dosage level.

Reproductive function: sperm measures:

not specified

Reproductive performance:

no effects observed

Description (incidence and severity):

No test substance-related effects on reproductive performance were observed at any dosage level. All males in the control, 15, 50, and 150 mg/kg/day groups sired litters, and all females in the same respective groups were gravid.
The mean numbers of days between pairing and coitus in the test substance-treated groups were similar to the control group value. The mean lengths of estrous cycles in these groups were also similar to the control group value.

Dose descriptor:

NOAEL

Remarks:

reproductive toxicity

Effect level:

150 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: overall reproductive toxicity parameters

Remarks on result:

other: Based on the absence of adverse effects, a dosage level of 150 mg/kg/day, the highest dose administered, was considered to be the NOAEL.

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

150 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: overall systemic toxictiy parameters

Remarks on result:

other: Based on the absence of adverse effects, a dosage level of 150 mg/kg/day, the highest dose administered, was considered to be the NOAEL.

Critical effects observed:

no

Clinical signs:

no effects observed

Description (incidence and severity):

F1 Litters: No test substance-related clinical observations were noted for F1 males and females at any dosage level. Clinical observations noted in the test substance-treated groups at the daily examinations and the 1–2 hour postdosing observations were noted infrequently, at similar frequencies in the control group, and/or in a manner that was not dose-related.

F1 Generation: The general physical condition (defined as the occurrence and severity of clinical observations) of all F1 pups in this study was unaffected by test substance administration. Zero (0), 4(4), 0(0), and 3(3) pups (litters) in the control, 15, 50, and 150 mg/kg/day groups, respectively, were found dead. Zero (0), 2(1), 1(1), and 1(1) pups (litters) in the same respective groups were missing and presumed to have been cannibalized.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

All F1 males and females in the control, 15, 50, and 150 mg/kg/day groups survived to the scheduled necropsies.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Mean F1 pup birth weights (PND 1) in the 150 mg/kg/day group were lower (10.3 % and 6.8 %, not statistically significant) for males and females, respectively, compared to the control group. The mean live litter size on PND 0 in the 150 mg/kg/day group (15.2/dam) was higher than the control group (12.6/dam). Mean body weight gains for male and female pups in the 150 mg/kg/day group were comparable to the control group during PND 1–17, but statistically significantly lower during PND 17–21. Mean absolute body weights for F1 males and females in this group remained lower (5.1 % to 13.5 %) than the control group on PND 4 and 7, reflecting the lower birth weights in this group, and again on PND 21; none of these differences were statistically significant. Because there was no effects on pup health or survival and body weight differences often occurred in a manner that was not clearly dose related, the effects on F1 body weights prior to weaning were not considered test substance-related. Mean F1 male and female pups body weights and body weight changes during PND 1–21 in the 15 and 50 mg/kg/day groups were unaffected by parental test substance administration. No statistically significant differences from the control were noted.

Males: Mean body weights and body weight gains in the 15, 50, and 150 mg/kg/day group F1 males were unaffected by test substance administration throughout the generation; the lower mean body weights on PND 21 and 24 in the 150 mg/kg/day group were considered a carryover from the preweaning body weight effects seen in this group and not a result of direct dosing with the test substance. Differences from the control group were slight, not statistically significant, and/or did not occur in a clear dose-responsive manner.

Females: Mean body weights and body weight gains in the 15, 50, and 150 mg/kg/day group F1 females were unaffected by test substance administration throughout the generation; the lower mean body weight on PND 21 in the 150 mg/kg/day group was considered a carryover from the preweaning body weight effects seen in this group and not a result of direct dosing with the test substance. Differences from the control group were slight, not statistically significant, and/or did not occur in a clear dose-responsive manner.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

F1 Generation: mean food consumption evaluated as g/animal/day, in the 15, 50, and 150 mg/kg/day group F1 males/females was unaffected by test substance administration during PND 21–35. None of the differences from the control group were statistically significant.

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:

no effects observed

Description (incidence and severity):

F1 Litters: There were no test substance-related effects on T4 concentrations in the F1 males and females at any dosage level on PND 21. Statistically significantly higher values were noted in the 150 mg/kg/day group males and 50 and 150 mg/kg/day group females compared to the control group. The differences were attributed to outliers within these groups that contributed to the higher mean values. All values were within the range of concentrations in the control data of the laboratory (version 2019.02); differences from the control group were, therefore, considered to be the result of normal biological variation and were not considered to be of toxicological significance.

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

The anogenital distances (absolute and relative to the cube root of pup body weight) in the 15, 50, and 150 mg/kg/day groups were similar to the control group values. Differences from the control group were slight and not statistically significant.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

Areolae/nipple anlagen in the F1 male pups was unaffected by parental administration of the test substance when evaluated on PND 13. The test substance-treated group values were not statistically different from the control group values.

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

no effects observed

Description (incidence and severity):

F1 Litters: Zero (0), 4(4), 0(0), and 3(3) pups (litters) in the control, 15, 50, and 150 mg/kg/day groups, respectively, were found dead. No internal findings that could be attributed to parental test substance administration were noted at the necropsies of pups that were found dead. Aside from the presence or absence of milk in the stomach, no other internal findings were noted.

PND 21 Nonselected Pups: No internal findings that could be attributed to parental test substance administration were noted at the necropsies of nonselected pups on PND 21. A single pup in the 15 mg/kg/day was noted with a dilated renal pelvis; however, this was noted for only a single pup and did not occur in a dose-related manner. No other internal findings were noted.

F1 Generation: No test substance-related gross findings were noted. A gross finding of dark red area(s) was observed in the stomach from 1 control group male and considered unrelated to administration of 2-Dimethylaminoethanol.

Histopathological findings:

no effects observed

Description (incidence and severity):

No test substance-related microscopic findings were noted at any dosage level (evaluation limited to the stomach and gross lesions per protocol). The microscopic findings observed were considered incidental, of the nature commonly observed in this strain and age of rat, and/or were of similar incidence and severity in control and treated animals and, therefore, were considered unrelated to administration of 2-Dimethylaminoethanol.

Other effects:

no effects observed

Description (incidence and severity):

- PND 0 Litter Data and Postnatal Survival: The mean number of pups born, live litter size and the percentage of males at birth in the 15, 50, and 150 mg/kg/day groups were largely unaffected by maternal test substance administration. A statistically significantly higher mean number of pups born was noted in the 150 mg/kg/day group; however, more pups born/litter is not considered to be of toxicological significance. Postnatal survival in the 15, 50, and 150 mg/kg/day groups were unaffected by test substance administration.

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

150 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: overall neonatal/developmental toxicity and systemic toxicity parameters

Remarks on result:

other: Based on the absence of adverse effects, a dosage level of 150 mg/kg/day, the highest dose administered, was considered to be the NOAEL.

Critical effects observed:

no

Reproductive effects observed:

no

Analyses of Dosing Formulations

The analyzed dosing formulations contained 92.3 % to 110 % of the test substance which was within the protocol-specified range of target concentrations for solutions (90 % to 110 %) with the following exceptions: The results of the initial assessment of concentration acceptability of the 13 Jan 2020 Group 2 formulation failed to meet the acceptance criteria. Subsequent analysis of the back-up samples confirmed the initial analysis and the overall mean concentration was reported as 82.2 % of target. A new Group 2 formulation was prepared on 15 Jan 2020 and analyzed, and the results met the protocol-specified acceptance criteria (100 % of target). The Group 2 formulation (prepared on 13 Jan 2020) which was below target concentration was used for a single day of dosing, and was replaced with the new formulation beginning on the second day; use of this formulation for dosing the low dose group on a single day is not considered to have had any negative impact on the study as the highest dose was NOAEL for male and female reproductive toxicity. The results of the initial assessment of concentration acceptability of the 24 Feb 2020 Group 2 formulations also failed to meet the acceptance criteria. Subsequent analysis of the back-up samples met the protocol-specified acceptance criteria and the overall mean concentration was reported as 110 % of target, which also meets protocol-specified acceptability criteria. No test substance was detected in the analyzed vehicle administered to the control group.

Conclusions:

Under the conditions of this screening study, based on the absence of adverse effects, a dosage level of 150 mg/kg/day, the highest dose administered, was considered to be the no-observed-adverse-effect level (NOAEL) for F0 male and female reproductive toxicity, F0 male and female systemic toxicity, and F1 neonatal/developmental toxicity and systemic toxicity of 2-Dimethylaminoethanol when administered orally by gavage to Crl:CD(SD) rats.

Executive summary:

The objective of this GLP-study was to provide preliminary information on the potential adverse effects of the test substance, 2-Dimethylaminoethanol, on male and female reproduction within the scope of an OECD 422 study. This encompassed gonadal function, mating behavior, conception, parturition, and lactation of the F0 generation, and the development of F1 offspring from conception through Day 37 of postnatal life. In addition, the F1 pups were directly administered 2-Dimethylaminoethanol for 2 weeks following weaning.

The study design was as follows:

Group Number	Test Substance	Dosage Level ^a (mg/kg/day)	Dose Concentration (mg/mL)	Dose Volume (mL/kg)	Number of Animals
					Males	Females
1	Vehicle Control	0	0	5	10	10
2	2-Dimethylaminoethanol	15	3	5	10	10
3	2-Dimethylaminoethanol	50	10	5	10	10
4	2-Dimethylaminoethanol	150	30	5	10	10

^a Not corrected for salt, purity and water content.

Animals were dosed via oral gavage once daily. F0 males were dosed for 14 days prior to mating and continuing through 1 day prior to euthanasia. F0 females were dosed for 14 days prior to mating and continuing throughout mating, gestation, and lactation until 1 day prior to euthanasia. F1 animals were potentially exposed to the test substance in utero and via maternal milk during lactation from birth until Postnatal Day (PND) 21. Following selection and weaning, F1 male and female weanlings (at least 1 pup/sex/litter) was dosed once daily from PND 22 through 36, inclusively.

The following parameters and end points were evaluated in this study: clinical signs, body weights, body weight gains, food consumption, estrous cycles, reproductive performance, parturition, gestation lengths, litter viability and survival, preweaning developmental landmarks, neurobehavior, thyroid hormones, clinical pathology, gross necropsy findings, organ weights, and histopathologic examinations.

There were no test substance-related effects on mortality, clinical observations, body weights, body weight gains, food consumption, neurobehavioral parameters (FOB and motor activity) and reproductive performance (male and female mating and fertility, male copulation, and female conception indices, mean estrous cycle, precoital intervals, gestation length, and process of parturition) in F0 males or females at any dosage level.

At the scheduled necropsy, there were no test substance-related macroscopic and microscopic findings in F0 males at any dosage level. Test substance-related lower thymus weights were noted in the 150 mg/kg/day group males, which occurred in the absence of correlating gross observations or microscopic findings, and were therefore not considered adverse. F0 females in all test substance-treated groups were noted with dark red areas in the stomach, with corresponding microscopic findings of erosion and mixed cell inflammation/infiltration. In the absence of corresponding effects on survival, body weight, and food consumption, and the absence of a clear dose response, the test substance-related gross observations and microscopic findings were considered nonadverse. Test substance-related higher liver weights were also noted in F0 females in the 50 and 150 mg/kg/day groups, which occurred in the absence of correlating gross observations or microscopic findings, and therefore were considered nonadverse.

No effects on mean number of pups born, live litter size, percentage of males at birth, postnatal survival, clinical observations, pup body weights, anogenital distance (absolute and relative to the cube root of pup body weight), areolae/nipple anlagen (males only), gross pathology of unscheduled deaths and nonselected pups on PND 21, and T4 concentrations were seen in F1 pups prior to weaning that were attributed to parental test substance administration at any dosage level.

For males and females assigned to the F1 generation following weaning, no test substance-related effects were seen on mortality, clinical observations, body weights, body weight gains, and food consumption at any dosage level. No macroscopic or microscopic findings were noted at the scheduled necropsy on PND 37 that were attributed to test substance administration.

Under the conditions of this screening study, based on the absence of adverse effects, a dosage level of 150 mg/kg/day, the highest dose administered, was considered to be the no-observed-adverse-effect level (NOAEL) for F0 male and female reproductive toxicity, F0 male and female systemic toxicity, and F1 neonatal/developmental toxicity and systemic toxicity of 2-Dimethylaminoethanol when administered orally by gavage to Crl:CD(SD) rats.

Reference 3

Endpoint:

screening for reproductive / 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:

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
- NOAEL 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

- NOAEL 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

Reference 4

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:

supporting 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.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

In the high-dose F0 females (5000 ppm), reduced food consumption during lactation (21% below control) was recorded.

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%** (**:p≤0.01) 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* (*:p≤0.05) 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%** (**:p≤0.01) 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.

Reference 5

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	23^a
^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 weight^3
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 change^3
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 ± 1.2^b	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 ± 25.4^b	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 ± 16.9^c
Live litter size	9.5 ± 1.7	7.6 ± 3.1*	8.5 ± 3.4	8.4 ± 2.9^c
Fetal body weight/litter (g)
All fetuses	4.47 ± 0.15	4.56 ± 0.26	4.53 ± 0.24	4.66 ± 0.27^c
Male fetuses	4.63 ± 0.16	4.67 ± 0.24	4.61 ± 0.26^d	4.82 ± 0.26*^c
Female fetuses	4.28 ± 0.13	4.38 ± 0.26^e	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

150 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Data are of good quality (OECD Guideline Tests conduced in compliance with GLP)

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

In an Extended One-Generation Reproductive Toxicity Study with Cohorts 1A, 1B without extension, 2A and 2B according to OECD 443 and GLP (Millard, 2021), DMAE was administered to groups of Sprague Dawley Crl:CD(SD) rats at graded dose levels of 0, 15, 50, or 150 mg/kg bw/day (p.o.). F0 males were dosed 70 consecutive days prior to mating and continuing through the day prior to euthanasia (for 19 weeks) and F0 females for 70 consecutive days prior to mating and continuing throughout mating, gestation, and lactation, through the day prior to euthanasia. Offspring selected for the F1 generation began dosing following weaning until the day prior to euthanasia.

In the F0 generation, there were no test substance-related effects on survival any dose level. Test substance-related, adverse clinical observations of convulsions (clonic and tonic) and rales were noted at the daily examinations and postdosing observations for F0 males and females in the 150 mg/kg/day group sporadically throughout the study. No other test substance-related clinical observations or other parameters of systemic toxicity were noted.

With regard to fertility, there were no test substance-related effects on F0 estrous cyclicity, precoital intervals, reproductive performance (male and female mating and fertility, male copulation, and female conception indices), gestation lengths, and the process of parturition. Mean numbers of implantation sites and unaccounted-for sites were comparable across groups. No test substance-related effects on F0 sperm parameters (mean epididymal sperm numbers, motility, progressive motility, and morphology) were noted at any dosage level. Based on these results, it was concluded that administration of the test substance was not associated with reproductive performance in F0 animals.

With regard to sexual maturity and fertility in the F1 generation, the following results were obtained: The mean ages of attainment for balanopreputial separation and vaginal patency and the mean body weights at that the age of attainment for F1 males and females, respectively, in the 15, 50, and 150 mg/kg/day groups were unaffected by test substance administration. In F1 generation females (Cohort 1A), there were no test substance-related effects on the mean age at first estrus or the mean duration from vaginal opening to first estrus, as well as mean estrous cycle length. Also, in F1 generation males (Cohort 1A), there were no test substance-related effects on sperm parameters. In the F1 reproductive cohorts, administration of the test substance was not associated with ovarian follicle counts.

In conclusion, based on the adverse clinical observations or rales and clonic and/or tonic convulsion noted for F0 and F1 animals, a dose level of 50 mg/kg/day was considered to be the NOAEL for F0 and F1 systemic toxicity. Based on the lack of adverse effects for all other tested parameters, a dose level of 150 mg/kg/day (the highest dose level tested) was considered to be the NOAEL for F0 reproductive toxicity.

 

 

In a Combined Repeat Dose and Reproductive/Developmental Screening Test according to OECD 422 and GLP (Millard, 2021), DMAE was administered to groups of Sprague Dawley Crl:CD(SD) rats at graded dose levels of 0, 15, 50, or 150 mg/kg bw/day (p.o.). F0 males were dosed for 14 days prior to mating and continuing through 1 day prior to euthanasia. F0 females were dosed for 14 days prior to mating and continuing throughout mating, gestation, and lactation until 1 day prior to euthanasia. F1 animals were potentially exposed to the test substance in utero and via maternal milk during lactation from birth until Postnatal Day (PND) 21. Following selection and weaning, F1 male and female weanlings (at least 1 pup/sex/litter) was dosed once daily from PND 22 through 36, inclusively.

There were no test substance-related effects on general toxicity and neurobehavioral parameters (FOB and motor activity). With regard to fertility, no substance-related effects on reproductive performance (male and female mating and fertility, male copulation, and female conception indices, mean estrous cycle, precoital intervals, gestation length, and process of parturition) in F0 males or females at any dosage level were recorded.

Under the conditions of this screening study, based on the absence of adverse effects, a dosage level of 150 mg/kg/day, the highest dose administered, was considered to be the NOAEL for F0 male and female reproductive toxicity and systemic toxicity.

 

 

In a Combined Repeated Dose and Reproductive/Developmental Screening Test according to OECD 422 and GLP (BASF, 2019), the test compound DMAE was administered to groups of Wistar rats at concentrations of 0 ppm, 200 ppm, 1000 ppm, or 5000 ppm in the drinking water. These concentrations resulted in achieved test substance intakes by females/males of approx. 19, 89, and 355/257 mg/kg bw/day respectively. 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 post-mating in males and approximately 4-weeks post-mating 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 post-implantation 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.

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 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.

 

In the Pre- and Postnatal Developmental Toxicity Screening test (BASF, 2008), 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) at 0, 300, 600 mg/kg bw/day. The following effects on reproduction performance were observed, but assessed statistically not significant and/or without a dose-response relationship: Slightly increased post-implantation loss) (compared to the control group and the historical control data), also slightly increased resorptions (compared to the control group and the historical control data) were recorded. These effects were not considered toxicological relevant, therefore the NOAEL for reproductive performance was determined to be 600 mg/kg bw/day. No substance related findings were observed in foetuses. With regard to maternal toxicity, at 300 mg/kg bw/day local effects were recorded (strong ulcera in the stomach) and at 600 mg/kg bw/day systemic effects. Hence, a LOAEL for local effects was set to 300 mg/kg bw/day and a NOAEL for systemic effects to 300 mg/kg bw/day.

 

 

In the developmental OECD 414 study (Leung et al., 1996) timed-pregnant Fischer 344 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 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. 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. Since no toxicological relevant effects on fertility parameter were noted, the corresponding NOAEC was set to 100 ppm.

 

In further supporting studies it was noted that 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, oral: gavage, 0, 30, 100, 250 mg/kg bw of 2 -Dimethylaminoethanol. NOAEL (maternal toxicity) 100 mg/kg bw/day , NOAEL (prenatal developmental toxicity) 250 mg/kg bw/day

- 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. NOAEL (maternal toxicity) 10 ppm, NOAEL (embryonal toxicity and teratogenicity) ≥ 100 ppm

- 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.  NOAEL (F0 systemic toxicity) 300 mg/kg bw/day, LOAEL (F0 reproductive toxicity) 600 mg/kg bw/day, NOAEL (F1 pups) 300 mg/kg bw/day

Link to relevant study records

Reference

Reference 1

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.

Reference 2

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	23^a
^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 ± 1.2^b	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 ± 25.4^b	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 ± 16.9^c
Live litter size	9.5 ± 1.7	7.6 ± 3.1*	8.5 ± 3.4	8.4 ± 2.9^c
Fetal body weight/litter (g)
All fetuses	4.47 ± 0.15	4.56 ± 0.26	4.53 ± 0.24	4.66 ± 0.27^c
Male fetuses	4.63 ± 0.16	4.67 ± 0.24	4.61 ± 0.26^d	4.82 ± 0.26*^c
Female fetuses	4.28 ± 0.13	4.38 ± 0.26^e	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.

Reference 3

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 increased 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
- NOAEL 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

- NOAEL 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

Quality of whole database:

Data are of good quality (OECD Guideline Tests conduced in compliance with GLP)

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

In a Prenatal Developmental Toxicity Study according to OECD 414 and GLP (Millard, 2021), DMAE 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.

Adverse effects on maternal survival, maternal body weight losses and/or lower body weight gain, reduced food consumption, and corresponding clinical observations (considered secondary to local corrosion of the gastric mucosa) were recorded in the 250 mg/kg/day dose group. Based on these finding a dosage level of 100 mg/kg/day was considered to be the NOAEL for maternal toxicity. 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. 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. 1996 exposed pregnant timed-pregnant Fischer 344 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 (following a study design equivalent to OECD 414). 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), DMAE 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 to 100 %), 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.

 

Furthermore, in the EOGRTS according to OECD 443 and GLP (Millard, 2021) DMAE when administered orally by gavage to male and female Crl:CD(SD) rats NOAELs for F1 neonatal toxicity, F1 developmental neurotoxicity, and F1 immunotoxicity were set to 150 mg/kg/day (the highest dose level tested).

In a screening study according to OECD 422 and GLP (Millard, 2021) the highest dose administered dose, was considered to be the NOAEL of F1 systemic and neonatal/developmental toxicity of 2-Dimethylaminoethanol when administered orally by gavage to Crl:CD(SD) rats.

Toxicity to reproduction: other studies

Additional information

DMAE and Choline

There is some evidence from old publications 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 old 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

1. Assessment of reprotoxic potential of 2-Dimethylaminoethanol based available key studies for 2-Dimethylaminoethanol

 

The Extended One-Generation Reproductive Toxicity Study according to OECD 443 (oral route) and GLP (Millard, 2021, CRL) is considered as key study for reproductive toxicity. Adverse clinical observations or rales and clonic and/or tonic convulsion noted for F0 and F1 animals were observed in the highest dose group, hence a dose level of 50 mg/kg/day was considered to be the NOAEL for F0 and F1 systemic toxicity of DMAE when administered orally by gavage to male and female Crl:CD(SD) rats. Based on the lack of adverse effects for all other tested parameters, a dose level of 150 mg/kg/day (the highest dose level tested) was considered to be the NOAEL for F0 reproductive toxicity, F1 neonatal toxicity, F1 developmental neurotoxicity, and F1 immunotoxicity. These results were in accordance with the outcome of the screening test for reproductive / developmental toxicity according to OECD 422 (Millard, 2021). In this study, based on the absence of adverse effects, a dosage level of 150 mg/kg/day (the highest dose administered) was considered to be the NOAEL for F0 male and female reproductive toxicity, F0 male and female systemic toxicity, and F1 neonatal/developmental toxicity and systemic toxicity of DMAE when administered orally by gavage to Crl:CD(SD) rats.

The Prenatal Developmental Toxicity Study, conduced in rabbits according to OECD 414 (oral route) and GLP (Millard, 2021, CRL) is considered as a key study for pre-natal developmental toxicity. 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.

Regarding the prenatal developmental toxicity testing in a second species, the available inhalation study in rats, equivalent to OECD 414 (Leung et al., 1996) is considered as a second key study. Inhaled DMAE during organogenesis resulted in maternal toxicity at 100 ppm, therefore the maternal NOAEC was set to 10 ppm. Since no toxicological relevant effects on fertility parameter were noted, the corresponding NOAEC was set to 100 ppm.

 

Taken together all of the reprotoxic findings, including supporting in vivo studies, it is clear that DMAE if administered orally/via inhalation to rats/rabbits exhibits its effects on reproductive performance and developmental parameters only in the presence of systemic parental toxicity (Millard, 2021, OECD 443; OECD 422, BASF, 2019; Millard, 2021, OECD 414; Leung et al., 1996 OECD 414, Zahniser et al., 1978, BASF, 2008 Modified Developmental Toxicity Screening Study; Katyal and Lombardi, 1977, Zahniser et al., 1978). In addition, neither systemic parental toxicity nor reproduction or developmental effects were reported in an OECD 422 conducted recently (Millard, 20201).

Prenatal effects seen in further supporting studies with DMAE administered orally (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.

 

Based on further available in vitro and in vivo studies, it was discussed that the observed effects of DMAE in offspring may be related to choline homeostasis, i.e. disruption of intracellular methylation processes.  

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.

Dainous and Kanfer (1988), studied the interrelationship between choline uptake, synthesis of phospholipids, sphyngomyelins and alkanolamines administration. In the study, the incorporation of DMAE into phospholipids was investigated in fetal rat brain aggregating cell cultures. It was reported that DMAE was rapidly phosphorilated after it entered the cell and served as a precursor of phosphatidyldimethylethanolamine (Dainous and Kanfer, 1988, cited in “Toxicological Summary for Dimethylamine and Selected Salts and Esters”, 2002).

Zahniser et al. (1978) found that in brains of pups those mothers were fed with choline deficient but DMAE or N-Methylethanolamine (MMEA) supplemented diet the phosphatidyl-Ch and hosphatidylaminoethanol (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. In a study by Katyal and lombardi 1978, pregnant rats that were fed with a choline-deficient diet, but 1% DMAE developed a respiratory distress syndrome due to altered metabolism of lung surfactant.

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).

Based on the above described studies it was suggested that that effects on fertility as well as the low viability of pups observed in testing animals exposed to DMEA might be related to perturbation of intracellular methylation processes. In general, however, these in vitro and in vivo experiments are considered to be of little significance as the conditions, including experiments in choline deficiency environments, does not reflect the situation as it occurs in animals and humans. Therefore these results are either disregarded or judged only very careful. The actual animal GLP guideline studies are considered to be most reliable and provide valuable insights which allow judging the hazard profile of DMAE.

 

Considerations in relation to CLP:

Based on the available data no classification is proposed according to Regulation (EC) No 1272/2008.

 

2. Assessment of convulsions in relation to DMAE treatment and classification (please find full statement in section 13.2)

 

2-Dimethylethanolamine (DMAE; CASRN 108-01-0) has been tested for effects upon reproduction in OECD TG 422 combined repeated-dose/reproductive toxicity screening studies and OECD TG 443 extended one-generation reproductive toxicity study. Convulsions have been reported for some treated female animals. The purpose of this document is to summarise the findings, and to evaluate them in relation to treatment and relevance for classification.

Because, these studies conformed to standard OECD test guidelines, methodological details and general observations are omitted, except where they have bearing on the interpretation of findings.

 

OECD TG 422 (BASF, 2019)

DMAE was administered to groups of Wistar rats at concentrations of 0 ppm, 200 ppm, 1000 ppm, or 5000 ppm in the drinking water. These concentrations resulted in achieved test substance intakes by females of approx. 19, 89, and 355 mg/kg bw/day respectively.

Relevant findings were limited to females in the high-dose group. Food and water consumption were substantially reduced, and body weight gain was impaired. The severe reduction in water consumption, by up to ~30 %, was attributed to the negative organoleptic characteristics of DMAE. One animal was found dead on PND17. Four females exhibited moderate-to-severe tonic/clonic convulsions, each on a single day of lactation. Histopathologic evaluation revealed axonal degeneration of the sciatic nerve (1/6), cervical cord (6/10), thoracic cord (5/10), and brain (8/10).

 

OECD TG 422 (Millard, 2021, CRL)

DMAE was administered to groups of Sprague Dawley Crl:CD(SD) rats at graded dose levels of 0, 15, 50, or 150 mg/kg bw/day (p.o.).

Relevant findings were limited to females in the high-dose group. One female exhibited a single event of clonic convulsions on day 59. No observations of clonic or tonic convulsions were made in any animals during the scheduled FOB test.

 

OECD TG 443 (Millard 2021, CRL)

DMAE was administered to groups of Sprague Dawley Crl:CD(SD) rats at graded dose levels of 0, 15, 50, or 150 mg/kg bw/day (p.o.). The study included cohort 2, for evaluation of effects upon the developing nervous system, including histopathology.

With the exception of one F₀ female of the mid-dose group, which showed a single event of clonic convulsion at dosing, the relevant findings were limited to males and females in the high-dose group. In this group, five F₀ males, twelve F₀ females, and four F₁ females exhibited clonic convulsions on one or more days. Two of the F₀ males also exhibited tonic convulsions alongside clonic convulsions on one occasion.

Functional tests in Cohort 2 revealed no difference between control and treatment groups. Histopathologic evaluation of the CNS indicated no structural or morphometric correlate to the clinical observations.

Consideration of the findings in relation to CLP

Classification for systemic target organ toxicity-repeated exposure (STOT RE) may be warranted on the “basis of evidence from studies in experimental animals can be presumed to have the potential to be harmful to human health following repeated exposure.” The determination of whether a substance should be classified into category 1 or category 2 is made on the basis of whether effects might be elicited at low exposures (≤ 10 mg/kg bw/day; Category 1) or moderate exposures (≤ 100 mg/kg bw/day; Category 2). These threshold values apply to oral studies in rat of 90 days’ duration.

The convulsions that have been noted for DMAE occur at doses in exceedance of 100 mg/kg bw/day, with one singular exception. The findings in oral gavage studies were sporadic and inconsistent, and there was no histopathologic correlate. Histopathological findings in the drinking water study were observed in high-dose animals in the presence of substantial toxicity: mortality; reduced body weight gain; reduced food intake; and extended and severely reduced water consumption, of which they may be a secondary response.

Consequently, the findings are not considered to support classification of DMAE for STOT-RE.