2-dimethylaminoethanol

Repeated dose toxicity: oral

Administrative data

Endpoint:

repeated dose toxicity: oral, other

Remarks:

Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test

Type of information:

experimental study

Adequacy of study:

key study

Study period:

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

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.

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Reference

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.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

29 July 2016

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

2-dimethylaminoethanol

EC Number:

203-542-8

EC Name:

2-dimethylaminoethanol

Cas Number:

108-01-0

Molecular formula:

C4H11NO

IUPAC Name:

2-(dimethylamino)ethanol

Test material form:

liquid

Details on test material:

- State of aggregation: colourless liquid

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

Test animals

Species:

rat

Strain:

other: Crl:CD(SD)

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

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

Deionized water

Details on oral 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

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

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Control

Doses / concentrations 2

Dose / conc.:

15 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

50 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

150 mg/kg bw/day (nominal)

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.

Examinations

Observations and examinations performed and frequency:

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).

Sacrifice and pathology:

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.

Statistics:

See 'Any other information'

Results and discussion

Results of examinations

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.

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

Description (incidence and severity):

Test substance-related higher mean liver weights were noted in the 50 and 150 mg/kg/day group females and test substance-related lower mean thymus weights were noted in the 150 mg/kg/day group males. Test substance-related organ weight changes are summarized in Text Table 26 (see attachment).
Test substance-related alterations in liver weights and thymus weights occurred without any correlating gross observations or microscopic findings. No other test substance-related organ weight changes were noted. There were other isolated organ weight values that were statistically different from their respective control group. There were, however, no patterns, trends, or correlating data to suggest these values were toxicologically relevant. Thus, other organ weight differences observed were considered incidental and/or related to difference of sexual maturity and unrelated to administration of 2-Dimethylaminoethanol.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Gross observations of dark red area(s) were noted in stomach of the females at 15, 50, and 150 mg/kg/day and 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. However, the relationship to the test substance was uncertain.
No other test substance-related gross findings were noted. The remaining gross findings observed were considered incidental and/or of the nature commonly observed in this strain and age of rat and, therefore, were considered unrelated to administration of 2-Dimethylaminoethanol.
The mean numbers of unaccounted-for sites and implantation sites in the 15, 50, and 150 mg/kg/day groups were unaffected by test substance administration. Statistically significantly higher mean numbers of implantation sites were noted in the test substance-treated groups; however, higher mean numbers of implantations were not considered to be of toxicological significance.

Neuropathological findings:

not examined

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

Effect levels

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

150 mg/kg bw/day (nominal)

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.

Target system / organ toxicity

Critical effects observed:

no

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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 systemic toxicity.

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 repeated dose toxicity within the scope of an OECD 422 study.

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.

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.

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 systemic toxicity.