2-dimethylaminoethanol

Endpoint summary

Description of key information

- Repeated dose toxicity, oral gavage: Millard 2021. Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test accoding to OECD 422 and GLP, conc. tested: 0, 15, 50, 150 mg/kg bw/day. No adverse effects observed. NOAEL is 150 mg/kg bw/day

- Repeated dose toxicity, inhalation: Klonne et al., 1987. Dimethylethanolamine: Acute, 2-week and 13-week Inhalation Toxicity Studies in Rats. Comparable to the OECD guideline 413, conc. tested: 8, 24 and 76 ppm (corresponding to 29.2, 87.5 and 277.1 mg/m³). NOEC is 87.5 mg/m³.

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

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

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other 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)

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 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

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Control

Dose / conc.:

15 mg/kg bw/day (nominal)

Dose / conc.:

50 mg/kg bw/day (nominal)

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.

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'

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

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.

Critical 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 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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

150 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Good quality: guideline study and accoding to GLP

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

not reported, published 1987

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Acceptable, well documented publication which meets basic scientific principles.

Reason / purpose for cross-reference:

reference to other study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)

Deviations:

not applicable

GLP compliance:

no

Limit test:

no

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Kingston, NY)
- Age at study initiation: approx. 9 weeks old.
- Weight at study initiation: 180 g for males and 130 g females
- Fasting period before study:
- Housing:
- Diet (e.g. ad libitum): Purina Certified Rodent Chow 5002, Ralston Purina Co., St. Louis, MO ad libitum except during exposures
- Water (e.g. ad libitum): yes, except during exposures
- Acclimation period: yes

ENVIRONMENTAL CONDITIONS
- Temperature (°C): not reported
- Humidity (%): not reported
- Air changes (per hr): not reported
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

inhalation

Type of inhalation exposure:

not specified

Vehicle:

other: no data

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: 4320-liter stainless-steel and glass chambers
- Method of holding animals in test chamber: not reported
- Source and rate of air: not reported
- Method of conditioning air: not reported
- System of generating particulates/aerosols: DMEA vapor was generated by metering the liquid into a heated, spiral-grooved evaporator with a countercurrent airstream, similar in design to the one used by Carpenter et al. (1975).
- Temperature, humidity, pressure in air chamber: 25°C and 46%,
- Air flow rate: 800-1000 liters/min
- Air change rate: not reported
- Method of particle size determination: not reported
- Treatment of exhaust air: not reported

TEST ATMOSPHERE
- Brief description of analytical method used: The CC column was a 5 ft X 1/4 in stainless-steel column packed with 20% SP-2100 on 80/ 100 mesh Supelcoport (Supelco, BeIlefonte, PA), maintained at 200°C.
- Samples taken from breathing zone: yes

VEHICLE (if applicable) no

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Chamber concentrations of DMEA were analyzed at approximately 40-min intervals with a Perkin-Elmer 3920B gas chromatograph (CC) equipped with a flame ionization detector. Chamber atmosphere samples were automatically injected into the GC with the use of a Perkin-Elmer environmental sampler.

Duration of treatment / exposure:

13 wk

Frequency of treatment:

6h/d, 5d/wk

Dose / conc.:

8 ppm (analytical)

Remarks:

corresponding to 29.2 mg/m³

Dose / conc.:

24 ppm (analytical)

Remarks:

corresponding to 87.5 mg/m³

Dose / conc.:

76 ppm (analytical)

Remarks:

corresponding to 277.1 mg/m³

No. of animals per sex per dose:

20

Control animals:

yes

Details on study design:

10 male rats were assigned to the control, middle and high concentration groups for possible ultrastructural evaluation of nerve tissue (not performed since no behavioral abnormalities or light microscopic lesions of nerve tissue were observed).

- Dose selection rationale: not reported
- Rationale for animal assignment (if not random): randomized
- Rationale for selecting satellite groups: to investigate recovery of effects of treatment
- Post-exposure recovery period in satellite groups: 5 week

One-half of all rats per sex per group were sacrificed after at least 2 days of exposure during the 14th week of the study; the remaining rats were sacrificed after 5 complete weeks of recovery.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations checked in table [No.3] were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: daily

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: were measured during the 16-hr urine collection period.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No data

WATER CONSUMPTION: were measured during the 16-hr urine collection period.

OPHTHALMOSCOPIC EXAMINATION: Yes, (evaluation of the eye with a light source and magnifying lens)
- Time schedule for examinations: not reported
- Dose groups that were examined: not reported

HAEMATOLOGY: Yes
- Time schedule for collection of blood: made the morning following the last DMEA exposure day (except for recovery animals).
- Anaesthetic used for blood collection: Yes (methoxyflurane)
- Animals fasted: No
- How many animals: not reported
- Parameters checked in table [No.1] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: made the morning following the last DMEA exposure day (except for recovery animals).
- Animals fasted: No
- How many animals: not reported
- Parameters checked in table [No.2] were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: during a 16-hr period prior to sacrifice from rats held in polycarbonate metabolism cages
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No data
- Parameters examined: Semiquantitative measurements on urine: included pH, protein, bilirubin, urobilinogen, glucose, ketones, and blood. In addition, osmolality determinations (Cryomatic osmometer, Advanced Instruments, Inc., Needham Heights, MA) and microscopic evaluations of the urine sediment were performed.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: not reported
- Dose groups that were examined: not reported
- Battery of functions tested: sensory activity / grip strength / motor activity / other: see Table 3

OTHER: Organ weight determinations, gross pathologic examination, and blood and urine sample collections were made the morning following the last DMEA exposure day (except for recovery animals).

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes (see Table 4). Additional histopathologic examinations were performed on rats from the control and high exposure groups, with nasal turbinates also being evaluated for the middle exposure group rats.

Other examinations:

Organ weights: brain, kidney, liver, lungs, testes, and adrenals.

Statistics:

Results of quantitative continuous variables were intercompared among the DMEA concentration levels and the control group by Bartlett’s homogeneity of variance (Sokal and Rohlf, 1969), analysis of variance (ANOVA), and Duncan’s multiple range test (Snedecor and Cochran, 1967). Duncan’s test was used when a significant F value from an ANOVA was observed. For heterogeneous group variances, the groups were compared by ANOVA for unequal variances (Brown and Forsythe, 1974) and either Student’s t test or Cochran’s t test (Snedecor and Cochran, 1967) was used. Corrected Bonferroni probabilities were used for t test comparisons (Miller, 1966). The fiducial limit of 0.05 (two-tailed) was used as the critical level of significance for all comparisons. For the calculation of the LC50, the method of Finney (1964) was used.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

lower in 76 ppm-group, returned to control during the recovery period

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

no effects observed

Ophthalmological findings:

effects observed, treatment-related

Description (incidence and severity):

corneal opacity which regressed during the night

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

nasal lesions

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
No animals died during the study.

BODY WEIGHT AND WEIGHT GAIN
The body weight gains for both sexes of the 76 ppm group were statistically significantlly lower than control values for most of the latter half of the 13 week exposure regimen (Table 1). The body weight gain valuesfor the 76 ppm group returned to control values during the recovery period. There were no exposure-related alterations of body weight gain for rats exposed to 8 or 24 ppm of DMEA.

OPHTHALMOSCOPIC EXAMINATION
Corneal opacity occurred in the 24 and 76ppm groups at the end of the daily exposure, beginning approximately 2-3 weeks after initiation of exposures. The opacity regressed during the night-time nonexposure hours. There was also a moderate incidence (approximately 25%) of audible respiration in rats of the 76 ppm group.

HISTOPATHOLOGY: NON-NEOPLASTIC
Exposure related nasal lesions were observed histologically at the termination of exposures in both sexes of the 76 ppm group, but were generally not observed in rats of the 24 ppm group (Table 2). The lesions were limited to the anterior nasal cavity and included squamous metaplasia, microcysts (cystic intraepithelial glands) mucous cell hyperplasia of the respiratory epithelium, mild rhinitis, and atrosoluphy of the dorsal olfactory epithelium. The incidence and severity of these lesions were decreased at the end of the recovery period, indicating some degree of repair. Additionally, 4/ 10 males had laryngitis and two of these rats also had tracheitis. No similar lesions were found in female rats. Vacuolization of the corneal epithelium was observed in 3/10 female rats of the 76 ppm group at the termination of exposures but not at the end of the recovery period.

Dose descriptor:

NOEC

Effect level:

24 ppm

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

body weight and weight gain

histopathology: non-neoplastic

mortality

Dose descriptor:

NOEC

Effect level:

87.5 mg/m³ air

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

body weight and weight gain

histopathology: non-neoplastic

mortality

Critical effects observed:

not specified

TABLE 1
Body Weight Gain for F-344 Rats Exposed to DMEA Vapor for 13 Weeks and Maintained for a 5-Week Recovery Period^a
Interval (weeks)	Mean exposure concentration (ppm)
	0	8	24	76
	Males
8^b	108 ± 12.2	112 ± 8.6	113 ± 11.1	102 ± 8.0*
14^c	154 ± 11.5	157 ± 9.9	157 ± 10.2	147 ± 10.6*
19^d	167 ± 16.7	166 ± 7.1	172 ± 13.2	169 ± 9.2
	Females
8	54 ± 4.7	53 ± 5.9	51 ± 5.0	49 ± 6.8**
14	73 ± 5.4	70 ± 5.8	70 ± 6.3	64 ± 6.6**
19	76 ± 6.8	73 ± 7.7	71 ± 5.1	71 ± 4.0
^aValues represent mean ± SD. ^bWeek 8 was the first week that a consistent statistically significant depression in body weight gain was observed for both sexes of the 76 ppm group. ^cFinal weight gain value obtained after two or three exposures during Week 14. ^dEnd of 5-week postexposure recovery period. * p<0.05 from controls. ** p <0.01 from controls.

TABLE 2
Incidence of Selected Nasal Tissue Histopathologic Lesions in F-344 Rats Exposed to DMEA Vapor for 2 or 13 Weeks and Sacrificed the Day after the Final Exposure
	2-week study^a						13-week study^b
	Males			Females			Males			Females
DMEA concentration (ppm):	0	98	288	0	98	288	0	24	76	0	24	76
Total No. examined	10	10	7	10	10	10	10	10	10	10	10	10
Unremarkable	10	1	0	9^c	5	0	9^d	8	0	9^d	8	1
Rhinitis	0	5	6	0	5	7	0	2	0	0	2	7
Squamous metaplasia	0	8	7	0	2	10	0	0	9	0	0	4
Epithelial erosion	0	1	4	0	0	0	0	0	0	0	0	0
Mucosal ulceration	0	4	6	0	2	8	0	0	0	0	0	0
Degeneration of olfactory mucosa/epithelium	0	0	1	0	0	9	0	0	0	0	0	0
Degeneration of respiratory epithelium	0	0	0	0	0	4	0	0	8	0	0	7
Atrophy of olfactory epithelium	0	0	0	0	0	0	0	0	10	0	0	3
Microcysts in respiratory epithelium	0	0	0	0	0	0	0	0	10	0	0	3
^aAll animals of the 586 ppm group died on study and were not histologically evaluated. Incidence values in the table also do not include those four male rats of the 288 ppm group which died on study. ^bRats of the 8 ppm group were not histologically evaluated as only minimal rhinitis was observed in 2 rats/sex of the 24 ppm group. ^cOne control had a hemorrhage in the nasolacrimal duct. ^dOne control had minimal mineralization of the olfactory epithelium.

Conclusions:

DMAE acts primarily as an ocular and upper respiratory tract irritant. 24 ppm is the NOEC.

Executive summary:

In the 13-week sub-chronic study, F-344 rats were exposed to 0, 8, 24, or 76 ppm DMEA for 6 hr/day, 5 days/week for 13 weeks. These doses correspond to 29.2, 87.5 and 277.1 mg/m³. The principal exposure-related changes were transient comeal opacity in the 24 and 76 ppm groups; decreased body weight gain for the 76 ppm group; and histopathologic lesions of the respiratory and olfactory epithelium of the anterior nasal cavity of the 76 ppm group and of the eye of several 76 ppm group females. Rats maintained for a 5-week recovery period only exhibited histological lesions of the nasal tissue, with the lesions being decreased in incidence and severity. DMEA acts primarily as an ocular and upper respiratory tract irritant and toxicant at vapor concentrations of 76 ppm, while 24 ppm or less produced no biologically significant toxicity in rats. Thus, 24 ppm (= 87.5 mg/m³) was considered to be the no-observable-effect level.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

87.5 mg/m³

Study duration:

subchronic

Experimental exposure time per week (hours/week):

30

Species:

rat

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

not reported, published 1987

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Acceptable, well documented publication which meets basic scientific principles.

Reason / purpose for cross-reference:

reference to other study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)

Deviations:

not applicable

GLP compliance:

no

Limit test:

no

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Kingston, NY)
- Age at study initiation: approx. 9 weeks old.
- Weight at study initiation: 180 g for males and 130 g females
- Fasting period before study:
- Housing:
- Diet (e.g. ad libitum): Purina Certified Rodent Chow 5002, Ralston Purina Co., St. Louis, MO ad libitum except during exposures
- Water (e.g. ad libitum): yes, except during exposures
- Acclimation period: yes

ENVIRONMENTAL CONDITIONS
- Temperature (°C): not reported
- Humidity (%): not reported
- Air changes (per hr): not reported
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

inhalation

Type of inhalation exposure:

not specified

Vehicle:

other: no data

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: 4320-liter stainless-steel and glass chambers
- Method of holding animals in test chamber: not reported
- Source and rate of air: not reported
- Method of conditioning air: not reported
- System of generating particulates/aerosols: DMEA vapor was generated by metering the liquid into a heated, spiral-grooved evaporator with a countercurrent airstream, similar in design to the one used by Carpenter et al. (1975).
- Temperature, humidity, pressure in air chamber: 25°C and 46%,
- Air flow rate: 800-1000 liters/min
- Air change rate: not reported
- Method of particle size determination: not reported
- Treatment of exhaust air: not reported

TEST ATMOSPHERE
- Brief description of analytical method used: The CC column was a 5 ft X 1/4 in stainless-steel column packed with 20% SP-2100 on 80/ 100 mesh Supelcoport (Supelco, BeIlefonte, PA), maintained at 200°C.
- Samples taken from breathing zone: yes

VEHICLE (if applicable) no

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Chamber concentrations of DMEA were analyzed at approximately 40-min intervals with a Perkin-Elmer 3920B gas chromatograph (CC) equipped with a flame ionization detector. Chamber atmosphere samples were automatically injected into the GC with the use of a Perkin-Elmer environmental sampler.

Duration of treatment / exposure:

13 wk

Frequency of treatment:

6h/d, 5d/wk

Dose / conc.:

8 ppm (analytical)

Remarks:

corresponding to 29.2 mg/m³

Dose / conc.:

24 ppm (analytical)

Remarks:

corresponding to 87.5 mg/m³

Dose / conc.:

76 ppm (analytical)

Remarks:

corresponding to 277.1 mg/m³

No. of animals per sex per dose:

20

Control animals:

yes

Details on study design:

10 male rats were assigned to the control, middle and high concentration groups for possible ultrastructural evaluation of nerve tissue (not performed since no behavioral abnormalities or light microscopic lesions of nerve tissue were observed).

- Dose selection rationale: not reported
- Rationale for animal assignment (if not random): randomized
- Rationale for selecting satellite groups: to investigate recovery of effects of treatment
- Post-exposure recovery period in satellite groups: 5 week

One-half of all rats per sex per group were sacrificed after at least 2 days of exposure during the 14th week of the study; the remaining rats were sacrificed after 5 complete weeks of recovery.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations checked in table [No.3] were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: daily

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: were measured during the 16-hr urine collection period.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No data

WATER CONSUMPTION: were measured during the 16-hr urine collection period.

OPHTHALMOSCOPIC EXAMINATION: Yes, (evaluation of the eye with a light source and magnifying lens)
- Time schedule for examinations: not reported
- Dose groups that were examined: not reported

HAEMATOLOGY: Yes
- Time schedule for collection of blood: made the morning following the last DMEA exposure day (except for recovery animals).
- Anaesthetic used for blood collection: Yes (methoxyflurane)
- Animals fasted: No
- How many animals: not reported
- Parameters checked in table [No.1] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: made the morning following the last DMEA exposure day (except for recovery animals).
- Animals fasted: No
- How many animals: not reported
- Parameters checked in table [No.2] were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: during a 16-hr period prior to sacrifice from rats held in polycarbonate metabolism cages
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No data
- Parameters examined: Semiquantitative measurements on urine: included pH, protein, bilirubin, urobilinogen, glucose, ketones, and blood. In addition, osmolality determinations (Cryomatic osmometer, Advanced Instruments, Inc., Needham Heights, MA) and microscopic evaluations of the urine sediment were performed.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: not reported
- Dose groups that were examined: not reported
- Battery of functions tested: sensory activity / grip strength / motor activity / other: see Table 3

OTHER: Organ weight determinations, gross pathologic examination, and blood and urine sample collections were made the morning following the last DMEA exposure day (except for recovery animals).

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes (see Table 4). Additional histopathologic examinations were performed on rats from the control and high exposure groups, with nasal turbinates also being evaluated for the middle exposure group rats.

Other examinations:

Organ weights: brain, kidney, liver, lungs, testes, and adrenals.

Statistics:

Results of quantitative continuous variables were intercompared among the DMEA concentration levels and the control group by Bartlett’s homogeneity of variance (Sokal and Rohlf, 1969), analysis of variance (ANOVA), and Duncan’s multiple range test (Snedecor and Cochran, 1967). Duncan’s test was used when a significant F value from an ANOVA was observed. For heterogeneous group variances, the groups were compared by ANOVA for unequal variances (Brown and Forsythe, 1974) and either Student’s t test or Cochran’s t test (Snedecor and Cochran, 1967) was used. Corrected Bonferroni probabilities were used for t test comparisons (Miller, 1966). The fiducial limit of 0.05 (two-tailed) was used as the critical level of significance for all comparisons. For the calculation of the LC50, the method of Finney (1964) was used.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

lower in 76 ppm-group, returned to control during the recovery period

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

no effects observed

Ophthalmological findings:

effects observed, treatment-related

Description (incidence and severity):

corneal opacity which regressed during the night

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

nasal lesions

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
No animals died during the study.

BODY WEIGHT AND WEIGHT GAIN
The body weight gains for both sexes of the 76 ppm group were statistically significantlly lower than control values for most of the latter half of the 13 week exposure regimen (Table 1). The body weight gain valuesfor the 76 ppm group returned to control values during the recovery period. There were no exposure-related alterations of body weight gain for rats exposed to 8 or 24 ppm of DMEA.

OPHTHALMOSCOPIC EXAMINATION
Corneal opacity occurred in the 24 and 76ppm groups at the end of the daily exposure, beginning approximately 2-3 weeks after initiation of exposures. The opacity regressed during the night-time nonexposure hours. There was also a moderate incidence (approximately 25%) of audible respiration in rats of the 76 ppm group.

HISTOPATHOLOGY: NON-NEOPLASTIC
Exposure related nasal lesions were observed histologically at the termination of exposures in both sexes of the 76 ppm group, but were generally not observed in rats of the 24 ppm group (Table 2). The lesions were limited to the anterior nasal cavity and included squamous metaplasia, microcysts (cystic intraepithelial glands) mucous cell hyperplasia of the respiratory epithelium, mild rhinitis, and atrosoluphy of the dorsal olfactory epithelium. The incidence and severity of these lesions were decreased at the end of the recovery period, indicating some degree of repair. Additionally, 4/ 10 males had laryngitis and two of these rats also had tracheitis. No similar lesions were found in female rats. Vacuolization of the corneal epithelium was observed in 3/10 female rats of the 76 ppm group at the termination of exposures but not at the end of the recovery period.

Dose descriptor:

NOEC

Effect level:

24 ppm

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

body weight and weight gain

histopathology: non-neoplastic

mortality

Dose descriptor:

NOEC

Effect level:

87.5 mg/m³ air

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

body weight and weight gain

histopathology: non-neoplastic

mortality

Critical effects observed:

not specified

TABLE 1
Body Weight Gain for F-344 Rats Exposed to DMEA Vapor for 13 Weeks and Maintained for a 5-Week Recovery Period^a
Interval (weeks)	Mean exposure concentration (ppm)
	0	8	24	76
	Males
8^b	108 ± 12.2	112 ± 8.6	113 ± 11.1	102 ± 8.0*
14^c	154 ± 11.5	157 ± 9.9	157 ± 10.2	147 ± 10.6*
19^d	167 ± 16.7	166 ± 7.1	172 ± 13.2	169 ± 9.2
	Females
8	54 ± 4.7	53 ± 5.9	51 ± 5.0	49 ± 6.8**
14	73 ± 5.4	70 ± 5.8	70 ± 6.3	64 ± 6.6**
19	76 ± 6.8	73 ± 7.7	71 ± 5.1	71 ± 4.0
^aValues represent mean ± SD. ^bWeek 8 was the first week that a consistent statistically significant depression in body weight gain was observed for both sexes of the 76 ppm group. ^cFinal weight gain value obtained after two or three exposures during Week 14. ^dEnd of 5-week postexposure recovery period. * p<0.05 from controls. ** p <0.01 from controls.

TABLE 2
Incidence of Selected Nasal Tissue Histopathologic Lesions in F-344 Rats Exposed to DMEA Vapor for 2 or 13 Weeks and Sacrificed the Day after the Final Exposure
	2-week study^a						13-week study^b
	Males			Females			Males			Females
DMEA concentration (ppm):	0	98	288	0	98	288	0	24	76	0	24	76
Total No. examined	10	10	7	10	10	10	10	10	10	10	10	10
Unremarkable	10	1	0	9^c	5	0	9^d	8	0	9^d	8	1
Rhinitis	0	5	6	0	5	7	0	2	0	0	2	7
Squamous metaplasia	0	8	7	0	2	10	0	0	9	0	0	4
Epithelial erosion	0	1	4	0	0	0	0	0	0	0	0	0
Mucosal ulceration	0	4	6	0	2	8	0	0	0	0	0	0
Degeneration of olfactory mucosa/epithelium	0	0	1	0	0	9	0	0	0	0	0	0
Degeneration of respiratory epithelium	0	0	0	0	0	4	0	0	8	0	0	7
Atrophy of olfactory epithelium	0	0	0	0	0	0	0	0	10	0	0	3
Microcysts in respiratory epithelium	0	0	0	0	0	0	0	0	10	0	0	3
^aAll animals of the 586 ppm group died on study and were not histologically evaluated. Incidence values in the table also do not include those four male rats of the 288 ppm group which died on study. ^bRats of the 8 ppm group were not histologically evaluated as only minimal rhinitis was observed in 2 rats/sex of the 24 ppm group. ^cOne control had a hemorrhage in the nasolacrimal duct. ^dOne control had minimal mineralization of the olfactory epithelium.

Conclusions:

DMAE acts primarily as an ocular and upper respiratory tract irritant. 24 ppm is the NOEC.

Executive summary:

In the 13-week sub-chronic study, F-344 rats were exposed to 0, 8, 24, or 76 ppm DMEA for 6 hr/day, 5 days/week for 13 weeks. These doses correspond to 29.2, 87.5 and 277.1 mg/m³. The principal exposure-related changes were transient comeal opacity in the 24 and 76 ppm groups; decreased body weight gain for the 76 ppm group; and histopathologic lesions of the respiratory and olfactory epithelium of the anterior nasal cavity of the 76 ppm group and of the eye of several 76 ppm group females. Rats maintained for a 5-week recovery period only exhibited histological lesions of the nasal tissue, with the lesions being decreased in incidence and severity. DMEA acts primarily as an ocular and upper respiratory tract irritant and toxicant at vapor concentrations of 76 ppm, while 24 ppm or less produced no biologically significant toxicity in rats. Thus, 24 ppm (= 87.5 mg/m³) was considered to be the no-observable-effect level.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

87.5 mg/m³

Study duration:

subchronic

Species:

rat

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Repeated dose toxicity: oral gavage

Recently, a combined 28 -day repeated dose oral (gavage) toxicity study with the reproduction/developmental toxicitxy screening test of 2 -Dimethylaminoethanol was conducted in Sprague Dawley Rats. The objective of this GLP-study was to provide preliminary information on the potential adverse effects of the test substance on male and female repeated dose toxicity within the scope of an OECD 422 study. Animals (10 / sex / dose group) were administered 0, 15, 50, 150 mg/kg bw/day via oral gavage.

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.

 

 

Repeated dose toxicity: inhalation:

In the two-week inhalation study (Klonne et al., 1987, supporting study), rats exposed to higher concentrations of DMAE vapour (98, 288 and 586 ppm) during an 11-day period exhibited severe signs of respiratory and ocular irritation (except the 98 ppm group). All animals of the 586 ppm group and 4 of 15 male rats of the 288 ppm group died. Body weight values for the 288 ppm group were reduced to about 75 % of preexposure values, while the 98 ppm group gained 35 % less weight than controls. Statistically significant differences in clinical pathology parameters (288 ppm group) and in organ weight values (288 and 98 ppm groups) probably resulted from the decreased food consumption and not from specific target organ toxicity. In the groups evaluated histologically (the 98 and 288 ppm groups) the eye and nasal mucosa were the primary target organs.

In the 13-week sub-chronic study (Klonne et al., 1987; Key study), F-344 rats were exposed to 0, 8, 24, or 76 ppm DMEA for 6 hr/day, 5 days/week for 13 weeks. No animals died during the study. The body weight gains for both sexes of the 76 ppm group were statistically significantly lower than control values for most of the latter half of the 13-week exposure time. The body weigh gain values for the 76 ppm group returned to control values during the recovery period. There were no exposure-related of body weight gain for rats exposed to 8 or 24 ppm of DMEA. There were no exposure-related effects on the neurobehavioral, food and water consumption, hematologic, serum chemistry, or urinalysis evaluations, on organ weights, or on the gross appearance of organs. Exposure-related nasal lesions were observed histologically at the termination of exposures in both sexes of the 76 ppm group, but were generally not observed in rats of the 24 ppm group. The lesions were limited to the anterior nasal cavity and included squamous metaplasia, microcysts and mucous cell hyperplasia of the respiratory epithelium, mild rhinitis and atrophy of the dorsal olfactory epithelium. The incidence and severity of these lesions were decreased at the end of the recovery period, indicating some degree of repair. Additionally, 4/10 males had laryngitis and two of these rats also had tracheitis. No similar lesions were found in female rats. Vacuolization of the corneal epithelium was observed in 3/10 female rats of the 76 ppm group at the termination of exposures but not at the end of the recovery period. Corneal opacity occurred in the 24 and 76 ppm groups at the end of the daily exposure, beginning approximately 2-3 weeks after initiation of exposures. The opacity regressed during the night-time nonexposure hours. There was also a moderate incidence (approximately 25 %) of audible respiration in rats of the 76 ppm group.

 

In conclusion, DMAE acts primarily as an irritant to the eyes and respiratory tract. In rats, repeated exposure to DMAE vapour up to 24 ppm represent no hazard. Thus, 24 ppm (87.5 mg/m³) is NOEC.

Repeated dose toxicity: inhalation - systemic effects (target organ) respiratory: nose; other: all gross lesions and masses

Justification for classification or non-classification

Classification is not warranted according to the criteria of EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008. 

The assessment of whether STOT-RE classification is required was carried out on the basis of available studies according to OECD 443 and 422 and can be found in the endpoint summary under 7.8 'Toxicity to reproduction'. The full justification for non-classification is attached in section 13.2.