Ethyldimethylamine

Endpoint summary

Description of key information

There are three studies available for DMEA. A 7-, 28- and 90-day inhalation studies performed in rats performed.

A two weeks oral toxicity repeated study was performed as a preliminary study for oral reprotoxicity studies (Renaud, Covance 2020).

 

1. 7-day inhalation toxicity study (van Triel et al., 2019)

The concentrations to be tested in the sub-acute study were selected on the basis of a 7-day range finding study in which five groups of five male and five female Wistar rats were exposed to target concentrations of 0, 10, 100, 500 or 1000 ppm for 6 hours/day, 5 days/week. Exposure at 1000 ppm resulted in marked toxicity, as evidenced by clinically observed breathing abnormalities and general signs of substantial distress, depressed growth and food intake, increased relative weight of several organs (heart, lungs, and in males also testes and adrenals), and histopathological changes along the entire respiratory tract indicating severe epithelial damage (e.g. ulceration, atrophy, necrosis) in the upper airways and primarily inflammatory changes in the lower airways. Exposure at 500 ppm resulted in transient clinical abnormalities, growth depression (mainly in males), atrophy of the nasal olfactory epithelium, and inflammatory changes in the lower airways of several animals. Minimal to mild atrophy of the olfactory epithelium was still observed in animals exposed at 100 ppm. Exposure at 10 ppm did not result in any exposure-related changes.

 

2. 28-day inhalation toxicity study (Van Triel et al., 2019)

The toxicity of Dimethylethylamine upon repeated exposure by inhalation was investigated in a sub-acute (28-day) study in Wistar rats perfomed according to OECD 412 guideline and GLP. Four main groups of five male and five female rats each were exposed (nose-only) to target concentrations of 0 (control), 10, 50 or 250 ppm for 6 hours/day, 5 days/week over a 28-day period. Animals of the main groups were sacrificed on the day after the last exposure. In addition, two recovery groups, also consisting of five male and five female animals each, were simultaneously exposed with the main study animals to the control or 250 ppm test atmosphere, and were sacrificed after a 28-day recovery period following the last exposure. Endpoints to assess toxicity included clinical and ophthalmoscopic observations, growth, food consumption, hematology, clinical chemistry and organ weights. Estrus cyclicity was evaluated during the last three weeks of the exposure period, and sperm analysis was conducted at sacrifice. In addition, the animals were macroscopically examined at necropsy, the right lung lobes were lavaged and used for determination of biochemical markers and cell differentials, and the left lung lobes together with the full respiratory tract and a number of organs and tissues were examined microscopically.

The target concentrations of Dimethylethylamine were accurately achieved as demonstrated by the results of total carbon analysis of the test atmosphere. The overall average (± standard deviation) actual concentrations were 10.0 (± 0.2), 49.7 (± 0.8) or 249.0 (± 4.0) ppm for the low-, mid- and high-concentration groups, respectively.

Mortality did not occur during the study. Clinical and ophthalmoscopic observations revealed no exposure-related abnormalities.

Body weight data showed a statistically significantly reduced growth in male animals of the high concentration group throughout the exposure period; average body weight was about 10% below controls at the end of the exposure period. Catch-up growth was observed during the 28- day recovery period. Body weight gain of males of the mid-, and females of the high concentration group was also below control level (-28%) during the first ~2 weeks of the exposure phase (though statistical significance was not reached).

Food consumption was slightly lower than controls in males of the high-concentration group throughout the exposure period (maximally approximately 15%) followed by normal food intake during the recovery period and in females of the high-concentration group during the first two weeks of the study (maximally approximately 10%).

Estrus cycle evaluation did not reveal any exposure-related abnormalities.

No exposure-related changes were observed on epidydimal and testicular sperm analysis.

Analysis of hematology parameters showed a decrease in absolute number of eosinophils in blood of males of the high-concentration group sacrificed at the end of the exposure period. This finding was not accompanied by any changes in other white blood cell parameters, by any changes in females, or in males of the recovery groups. No exposure-related changes were observed in red blood cell and coagulation parameters.

Investigation of clinical chemistry parameters at the end of the exposure period revealed a statistically significant increase in plasma thyroxine (T4) concentration in females of the high concentration group when compared to concurrent controls (but which was within the historical control range, and in line with results obtained in control animals of the recovery group.

Therefore, is was not considered as treatment-related). No further changes in clinical chemistry parameters were observed in animals of the main and recovery groups.

Analysis of cellular parameters in bronchoalveolar lavage (BAL) fluid did not reveal any adverse changes in response to the exposure to the test material. An increase in the absolute number of macrophages in BAL fluid of males of the high-concentration group was considered to be of no toxicological significance, because changes in the percentage distribution of white blood cells in BAL fluid (which normally contains ~100% macrophages in healthy animals) or any histopathological changes in the lungs were absent. Investigation of biochemical parameters revealed a slight, but statistically significant increase in gamma-glutamyltransferase (GGT) activity in BAL fluid of females of the mid- and high-concentration main groups, which was fully reversible within the 28-day recovery period.

Organ weight data obtained at necropsy of animals of the main groups showed an increase in relative (to body weight) weight of the testes (+15%) and adrenals (+28%) in males of the high-concentration, and a decreased weight of the thymus in males (-28%) and females (-27%) of the high-concentration group. At the end of the recovery period, no exposure-related changes were observed in the weight of selected organs (seminal vesicles, testes, epididymides, and thymus).

Macroscopic examination at scheduled termination revealed no exposure-related gross pathology.

Microscopic examination revealed exposure-related histopathological changes in the nasal tissues of animals of the mid- and high-concentration group, characterized by degeneration of the respiratory and olfactory epithelium. The incidence and severity of these lesions were evidently concentration-dependent. In the mid-concentration group, animals displayed minimal to mild degeneration of the respiratory epithelium (in 4/10 animals at level 2 of the nose), and minimal to mild degeneration of the olfactory epithelium at levels 3 (10/10 animals), 4 (9/10 animals) and 5 (2/10 animals) of the nose2. In the high-concentration group, minimal to mild degeneration of the respiratory epithelium was observed in all animals at level 2, and mild to moderated degeneration of the olfactory epithelium was found in all animals throughout levels 3 to 6 of the nasal tissues. At the end of the 28-day recovery period, minimal to mild degeneration of the olfactory epithelium was observed at level 3 in 7/10 animals and at level 4 in only one animal of the high-concentration group (changes in the respiratory epithelium were no longer found), indicating substantial - but not full - recovery of the nasal pathology. No exposure-related microscopic changes were observed in the lower respiratory tract or in any of the other organs and tissues examined.

Under the conditions of the current study, sub-acute inhalation exposure to Dimethylethylamine at actual concentrations of:

• 249.0 ppm resulted in local toxicity in the upper respiratory tract as characterized by degeneration of the respiratory and olfactory epithelium in the nasal tissues and systemic toxicity, as evidenced by significant decrease in body weight and food consumption;

• 49.7 ppm resulted in no systemic toxicity but presence of adverse local changes in the upper respiratory tract, as characterized by degeneration of the respiratory and olfactory epithelium in the nasal tissues;

• Both in the 49.7 and 249.0 ppm groups, substantial, but not full recovery of the nasal pathology was observed within a 4-week post-exposure recovery period;

• 10 ppm resulted in no systemic or local adverse changes.

Based on these findings, the No-Observed-Adverse-Effect-Concentration (NOAEC) for systemic toxicity upon sub-acute inhalation exposure to Dimethylethylamine in rats was placed at 49.7 ppm, and the NOAEC for local toxicity was placed at 10.0 ppm.

 

3. 90-day toxicity study by inhalation (Beebe, 2021)

Four main groups of 10 male and 10 female Wistar Han rats each were exposed (nose-only) to target concentrations of 0 (control), 10, 30 or 100 ppm for 6 hours/day, 5 days/week over a 13-week period according to OECD 413 guideline and GLP. Animals of the main groups were sacrificed on the day after the last exposure. In addition, two recovery groups, also consisting of 10 male and 10 female animals each, were simultaneously exposed with the main study animals to the control or 100 ppm test atmosphere, and were sacrificed after a 6-week recovery period following the last exposure.Animals received the air control, or the test item, Dimethylethylamine by inhalation for 13 weeks. Recovery animals were similarly treated for 13 weeks followed by a 6 week off dose period. During the study, clinical condition, detailed physical and arena observations, sensory reactivity, grip strength, motor activity, estrous cycle, body weight, food consumption, ophthalmoscopy, hematology (peripheral blood), blood chemistry, thyroid hormone (T3 and T4), thyroid hormone (TSH), organ weight, sperm analysis, bronchoalveolar lavage, macropathology and histopathology investigations were undertaken.

The mean achieved atmosphere concentrations were 10.3, 29.9 and 106 ppm (103, 100 and 106% of target) for Groups 2, 3 and 4, respectively and was clinically well tolerated, recovery was assessed during a 6 week off-dose period.  There were no test item-related deaths or effects on clinical signs, food consumption, sensory reactivity and grip strength or motor activity.  There were also no effects on ophthalmoscopy, haematology, blood chemistry, thyroid hormone levels, sperm motility, estrous cycle, bronchoalveolar lavage, organ weights or macroscopic pathology.

Test item-related changes were evident in the nasal turbinates of animals exposed to 29.9 or 106 ppm and consisted of minimal to moderate degeneration/atrophy of the olfactory epithelium mainly affecting the dorsal parts of the nasal vestibules and was associated with loss of axon bundles in the sub adjacent lamina propria.  There was evidence of partial recovery after 6 weeks without exposure to DMEA; however, based on the incidence and severity these findings were considered adverse.

Reduced body weight gain evident at the end of the treatment period for both sexes exposed to 106 ppm resolved following 6 weeks of recovery. 

Based on the findings in this study a No Observed Adverse Effects Concentration (NOAEC) for the local toxicity is considered to be 10.3 ppm based on the minimal to moderate nasal degeneration/atrophy of the olfactory epithelium recorded at 29.9 or 106 ppm and the NOAEC for systemic toxicity is considered to be 106 ppm.

 

 

Key value for chemical safety assessment

Toxic effect type:

concentration-driven

Repeated dose toxicity: via oral route - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08 November 2018 - 01 July 2019
report finalized 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.29 (Sub-Chronic Inhalation Toxicity:90-Day Study)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: SAMP160321
- Expiration date of the lot/batch: 17 April 2019


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: In a refrigerator (2 to 8°C), desiccated, in the dark.

Species:

rat

Strain:

Wistar

Details on species / strain selection:

The rat was chosen as the test species because it is accepted as a predictor of toxic change in man and the requirement for a rodent species by regulatory agencies. The Crl:WI(Han) strain was used because of the historical control data available at this laboratory.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Ltd.
- Females (if applicable) nulliparous and non-pregnant: yes]
- Age at study initiation: 53 to 59 days
- Weight at study initiation: Males :222 to 318 g, Females: 143 to 203 g
- Fasting period before study: N/A
- Housing: Cages-Polycarbonate body with a stainless steel mesh lid, changed at appropriate intervals. The cages constituting each group were blocked together by sex on separate batteries. Five of the same sex per cage (main study and recovery), unless reduced by mortality or isolation. Wood based bedding which was changed at appropriate intervals each week.
- Diet (e.g. ad libitum): Teklad 2014C Diet. Non-restricted (removed overnight before blood sampling for hematology or blood chemistry and during the period exposure).
- Water (e.g. ad libitum): Potable water from the public supply via polycarbonate bottles with sipper tubes. Bottles were changed at appropriate intervals. Non-restricted (except during exposure).
- Acclimation period: 11 days before commencement of treatment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24ºC
- Humidity (%): 40-70%.
- Air changes (per hr): Filtered fresh air which was passed to atmosphere and not recirculated
- Photoperiod (hrs dark / hrs light): Artificial lighting, 12 hours light : 12 hours dark.

IN-LIFE DATES (Main study): From: 08 November 2018 To: 18 to 19 February 2019
IN-LIFE DATES (Recovery study): From: 08 November 2018 To: 01 April 2019

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

air

Details on inhalation exposure:

Route: Inhalation - snout only exposure.
Control (Group 1): Air only.
Duration of daily exposure: 6 hours. Weeks 1 to 12: 5 days each week; Week 13: 7 days.
Training for dosing: The animals on study were acclimated to the method of restraint, over a 3 day period immediately preceding the first test item exposure.

Exposure System:
Flow through nose-only chamber
Aluminum alloy construction comprising a base unit, three animal exposure sections, a top section and a pre-chamber

Animal Restraint:
Plastic nose-only restraint tube

Atmosphere Generation:
Glass sintered vaporizer
The test item was supplied to the generator, via a feed line, from a syringe driven at a constant rate by a syringe pump

Inlet Airflow:
From in-house compressed air system – breathing quality
Generator flow: 10-60 L/minute

Extract Airflow:
Drawn by in-house vacuum system
Filtered locally
Extract flow: 40-160 L/minute

Airflow Monitoring:
High quality tapered tube flowmeters - calibrated daily
In-line flowmeters monitored continuously

System Containment:
Systems housed in separate ventilated cabinets.

Administration:
Test group animals (Groups 2 – 4) were exposed to an atmosphere containing dimethylethylamine.
Group 1 animals were exposed to compressed air only
Animals were exposed on five days each week for 13 weeks. Additional animal exposures were conducted on Week/Day 13.6, 13.7 and 14.1 to cover end of study
investigations.
Duration of exposure was 6 hours each day
Exposures commenced on 19 November 2018
Different exposure levels were achieved by varying the concentration of test item in the exposure systems, whilst keeping the duration of exposure constant
The animals on study were acclimatized to the method of restraint for three consecutive days preceding their first exposure
System operating conditions were amended at the discretion of the Study Director to maintain achieved atmosphere concentrations close to target.

Concentration:
Atmosphere samples collected as follows:
Collection media: Dreschel head and solvent trap (bubbler)
Sample solvent: Methanol
Sample flow: 2.0 L/minute
Sample volume: Measured by wet-type gas meter
Sample frequency: 1 sample from Group 1/day (taken at approximately 180 minutes into exposure)
Minimum of 3 samples from Group 2, 3 and 4/day (taken at approximately 60, 180 and 300 minutes during exposure)
Sample location: Animal exposure port
Sample analysis: Chemical
During preliminary characterization trials an assessment was made of the percentage breakthrough of test item through the sample collection media; this was achieved by setting up two bubblers in series and collecting a sample of test atmosphere. The acceptable breakthrough limit to the second solvent trap is = 10%. The percentage break through the sample collection media was less than 10%, therefore one bubbler was used on study to collect chamber atmosphere samples.

Chamber air temperature was measured throughout exposure using an electronic thermometer probe placed in the breathing zone of the animals via an unused exposure port. Chamber air temperature was monitored continuously and recorded at 60-minute intervals.

Chamber relative humidity was measured throughout exposure using an electronic hygrometer probe placed in the breathing zone of the animals via an unused exposure port.
Chamber relative humidity was monitored continuously and recorded at 60-minute intervals.

The mean achieved atmosphere concentrations were 103, 100 and 106% of target for Groups 2, 3 and 4, respectively. Initially the inter and intra exposure variation was higher than anticipated. Bubbles were observed to be forming in the syringes and feed lines containing the test item. This was attributed to the test item expanding as it warmed following refrigerated storage and was
remedied by allowing the test item to warm to ambient temperature prior to generation. Subsequently the test item was observed to be vaporizing in the feed lines with an inversely
proportional relationship to target atmosphere concentration. This was considered to be a consequence of the very low feed rates required to achieve the target atmosphere concentrations and was remedied by reducing the diameter of the feed lines.

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

13 Weeks.

Frequency of treatment:

Dimethylethylamine was administered to Wistar Han rats by snout-only inhalation exposure for 6 hours per day (5 days per week) for 13 weeks.

Dose / conc.:

0 ppm

Dose / conc.:

10 ppm

Dose / conc.:

30 ppm

Dose / conc.:

100 ppm

No. of animals per sex per dose:

10 animals, per sex, per dose.

Control animals:

yes

Details on study design:

The target doses used in this study (0, 10, 30 and 100 ppm) were selected in conjunction with the Sponsor.
In a previous 28-day inhalation study in rats with Dimethylethylamine at 10, 50 or 250 ppm, degeneration of respiratory and olfactory epithelium in the nose was evident at 50 or 250 ppm with additional effects evident at 250 ppm relating to body weight and food consumption. For this study, a high exposure level of 100 ppm was selected and local effects were expected in the nose but it was considered these would be tolerated. Intermediate and low exposure levels of 30 or 10 ppm were selected to assess any relationship to exposure level.

The rat was chosen as the test species because it is accepted as a predictor of toxic change in man and the requirement for a rodent species by regulatory agencies. The Crl:WI(Han) strain was used because of the historical control data available at this laboratory.

Please see study design section under material and methods section below.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
Cages were inspected daily for evidence of animal ill-health amongst the occupants. Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment.

DETAILED CLINICAL OBSERVATIONS: Yes
Signs Associated with Dosing
Daily during the first four weeks of treatment on exposure days and weekly thereafter, detailed observations were recorded at the following times in relation to dose administration:
Pre-exposure observation
As each animal was returned to its home cage
As late as possible in the working day

In addition observations were made in the treatment period, on days without exposures during Weeks 1 to 4, at the following times during the day:
Early in the working day (equivalent to pre-exposure observation)
As late as possible in the working day

Observations during exposure is severely restricted due to tube restraint.

Detailed Physical Examination and Arena Observations
Before treatment commenced and during each week of treatment and recovery, detailed physical examination and arena observations were performed on each animal. On each occasion, the examinations were performed at approximately the same time of day (before dosing during the treatment period), by an observer unaware of the experimental group identities.
After removal from the home cage, animals were assessed for physical condition and behavior during handling and after being placed in a standard arena. Any deviation from normal was recorded with respect to the nature and, where appropriate, degree of severity. Particular attention was paid to possible signs of neurotoxicity, such as convulsions, tremor and abnormalities of gait or behavior.
Findings were either reported as "present" or assigned a severity grade - slight, moderate or marked.

BODY WEIGHT: Yes
The weight of each animal was recorded twice weekly from one week before treatment commenced, on the day that treatment commenced (Day 1) and during Weeks 1 to 4. Weekly body weights were recorded during Weeks 5 to 13, during recovery and on the day of necropsy.

FOOD EFFICIENCY: Not specified

FOOD CONSUMPTION
The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded for the week before treatment started and for each week throughout the study.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Not specified

OPHTHALMOSCOPIC EXAMINATION: Yes
The eyes of all animals were examined by means of a binocular indirect ophthalmoscope during pretreatment and Week 13.

HAEMATOLOGY: Yes
Blood samples were collected after overnight withdrawal of food duing week 13 from all animals.
Animals were held under light general anesthesia induced by isoflurane. Blood samples (nominally 0.5 mL) were withdrawn from the sublingual vein, collected into tubes containing EDTA anticoagulant and examined for the following characteristics using a Bayer Advia 120 analyzer:
Hematocrit (Hct)*
Hemoglobin concentration (Hb)
Erythrocyte count (RBC)
Absolute reticulocyte count (Retic)
Mean cell hemoglobin (MCH)*
Mean cell hemoglobin concentration (MCHC)*
Mean cell volume (MCV)
Red cell distribution width (RDW)
Total leucocyte count (WBC)
Differential leucocyte count:
Neutrophils (N)
Lymphocytes (L)
Eosinophils (E)
Basophils (B)
Monocytes (M)
Large unstained cells (LUC)
Platelet count (Plt)

*Derived values calculated in ClinAxys

Blood film (prepared for all samples) - Romanowsky stain, examined for abnormalities by light microscopy, in the case of flags from the Advia 120 analyzer. Confirmation or a written description from the blood film was made where appropriate. Additional blood samples (nominally 0.5 mL) were taken into tubes containing citrate anticoagulant and examined using a Stago STA Compact Max analyzer and appropriate reagent in respect of:
Prothrombin time (PT) - using IL PT Fibrinogen reagent. Activated partial thromboplastin time (APTT) - using IL APTT reagent.

BLOOD CHEMISTRY: Yes
Blood samples were collected after overnight withdrawal of food duing week 13 from all animals.
Animals were held under light general anesthesia induced by isoflurane. Blood samples (nominally 0.7 mL) were withdrawn from the sublingual vein and collected into tubes containing lithium heparin as anticoagulant. After separation, the plasma was examined using a Roche P Modular Analyzer in respect of:
Alkaline phosphatase (ALP)
Alanine aminotransferase (ALT)
Aspartate aminotransferase (AST)
Total bilirubin (Bili)
Urea*
Blood urea nitrogen (BUN)
Creatinine (Creat)
Glucose (Gluc)
Total cholesterol (Chol)
Triglycerides (Trig)
Sodium (Na)
Potassium (K)
Chloride (Cl)
Calcium (Ca)
Inorganic phosphorus (Phos)
Total protein (Total Prot)
Albumin (Alb)

*Numerically equivalent to blood urea nitrogen (BUN)

Albumin/globulin ratio (A/G Ratio) was calculated from total protein concentration and analyzed albumin concentration.

URINALYSIS: Not specified

NEUROBEHAVIOURAL EXAMINATION: Yes
Sensory reactivity and grip strength assessments were performed (on non-dosing days) on all main study animals in Groups 2 and 3 and all recovery phase animals during Week 12 of treatment. Animals were tested by an observer who was unaware of the treatment group to which each animal belonged. Before the start of observations, cage labels showing the treatment group were replaced by labels stating only the study, animal and cage numbers. Animals were not necessarily all tested on the same day, but the numbers of animals and the times of testing were balanced across the groups on each day of testing.
The following measurements, reflexes and responses were recorded:

Approach response
A blunt probe was brought towards the animal’s head until it was close to the animal’s nose (but not touching the whiskers).
Pinna reflex
The inside of one ear was touched lightly with a nylon filament and the reaction recorded.
Auditory startle reflex
The animal’s response to a sudden sharp noise was assessed.
Tail pinch response
The animal’s tail was pinched sharply with forceps approximately one third from the tip and the response graded.
Grip strength
Forelimb and hindlimb grip strength was measured using Mecmesin Basic Force Gauges. Three trials were performed.

Motor Activity
During Week 12 of treatment (on non-dosing days), the motor activity of all main study animals in Groups 2 and 3 and all recovery phase animals was measured using a Rodent Activity Monitoring System (Version 2.0.6), with hardware supplied by Pearson Technical Services and software developed and maintained by Envigo.
Animals were tested individually in clear polycarbonate cages and motor activity was measured by counting infra-red beam breaks over ten 6-minute intervals (one hour total). Ten beams were set at two height levels (five low and five high) to detect cage floor and rearing activity respectively. Animals were not necessarily all tested on the same day, but the numbers of animals and the times of testing were balanced across the groups on each day of testing.


IMMUNOLOGY: Not specified

OTHER:
Estrous Cycles – Vaginal Smears: Dry smears were taken For 14 days during Weeks 12 and 13 of treatment and during the recovery phase, using cotton swabs.

Mortality: A viability check was performed near the start and end of each working day. Animals were isolated or killed for reasons of animal welfare where necessary.
A complete necropsy was performed in all cases.

All observations regarding Thyroid hormone analysis please see materials and methods section below.

Sacrifice and pathology:

All main study and recovery animals were subject to a detailed necropsy. After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative. The retained tissues were checked before disposal of the carcass.

Other examinations:

Bone Marrow
Bone marrow smears were prepared immediately following death, on completion of the scheduled treatment or recovery periods and from animals killed prematurely during the study

Bronchoalveolar Lavage (BAL)
The right lung was used for bronchoalveolar lavage sampling and the left lung was processed for histology and light microscopy.

Sperm Analysis
Immediately after scheduled sacrifice of each male and collection of blood and bone marrow, the left vas deferens, epididymis and testis were removed and the epididymis and testis were weighed.
The following tests were performed:
Sperm motility – all groups
Sperm morphology – Groups 1 and 4
Sperm count – all groups
Homogenisation-resistant spermatid count – all groups

Stage-dependent Evaluation of Spermatogenesis
Stage dependent evaluation of spermatogenesis was conducted on sections of testes from all animals of Groups 1 (Control) and 4 (106 ppm) sacrificed on completion of the scheduled treatment period prepared and stained using the PAS method. A qualitative examination of spermatogenic stages was made for normal progression of the stages of the spermatogenic cycle, cell associations, and proportions expected to be present during normal spermatogenesis.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related clinical signs or dosing observations during the 13 weeks of treatment or during the 6 week recovery period.
Signs associated with the administration procedure included wet fur and/or red staining of the head, nose and eyes on return to home cage, in which the majority had resolved by end of working day. These signs were seen in animals from all groups including control, therefore are considered to be due to the method and duration of restraint and are commonly seen on inhalation studies of this study design. There were no test-item related effects observed during the physical examination and arena observations.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

There was one unscheduled death. A Group 2 male, number 20, died under anaesthetic during blood sample collection for hematology and blood chemistry during Week 13. The reason for death is unknown as the animal was considered normal prior to induction of anaesthesia and no macroscopic abnormalities were seen at necropsy.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

After thirteen weeks of treatment, group mean body weight gain was statistically lower than control for both sexes exposed to 106 ppm (0.77X and 0.79X control, males and females respectively).
There were no test-item related effects on body weight gain for either sex exposed to 10.3 or 29.9 ppm.
After 6 weeks of recovery, group mean body weight gain was higher than control for males previously exposed to 106 ppm (1.36X control).

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There were no test item-related effects on food consumption after 13 weeks of treatment or 6 weeks of recovery.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There was a higher incidence of superficial opacities in males that received 106 ppm after 13 weeks of treatment, evaluation of animals in groups that received 29.9 or 10.3 ppm did not reveal a similar effect. This finding is considered incidental in absence of a similar effect in females or animals in the lower exposure levels.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related effects on haematology.
All differences from control were minor, lacked exposure relationship or were inconsistent between the sexes. Therefore, these were considered to be due to individual variation and unrelated to treatment.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related effects on blood chemistry.
All differences from control were minor, lacked exposure relationship or were inconsistent between the sexes. Therefore, these were considered to be due to individual variation and unrelated to treatment.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related effects.
Group mean hindlimb grip strength was higher than control for all treated groups (not exposure related), however all were within the range of the historical data therefore this was considered incidental. A small number of differences attained statistical significance, however these were isolated and are attributed to normal variation.

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related effects.
After 13 weeks of treatment mean adjusted ovary weights were lower than control for females exposed to 106 ppm (0.86X), however there was no exposure-related effect evident and statistical significance was not achieved.
All other differences from control were minor, lacked exposure relationship or were inconsistent between the sexes. Therefore, these were considered to be due to individual variation and unrelated to treatment.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Animals Killed After 13 Weeks of Treatment: The macroscopic examination performed after 13 weeks of treatment revealed no intergroup differences of note. The incidence and distribution of all findings were considered to be unrelated to treatment.

Animals Killed After 6 Weeks of Recovery: The macroscopic examination performed after 13 weeks of treatment and 6 weeks of recovery revealed no intergroup differences of note.
The incidence and distribution of all findings were considered to be unrelated to treatment.

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Animals Killed After 13 Weeks of Treatment: Changes related to treatment with the test item were seen in the nose/turbinates. Degeneration/atrophy of the olfactory epithelium mainly affecting the dorsal parts of the nasal vestibules was observed in animals receiving 29.9 or 106 ppm and was associated with loss of axon bundles in the sub adjacent lamina propria. Incidence and severity of these changes showed an exposure level response.

Animals Killed After 6 Weeks of Recovery: Minimal degeneration/atrophy of the olfactory epithelium associated with loss of axon bundles in the sub adjacent lamina propria was observed in animals previously exposed to 106 ppm. These changes were mainly distributed in the dorsal part of the nasal cavities.

Histopathological findings: neoplastic:

no effects observed

Details on results:

Estrus cycle: When compared with control, estrus cycles in females exposed to 106 ppm during Weeks 12 and 13 showed a higher number of irregular cycles, 5 versus 1; there were also two individuals exposed to 106 ppm that either had extended estrus or were determined to be acyclic. During the recovery period, there was one control female with an irregular cycle and extended estrus for two females previously exposed to 106 ppm.

T3/T4 analysis: There were no test item-related effects. All samples taken from all groups, including control, at termination and from control and animals previously exposed to 106 ppm at the end of the recovery phase showed T3 and T4 concentrations were consistent among groups.

Thyroid Stimulating Hormone analysis: Individual serum TSH concentrations were found to be variable. Group mean TSH concentrations were lower for males exposed to 10.3 ppm when compared with control. Group mean TSH concentrations for males exposed to 29.9 or 106 ppm were similar to control. There was a slight increase in TSH concentrations for males previously exposed to 106 ppm when compared with control. Females showed an increase in TSH concentrations with increasing concentration of Dimethylethylamine when compared with control. There was a slight decrease in TSH concentrations for females previously exposed to 106 ppm when compared with control.
Given the high degree of variability, lack of exposure related response in males and inconsistency between the sexes, it is considered the observed changes are likely to be a result of biological variation rather than a test-item related effect.
For information regarding Thyroid hormones measurements please see the attached full study report

Sperm Analysis: No adverse effects on sperm motility, testicular spermatid numbers, cauda epididymal sperm numbers or sperm morphology were observed following treatment with Dimethylethylamine compared with control.

Bronchoalveolar Lavage (BAL): There were no test item-related effects.
Group mean cell counts were variable when compared with control, however individual values for test animals were within the control range and therefore all differences were attributed to normal biological variation.

Total Protein and Lactate Dehydrogenase: Although group mean data may suggest lower total protein and lactate dehydrogenase concentrations in treated males and higher total protein and lactate dehydrogenase concentrations in females, there was a large degree of variation and overlap in individual data when comparing test data with control, so there is no convincing test item-related effect.
After 6 weeks of recovery, higher group mean total protein (up to 1.82X control) and lactate dehydrogenase concentrations (up to 1.64X control) were observed in both sexes exposed to 106 ppm when compared with control.

Dose descriptor:

NOAEC

Remarks:

nasal local effects

Effect level:

10.3 ppm (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Remarks on result:

other:

Remarks:

30 mg/m3

Dose descriptor:

NOAEC

Remarks:

systemic toxicity

Effect level:

>= 106 ppm (analytical)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Remarks:

318 mg/m3

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

106 ppm (analytical)

System:

respiratory system: upper respiratory tract

Organ:

nasal cavity

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

The test item, Dimethylethylamine (DMEA), was administered by snout-only inhalation administration to Wistar Han rats, for 6 hours a day, 5 days a week, for 12 weeks and for 7 days in Week 13 at achieved exposure levels of 10.3, 29.9 or 106 mg/L and was clinically well tolerated, recovery was assessed during a 6 week off-dose period. There were no test item-related deaths or effects on clinical signs, food consumption, sensory reactivity and grip strength or motor activity. There were also no effects on ophthalmoscopy, haematology, blood chemistry, thyroid hormone levels, sperm motility, bronchoalveolar lavage, organ weights or macroscopic pathology.

 

After 13 weeks of exposure to DMEA, test item-related histopathological changes were evident in the nasal turbinates of animals exposed to 29.9 or 106 ppm and consisted of minimal to moderatedegeneration/atrophy of the olfactory epithelium mainly affecting the dorsal parts of the nasal vestibules and was associated with loss of axon bundles in the sub adjacent lamina propria and was considered adverse at 106 ppm. This finding was only present at minimal severity in a proportion animals exposed to 29.9 ppm, 2 out of 10 males and 4 out of 10 females, compared with all animals being affected up to moderate severity at 106 ppm. After 6 weeks of recovery this finding was observed in 8 out of 10 males and 4 out of 10 females previously exposed to 106 ppm and the severity was reduced compared with animals killed after 13 weeks of exposure to DMEA, only achieving slight severity in 3 males, indicating that partial recovery had occurred following 6 weeks without exposure to the test item. 

 

Statistically Reduced body weight gain evident in both sexes exposed to 106 ppm at the end of the treatment phase was not accompanied by reduced food consumption. Body weight gain returned to similar values, or exceeded those, seen in control during the recovery period; therefore, in the absence of any histopathological correlate the reduced bodyweight gain is considered to be non-adverse.

 

A higher incidence of irregular estrus cycles, extended estrus or acyclic animals were apparent for females exposed to 106 ppm when compared with control; extended estrus was still evident for females previously exposed to 106 ppm during the recovery period. Irregular and extended cycles were also observed in the control group, albeit at a lower incidence; however in the absence of any relationship to exposure and the absence of any findings correlating with these observations, they are considered to be non-adverse.

Conclusions:

The test item, Dimethylethylamine (DMEA), was administered by snout-only inhalation administration to Wistar Han rats, for 6 hours a day, 5 days a week, for 12 weeks and for 7 days in Week 13 at achieved exposure levels of 10.3, 29.9 or 106 ppm and was clinically well tolerated, recovery was assessed during a 6 week off-dose period. There were no test item-related deaths or effects on clinical signs, food consumption, sensory reactivity and grip strength or motor activity. There were also no effects on ophthalmoscopy, haematology, blood chemistry, thyroid hormone levels, sperm motility, estrous cycle, bronchoalveolar lavage, organ weights or macroscopic pathology.
Test item-related changes were evident in the nasal turbinates of animals exposed to 29.9 or 106 ppm and consisted of minimal to moderate degeneration/atrophy of the olfactory epithelium mainly affecting the dorsal parts of the nasal vestibules and was associated with loss of axon bundles in the sub adjacent lamina propria. There was evidence of partial recovery after 6 weeks without exposure to DMEA; however, based on the incidence and severity these findings were considered adverse.
Reduced body weight gain evident at the end of the treatment period for both sexes exposed to 106 ppm resolved following 6 weeks of recovery.
Based on the findings in this study a No Observed Adverse Effects Concentration (NOAEC) for the local toxicity is considered to be 10.3 ppm based on the minimal to moderate nasal degeneration/atrophy of the olfactory epithelium recorded at 29.9 or 106 ppm and the NOAEC for systemic toxicity is considered to be 106 ppm.

Executive summary:

Four main groups of 10 male and 10 female Wistar Han rats each were exposed (nose-only) to target concentrations of 0 (control), 10, 30 or 100 ppm for 6 hours/day, 5 days/week over a 13-week period. Animals of the main groups were sacrificed on the day after the last exposure. In addition, two recovery groups, also consisting of 10 male and 10 female animals each, were simultaneously exposed with the main study animals to the control or 100 ppm test atmosphere, and were sacrificed after a 6-week recovery period following the last exposure.Animals received the air control, or the test item, Dimethylethylamine by inhalation for 13 weeks. Recovery animals were similarly treated for 13 weeks followed by a 6 week off dose period. During the study, clinical condition, detailed physical and arena observations, sensory reactivity, grip strength, motor activity, estrous cycle, body weight, food consumption, ophthalmoscopy, hematology (peripheral blood), blood chemistry, thyroid hormone (T3 and T4), thyroid hormone (TSH), organ weight, sperm analysis, bronchoalveolar lavage, macropathology and histopathology investigations were undertaken.

The mean achieved atmosphere concentrations were 10.3, 29.9 and 106 ppm (103, 100 and 106% of target) for Groups 2, 3 and 4, respectively.

Statistically significant test item-related reduced body weight gain was evident for both sexes exposed to 106 ppm (0.77X and 0.79X control, males and females respectively).  After 6 weeks of recovery, group mean body weight gain was higher than control for males previously exposed to 106 ppm (1.36X control).

Histopathological changes were evident in the nasal turbinates in animals that received 29.9 or 106 ppm.  Minimal to moderate degeneration/atrophy of the olfactory epithelium mainly affecting the dorsal parts of the nasal vestibules was observed in animals receiving 29.9 or 106 ppm and was associated with loss of axon bundles in the sub adjacent lamina propria.  Incidence and severity of these changes showed an exposure level response.  There was evidence of partial recovery following 6 weeks without exposure to DMEA.  No histopathological changes were evident in animals that received 10.3 ppm.

Reference 2

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

First exposure: 03/04/18 Sacrifice of recovery animals: 29/05/19

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

Name1 : Dimethylethylamine
Chemical name 1 : Ethyldimethylamine; N,N-Dimethylethylamine
CAS registry number1 : 598-56-1
Molecular formula1 : C4H11N
Structural formula1 :
Molecular weight1 : 73.13 g/mol
Batch/lot number1 : SAMP160321
Purity1 : 99.41%
Solubility in water1 : completely soluble; exothermic reaction!
Melting point1 : -140 °C
Boiling point1 : 36.3 °C
Vapor pressure 1 : 527 hPa at 20° C
Density 1 : 660 kg/m3 at 20 °C
Viscosity1 : 0.67 mPa.s (OECD Test Guideline 114)
Flash Point1 : -25°C closed cup (ISO 3679)
Quantity received : 6.75 kg
Supplier : Arkema
Approved for use until1 : 20 November 2018
Storage conditions : ambient temperature (15-25°C)
Date of receipt : 23 November 2017
1Characteristics provided by the sponsor
The Triskelion dispense reference number is 17025D.

Species:

rat

Strain:

Wistar

Details on species / strain selection:

For this study rats were chosen as test system, because this animal species is normally used in toxicity studies of this type and is accepted by the relevant regulatory authorities. Young adult, male and female Wistar outbred (Crl:WI(Han)) rats were obtained from a colony maintained under specific pathogen free (SPF) conditions by Charles River Laboratories. The Wistar rat strain was used because it is routinely used at the test facility for this type of study.

Sex:

male/female

Details on test animals or test system and environmental conditions:

The age of the rats was about 7-8 weeks on the day of randomization (8 January and 29 March 2018 for animals of the range finding and main study, respectively). Body weight at allocation was within ±20% of the mean weight for each sex. Mean body weights at the start of exposure (day 0) in the range finding study were 251 and 166 grams for male and female animals, respectively. Mean body weights at the start of exposure in the main study were 235 and 170 grams for male and female animals, respectively.

Upon arrival on 21 March 2018, the rats (33 males and 33 females) were taken in their unopened shipping containers to a quarantine room (animal room 5.1.14) and were checked for overt signs of ill health and anomalies. During the quarantine period, serological investigation of the microbiological status was conducted in blood samples taken from five randomly selected animals. On 23 March 2018, the results of the serological examinations were received and indicated an acceptable microbiological status. The animals were subsequently released for experimental use, and were transferred to their definitive room (animal room 6.0.06). The duration of the acclimatization period to the laboratory conditions prior exposure (period between arrival and the start of the exposure period) was 13 days.

Shortly before initiation of exposure (on 29 March 2018), the animals were allocated to the various groups by computer randomization proportionally to body weight (males and females separately). The surplus animals (3 males, 3 females) were kept in reserve to serve as sentinels. These animals were not used in the present study.

Animal husbandry
Animal room
From their arrival, the animals were housed under conventional conditions in one animal room separated by sex. No other test system was housed in the same room during the study. The room was ventilated with about 10 air changes per hour and was maintained at a temperature of 20-24oC and a relative humidity of 45-65%. The upper limit of relative humidity was, however, higher than 65% for short periods of time because of wet cleaning activities. Relative humidity was below 45% on 31 March and 3 April 2018 (briefly, with a minimum of 44%) and on multiple occasions for prolonged durations in the period 5 April – 24 May 2018 (minimum of 39%). Lighting was artificial with a sequence of 12 hours light and 12 hours dark.

Caging
During exposure, the animals were housed individually in the inhalation unit. Immediately after each exposure, the animals were returned to their home cages. When not exposed, the animals were housed five animals to a cage, separated by sex. All animals were housed in macrolon cages with wood shavings (Lignocel, Rettenmaier, Rosenberg, Germany) as bedding material and strips of paper (Enviro-dri, Shepherd Specialty Papers, Michigan, USA) and a wooden block (ABEDD, Vienna, Austria) as environmental enrichment. The cages and bedding were changed at least weekly.

Food and drinking water
Food was provided ad libitum from the arrival of the animals until the end of the study, except during exposure and – for animals of the main study – the overnight fasting period before sacrifice. The animals received a cereal-based (closed formula) rodent diet (VRF1 (FG)) from a commercial supplier (SDS Special Diets Services, Whitham, England). Each batch of VRF1 (FG)) diet is analyzed by the supplier for nutrients and contaminants. The food was provided as a powder in stainless steel cans, covered by a perforated stainless steel plate to prevent spillage. The food in the feeders was replaced with fresh portions once weekly and filled up as needed.

Drinking water was provided ad libitum from the arrival of the animals until the end of the study, except during exposure. Each cage was supplied with domestic mains tap-water suitable for human consumption (quality guidelines according to Dutch legislation based on EC Council Directive 98/83/EC). The water was given in polypropylene bottles, which were cleaned weekly and filled as needed. Results of the routine physical, chemical and microbial examination of the drinking water as conducted by the supplier are made available to the test facility. In addition, the supplier periodically (twice per year) analyses water samples taken on the premises of the
test facility for a limited number of variables.

Route of administration:

inhalation: vapour

Type of inhalation exposure:

nose only

Vehicle:

air

Details on inhalation exposure:

Exposure equipment
The animals were exposed to the test atmosphere in nose-only exposure units, in an illuminated laboratory room different from the room where the animals were housed. Animals of groups 2, 3, 4 and – for the range finding – 5 were exposed in inhalation chambers consisting of a cylindrical aluminium column, surrounded by a transparent cylinder (a modification of the chamber made by ADG Developments Ltd., Codicote, Hitchin, Her ts, SG4 8UB, United
Kingdom). The column had a volume of 46.7 L and consisted of a top assembly with the entrance of the unit, a mixing section, two rodent tube sections and at the bottom the base assembly with the exhaust port. Each rodent tube section had 20 ports for animal exposure. Control animals (group 1) were exposed to clean air in a polypropylene nose-only inhalation chamber with a volume of 48.2 L (manufactured by P. Groenendijk Kunststoffen BV) which was very similar in construction to the aluminium chambers described above.

Empty ports were used for test atmosphere sampling (for analysis of the actual concentration) and measurement of oxygen, carbon dioxide, temperature and relative humidity. The animals were secured in plastic animal holders (Battelle), positioned radially through the outer cylinder around the central column. Animals were rotated with respect to their position in the column every week (main study) or two days (range finding study). The remaining ports were closed. Only the nose of the rats protruded into the interior of the column. Habituation to the restraint in the animal holders was not performed because in our experience habituation does not help to reduce possible stress (Staal et al., 2012).

In our experience, the animal’s body does not exactly fit in the animal holder which always results in some leakage from the high to the low pressure side. By securing a positive pressure in the central column and a slightly negative pressure in the outer cylinder, which encloses the entire animal holder, dilution of test atmosphere by air leaking from the animals’ thorax to the nose was avoided. The unit was illuminated externally by normal laboratory fluorescent tube lighting. The total air flow through the unit was at least 1 liter/min for each rat. The air temperature and relative humidity in the unit were maintained at 22 ± 3°C and between 30 and 70%, as far as possible.

Generation of the test atmosphere
The inhalation equipment was designed to expose the animals to a continuous supply of fresh test atmosphere. A schematic diagram of the generation and the exposure system is presented in Figure 1. To generate the test atmospheres, a continuous flow of liquid test material3, controlled by a peristaltic pump (Minipulse 3, Gilson, Velliers le Bel, France) was allowed to evaporate in a mass flow controlled (Bronkhorst Hi Tec, Ruurlo, the Netherlands) stream of compressed dry air, by directing it through a glass evaporator which was kept at a constant temperature of 48°C by circulating heated water. The resulting single stream of concentrated vapor was led through a condense trap (not used during the range finding study) and was subsequently divided for the different groups using mass flow controllers (Bronkhorst Hi Tec) and mixed with a controlled stream of humidified compressed air via an eductor (Fox Valve Development Corp., Dover, NJ, USA). The eductors were calibrated by measuring the total air flow at a range of driving air pressures of the eductors encompassing the driving pressures used during the study; the driving air pressure was used to monitor the total flow. The resulting test atmosphere was led to the inlet at the top of the exposure chamber and directed downward towards the noses of the animals; the atmosphere was exhausted at the bottom. As of 9 April 2018, a PI feedback system was installed to automatically control the feedrate of the peristaltic pump, based on the measured actual concentration (see paragraph 4.10.1). The feedback system took into account the proportional (P) and the integrated deviations (I) of the concentrations from the setpoint.

The exposure chamber for the control animals (group 1) was supplied with a stream of humidified compressed air only, which was controlled by a reducing valve and measured by mass view meter (Bronkhorst Hi Tec).
The animals were placed in the exposure unit after stabilization of the test atmospheres. Test atmosphere generation and animal exposure were performed in an illuminated laboratory at room temperature.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analysis of exposure conditions
Actual concentration
The actual concentration of the test material in the test atmospheres was measured by total carbon analysis (groups 2 and 3 of the main study and group 3 of the range finding study: Ratfisch RS55T, Munich, Germany; remaining groups: Sick Maihak GMS 810 EuroFID Total Hydrocarbon Analyzer; Sick Instruments Benelux, Hedel, the Netherlands). Test atmosphere samples were taken continuously from the exposure chamber at the animals’ breathing zone and were passed to the total carbon analyzer (TCA) through a sample line. The response of the analyzers was recorded on a PC every minute using a Data acquisition system (range finding study: CAN transmitters, G. Lufft Mess- und Regeltechnik GmbH, 70719 Felbach, Germany; main study: MyriaNed, van Mierlo Ingenieursbureau BV, Eindhoven, the Netherlands). The responses of the analyzers were converted to concentrations by means of calibration graphs (the formulas used to convert responses into concentrations are given below). For each exposure day, the mean concentration was calculated from the values determined every minute.

Prior to the first exposure, the output of the flame ionization detector of the TCA was calibrated using gas sample bags. To this end, sample bags were filled with an accurate (mass flow controlled) volume of air and an accurate (weighed) amount of test material, injected into the bag through a septum. Three concentrations were thus prepared (at least in duplicate) – at approximately 80%, 100% and 120% of the target concentration of each group – and analyzed by the TCA. Linear relations were found between the response of the analyzers and the concentration of the test material.

The calibrations were checked weekly during the study. To this end, gas sample bags were prepared at each target concentration as described above, and were subsequently analyzed by the TCA. If the measured concentration deviated more than 5% from the calculated concentration, the calibration check was repeated. If the deviation was more than 5% at the re-check, a complete re-calibration was to be carried out (which was not necessary during the range finding nor the main study).

Duration of treatment / exposure:

6 hours per day

Frequency of treatment:

5 days per week, over a 28-day study period (20 exposure days in total)

Dose / conc.:

0 ppm (analytical)

Dose / conc.:

10 ppm (analytical)

Remarks:

30 mg/m3

Dose / conc.:

49.7 ppm (analytical)

Remarks:

149.1 mg/m3

Dose / conc.:

249 ppm (analytical)

Remarks:

747 mg/m3

No. of animals per sex per dose:

The 28-day study comprised four test groups of five male and five female rats each, i.e. a control group exposed to clean air and three groups exposed to different concentrations of the test material.

Five additional males and 5 additional females were included in the control and high-concentration group, which were exposed similarly and kept for a recovery period of 28 days after the last exposure.

Control animals:

yes, concurrent no treatment

Details on study design:

7-day concentration range finding study:
The range finding study consisted of five test groups of five male and five female rats each, i.e. a control group exposed to clean air, and four groups exposed to different concentrations of the test material. The highest concentration was intended to result in clear toxicity but not death or moribundity. The lowest concentration was intended to produce little or no evidence of toxicity. The animals of the control group were handled identically as those of the other groups, except for exposure to the test material. The animals were exposed for 6 hours per day, 5 days per week, over a 7-day study period (5 exposure days in total) and sacrificed on the day after the last exposure. The date of the first exposure (10 January 2018) was named day 0.

Concentrations (ppm): 0, 10, 100, 500 and 1000

Selection of target concentrations for the concentration range finding study were based on the 4h LC50 that was found to be ca. 3400 ppm; no mortality was observed at a lower concentration of 2300 ppm. Although no repeated-dose inhalation toxicity data were available for Dimethylethylamine, there were data for the toxicity of the comparable substance Trimethylamine (CAS no. 75-50-3). The 4h LC50 was comparable at ca. 2400-3500 ppm. In a subacute study, mild toxicity was observed at the low concentration of 75 ppm, while mild toxicity was observed at a concentration as low as 10 ppm in a chronic study (all information available at http://www.echa.eu); thus, to enable identification of a NOAEC and characterize the concentration-response relationship, a broad concentration range was chosen for this range finding study.


Animal allocation
7-day range finding study
27 male and 27 female rats were ordered, and upon arrival on 20 December 2017 they were taken in their unopened shipping containers to a quarantine room (animal room 5.1.20) and were checked for overt signs of ill health and anomalies. During the quarantine period, serological investigation of the microbiological status was conducted in blood samples taken from four randomly selected animals. On 22 December 2017, the results of the serological examinations were received and indicated an acceptable microbiological status. The animals were subsequently released for experimental use, and they were transferred to their definitive room (animal room 6.0.06) on the same day. The duration of the acclimatization period to the laboratory conditions prior exposure (period between arrival and the start of the exposure period) was 21 days.

On 8 January 2018, shortly before initiation of exposure of the rats, they were allocated to the various groups by computer randomization proportionally to body weight. . The surplus animals (2 males, 2 females) were kept in reserve to serve as sentinels. These animals were not used in the present study.

28-day main study
Upon arrival on 21 March 2018, the rats (33 males and 33 females) were taken in their unopened shipping containers to a quarantine room (animal room 5.1.14) and were checked for overt signs of ill health and anomalies. During the quarantine period, serological investigation of the microbiological status was conducted in blood samples taken from five randomly selected animals. On 23 March 2018, the results of the serological examinations were received and indicated an acceptable microbiological status. The animals were subsequently released for experimental use, and were transferred to their definitive room (animal room 6.0.06). The duration of the acclimatization period to the laboratory conditions prior exposure (period between arrival and the start of the exposure period) was 13 days.

Shortly before initiation of exposure (on 29 March 2018), the animals were allocated to the various groups by computer randomization proportionally to body weight (males and females separately). The surplus animals (3 males, 3 females) were kept in reserve to serve as sentinels. These animals were not used in the present study.

4.6 Identification
The study was identified as Triskelion study number 21124, with a subcode /01 for the range finding study and /02 for the main study.

Before and during allocation, the individual rats were identified by a transient mark on their tail.

After allocation, they were identified by an identification number (even for males and odd for females), using subcutaneous transponders. During the study each group of rats was coded by a number and a color. Each cage was provided with a card showing the color code, the animal identification numbers, the group number and the study number.

Positive control:

None

Observations and examinations performed and frequency:

Clinical observations
Animals were observed daily in the morning hours by cage-side observations and, if necessary, handled to detect signs of toxicity. The observations included – but were not limited to – the signs listed in Annex 6. The animals were also observed about halfway through the 6-hour exposure period, in particular to monitor any breathing abnormalities and restlessness; observation of other abnormalities was hindered due to the animals’ stay in restraining tubes. All animals were thoroughly checked again in the afternoon. All abnormalities, signs of ill health, and reactions to treatment were recorded.

Ophthalmoscopic examination (28-day sub-acute study)
Ophthalmoscopic observations were made prior to the start of exposure in all animals (on day -7) and towards the end of the exposure period in the animals of the control and high-concentration groups (on day 23). Eye examinations were carried out using an ophthalmoscope after induction of mydriasis by a solution of atropine sulphate. Since no evident exposure-related ocular changes were observed, eye examinations were not extended to the animals of the intermediate concentration groups at the end of the exposure period, or to animals of the recovery groups.

Body weights
In the range finding study, the body weight of each animal was recorded 2 days before the start of the exposure. These pre-test weights served as a basis for animal allocation. Subsequently, the animals were weighed prior to the first exposure on day 0, on day 4 of exposure and on their scheduled sacrifice data in order to calculate the correct organ to body weight ratios.

In the main study, the body weight of each animal was recorded 5 days before the start of exposure. These pre-test weights served as a basis for animal allocation. Subsequently, the animals of the main study were weighed prior to exposure on the first day (day 0), and twice a week thereafter. The animals were also weighed on the day before overnight fasting prior to necropsy, and on their scheduled sacrifice date in order to calculate the correct
organ to body weight ratios.

Food consumption
Food consumption of the animals was measured per cage by weighing the feeders. The results are expressed in g per animal per day. Food consumption was measured twice weekly for animals of the range finding study. For animals of the main study, food consumption was measured once weekly.

Estrus cycle evaluation (28-day sub-acute study)
Vaginal smears to evaluate the estrus cycle length and normality were made daily in the three weeks prior to sacrifice, including the day of sacrifice, in all female animals of the main study. Since no exposure-related abnormalities were observed in animals of the main study, estrus cycle evaluation was not extended to animals of the recovery groups.

Sacrifice and pathology:

Sperm analysis (28-day sub-acute study)
Epididymal sperm motility, count and morphology At scheduled necropsy, epididymal sperm was derived from the left cauda epididymis of all male animals from both the main and recovery groups (30 animals in total). For this purpose the cauda epididymis was dissected, weighed and thereafter minced in M199 medium containing 0.5% bovine serum albumin. Sperm motility and, after sonification and DNA staining, the cauda epididymal sperm reserves (sperm count) were measured for all males, using the Hamilton Thorne Integrated Visual Optical System (IVOS). In addition, a smear of the sperm solution was prepared and stained, and two hundred spermatozoa of this smear were examined for morphology.

Testicular sperm count
At scheduled necropsy, the left testis of all males were placed on dry ice and subsequently stored in a freezer (<-70°C) for later determination of the number of homogenization-resistant spermatids. The testes to be analysed were thawn just before further processing. Following removal of the tunica albuginea, the testicular parenchyma were weighed, minced and homogenized in Saline Triton X-100 solution. Following DNA-staining, the homogenizationresistant sperm heads were enumerated using the IVOS. The daily sperm production was calculated. The evaluation of homogenization-resistant spermatids was performed in the males of the control and high-concentration groups from both the main and recovery study. Since no exposure-related changes were observed in animals of the high-concentration group, the evaluation of homogenization-resistant spermatids was not extended to animals of the intermediate-concentration groups.

Hematology (28-day sub-acute study)
Hematology was conducted at the end of the treatment period on all surviving animals of the main groups. Blood samples were taken from the abdominal aorta of overnight fasted rats (water was freely available) whilst under pentobarbital anesthesia at sacrifice. Citrate (for prothrombin time) or EDTA (for other parameters) were used as anticoagulant. Blood samples were discarded after analysis. In each sample the following determinations were carried out.
- hemoglobin
- packed cell volume
- red blood cell count
- reticulocytes
- total white blood cell count
- differential white blood cell count
(lymphocytes, neutrophils, eosinophils, basophils and monocytes)
- prothrombin time
- thrombocyte count (platelet count)

The following parameters were calculated:
- mean corpuscular volume (MCV)
- mean corpuscular hemoglobin (MCH)
- mean corpuscular hemoglobin concentration (MCHC)

Since (possible) exposure-related changes were observed in animals of the main groups, investigation of all hematology parameters (except prothrombin time) was extended to animals of the recovery groups.

4.11.8 Clinical chemistry (28-day sub-acute study)
Clinical chemistry was conducted at the end of the exposure period on all surviving rats after overnight fasting, at the same time blood samples for hematology were collected. Blood samples were taken from the abdominal aorta of the rats whilst under pentobarbital anaesthesia. The blood was collected in heparinized plastic tubes and plasma was prepared by centrifugation. Plasma samples were stored frozen (<-18°C) until analysis and
discarded afterwards.
- alkaline phosphatase activity (ALP) - bilirubin (total)
- aspartate aminotransferase activity (ASAT) - cholesterol
- alanine aminotransferase activity (ALAT) - triglycerides
- gamma glutamyl transferase activity (GGT) - calcium (Ca)
- total protein - sodium (Na)
- albumin - potassium (K)
- ratio albumin to globulin (calculated) - chloride (Cl)
- urea - inorganic phosphate (PO4)
- creatinine - thyroxine (T4)
- glucose (fasting)

Since (possible) exposure-related changes were observed in animals of the main groups, investigation of clinical chemistry parameters was extended to animals of the recovery groups.

Pathology
Sacrifice, organ weights and macroscopic examination
At the end of the exposure period, the animals were sacrificed in such a sequence that the average time of sacrifice was approximately the same for each group. Similarly, animals of the recovery groups were sacrificed at the end of the 28-day post-exposure recovery period included in the sub-acute study. The animals were sacrificed by exsanguination from the abdominal aorta under pentobarbital anesthesia (intraperitoneal injection of sodium pentobarbital) and then examined grossly for pathological changes.

7-day concentration range finding study
The following organs of all animals were weighed (paired organs together) as soon as possible after dissection to avoid drying. Relative organ weights (g/kg body weight) were calculated from the absolute organ weights and the terminal body weight:
- heart
- adrenals
- kidneys
- liver
- spleen
- testes
- ovaries
- lungs with trachea

28-day sub-acute study
The following organs of all animals were weighed (paired organs together) as soon as possible after dissection to avoid drying. Relative organ weights (g/kg body weight) were calculated from the absolute organ weights and the terminal body weight:
- adrenals
- brain
- heart
- kidneys
- liver
- lungs (left lung only, because of lavage of the other lobes)
- spleen
- testes
- ovaries
- thymus

For assessment of fertility in male rats of the main groups (20 animals in total), the following additional weights were determined:
- epididymides
- prostate
- seminal vesicles

Since (possible) exposure-related changes were observed in animals of the main groups, the seminal vesicles (+ coagulation glands), testes, epididymides (all three organs for males only) and thymus (both sexes) were also weighed in animals from the recovery groups of the main study; the other organs included for main study animals were not weighed.

Bronchoalveolar lavage and measurements (28-day sub-acute study)
The lungs of all animals of the main groups were lavaged at necropsy according to a standardized method. In short: the right half of the lungs (after binding off the left lung lobe, used for histopathology) from these animals was rinsed three times with a single volume of 26.7 ml saline per kg body weight (one value for each group based on mean body weight). The final amount of lung lining fluid and cells collected was weighed and retained on ice. The
bronchoalveolar lavage cells were recovered by centrifugation (250xG) for 5 minutes. The temperature control of the centrifuge was set at 4ºC. Each cell pellet thus obtained per animal was resuspended in 0.5 ml saline and used for total white blood cell numbers, viability and cell differentials. The supernatant was stored frozen (<-18°C) until analysis of biochemical parameters. BAL fluid samples were discarded after analysis.

Cellular determinations
Total white blood cell numbers were counted using an AdVia 2120i analyzer (Siemens N.V., the Netherlands; Reference: “Training manual 04/11/99, chapter 5 Impedance”.) The number of viable cells was determined using an acridine orange / ethidium bromide staining method in combination with fluorescent microscopic evaluation. The cytospins for cell differentials were made using a Cyto-Tek (Sakura, Netherlands) and stained by May-Grunwald Giemsa. The differential cells (monocytes, macrophages, neutrophils, eosinophils, lymphocytes) were evaluated by light microscopy (absolute numbers were calculated from total white blood cell number and percentage distribution of the different cell types).

Since exposure-related changes were observed in animals of the main groups, investigation of bronchoalveolar lavage parameters was extended to animals of the recovery groups of the sub-acute study.


Histopathology
7-day concentration range finding study
For histopathological examination, samples of the following tissues and organs from all animals, and all gross lesions were preserved in a neutral aqueous phosphate-buffered 4% solution of formaldehyde (10% solution of Formalin). The lungs (after weighing) were infused with the fixative under ca. 15 cm water pressure to ensure fixation). Tissues were processed and examined using the same methods as described below for the relevant tissues examined during the 28-day study. The carcass containing any remaining tissues was retained in the fixative until completion of the histopathological examination and then discarded.
- kidneys
- nasal turbinates
- larynx
- trachea
- lungs
- tracheobronchial lymph nodes

All preserved tissues of all animals of the control and top-concentration groups were examined histopathologically (by light microscopy). Since exposure-related changes were observed in the nasal tissues (all 6 levels), larynx, trachea and lungs of the top-concentration group, histopathological examination of these tissues was extended to animals of the intermediate concentration groups of the range finding study.

28-day sub-acute study
For histopathological examination, samples of the following tissues and organs from all animals, and all gross lesions were preserved in a neutral aqueous phosphate-buffered 4% solution of formaldehyde (10% solution of Formalin). The lungs (after weighing) were infused with the fixative under ca. 15 cm water pressure to insure fixation).
- adrenals
- bone marrow
- brain (including sections of cerebrum, cerebellum, and medulla/pons)
- eyes (with specific focus on adverse corneal changes)
- heart
- kidneys
- liver
- esophagus
- ovaries
- seminal vesicles
- spinal cord (cervical, mid-thoracic, and lumbar)
- spleen
- stomach
- testes (including testicular staging)
- thymus
- thyroid
- tracheobronchial lymph nodes
- uterus
- respiratory tract including larynx, trachea, left lung lobe (the right lobes were lavaged) and
nasopharyngeal tissues
- all gross lesions

The carcass containing any remaining tissues was also retained in Formalin, but discarded after completion of the histopathological examination.

Slide preparation
Tissues to be examined were embedded in paraffin wax, sectioned and stained with hematoxylin and eosin. Unless required for histopathological examination, the tissues of the animals of the intermediate concentration groups (groups 2 and 3) were not processed, except for the noses of animals of groups 2 and 3 which were decalcified and embedded in paraffin concurrently with the noses of the animals of groups 1 (control) and 4 (high concentration).

Histopathological examination
All preserved tissues of all animals of the control and high-concentration main groups were examined histopathologically (by light microscopy). In addition, all gross lesions observed in rats of the intermediate-concentration main groups were examined microscopically. Histopathology was subjected to a peer review system.

The nasopharyngeal tissues were examined at six levels (Woutersen et al., 1994; an illustration of these levels is shown in Annex 9) with one level to include the nasopharyngeal duct and the Nasal Associated Lymphoid Tissue (NALT), the larynx at three levels (one level to include the base of the epiglottis). The trachea was examined at three levels (including the bifurcation, and one longitudinal section through the carina), and the left lung lobe at three levels. When examining the eye, particular focus was directed towards assessment of corneal changes, which have been observed in exposed workers who have experienced “blue haze”, associated with
corneal swelling.

Since exposure-related changes were observed in the nasal tissues of animals of the highconcentration group, histopathological examination of these tissues was extended to animals of the intermediate concentration main groups (nose levels 2-6) and to animals of the recovery groups (nose levels 1-6) of the 28-day sub-acute study.

Statistics:

Ancova/Anova & Dunnett
Pretreatment body weight, body weight after initiation of treatment, clinical pathology (hematology, clinical chemistry), bronchoalveolar lavage parameters, organ weights, sperm motility
(numerical), sperm count, testicular sperm count.
Food consumption - no statistics were applied (only one cage per sex per group).
Kruskal-Wallis & Wilcoxon: Estrus cyclicity: mean length of the longest cycle, number of complete cycles in the test period; sperm motility (expressed as %), sperm morphology
Chie-squared & Fisher's Exact: Incidences of histopathological changes; Estrus cyclicity: number of acyclic females, number of animals with prolonged estrus period

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No exposure-related clinical abnormalities were observed. The few signs noted were considered unrelated to the exposure to the test material. Abnormalities of the skin or fur (sparsely haired areas, encrustations) were observed in a few animals across the groups. These are common findings, possibly caused by slight movement of the animals in the restraining tubes during exposure, resulting in slight irritation of the skin.

Mortality:

no mortality observed

Description (incidence):

All animals survived until scheduled sacrifice.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Male animals of the high-concentration group showed a statistically significantly lower body weight than controls from day 3 until the end of the exposure period (up to ~10% lower average body weight by day 24), which was the result from body weight loss or decreased growth on days of exposure; normal growth was observed during periods including the exposure-free weekends. The difference in body weight with controls gradually decreased during the recovery period; males of the high-concentration group displayed catch-up growth after cessation of exposure. A trend towards decreased body weight gain was also observed in females of the
high-concentration group and in males of the mid-concentration group during the first ~2 weeks of exposure, but statistical significance was not reached.

PLEASE REFER TO ATTACHED TABLES - 'Body weight' and Body weight change'

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Although statistical analysis could not be performed (as only one cage per sex per group was used), food consumption of male animals of the high-concentration group was slightly lower (maximally approximately 15%) than controls throughout the exposure period; normal food intake was observed during the recovery period. Food consumption in females of the high-concentration group also seemed slightly decreased (maximally approximately 10%) during the first half of the exposure period, when compared to controls. No treatment-related changes in food intake were observed in animals of the low- and midconcentration group.

PLEASE REFER TO ATTACHED TABLE - 'Food consumption'

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Ophthalmoscopic examination near the end of the exposure period revealed unilateral focal corneal opacity in two (of five) male animals of the high-concentration main group. This – usually transient, reversible – lesion is occasionally observed as a background finding, possibly related to the nose-only mode of exposure in restraining tubes (sometimes causing minor superficial damage to the cornea, because the animal’s head may be
somewhat compressed against the front of the tube). For this reason, and because the lesion was observed only focally (rather than diffusely throughout the cornea), in just two animals of the high-concentration group, and was not associated with any microscopic changes in the eye, a relation with the exposure to the test material was considered to be highly unlikely. No further ophthalmoscopic abnormalities were observed at the end of the exposure period.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Analysis of hematology parameters revealed the following statistically significant differences between animals of the main groups exposed to the test material and unexposed controls:
- Decreased absolute number of eosinophils in males of the high-concentration group. This finding was not accompanied by any changes in other white blood cell parameters, or by a shift in the percentage distribution of differential white blood cell numbers.
- Increased packed cell volume (PCV) in males of the low-concentration group, which was considered to be a chance finding (unrelated to the exposure), because a concentrationresponse relationship was absent.
There were no statistically significant changes in coagulation parameters of animals of the main groups.

Investigation of hematology parameters in animals of the recovery groups, performed in response to the statistically significant differences observed in animals of the main groups, did not reveal any exposure-related changes. In the absence of any corresponding changes at the end of the exposure period or any corroborative findings in animals of the recovery groups, a slightly increased mean corpuscular volume (MCV) in males of the high-concentration (without any changes in parameters from which MCV is calculated, i.e. PCV and total red blood cell numbers) and slightly lower lymphocyte and total white blood cell numbers in females of the
high-concentration recovery group were considered to be chance findings, unrelated to the exposure to the test material.

PLEASE REFER TO ATTACHED TABLES - 'Red blood cell and coagulation parameters (main and recovery)', 'Total and differential white blood cell counts (main and recovery)

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Analysis of clinical chemistry parameters revealed the following statistically significant differences between animals of the main groups exposed to the test material and unexposed controls:
- Decreased plasma activity of alkaline phosphatase (ALP) in males of all dose groups. This difference was considered to be a chance finding (unrelated to the exposure), caused by a few relatively high values recorded for concurrent control animals, which was substantiated by the results obtained in recovery animals (showing a control range of plasma ALP activity similar to exposed animals of the main groups). Moreover, an increase – rather than a decrease – in ALP activity is commonly associated with pathological changes. Thus, the apparent decrease in ALP activity in exposed males of the main groups was considered to be of no toxicological relevance.

- Increased thyroxine (T4) concentration in plasma of females of the high-concentration group. Though increased relative to concurrent controls, the average T4 concentration (466.22 ± 133.43 ng/mL) was within the range of recent historical controls4, and in line with results obtained in animals of the recovery group.

- Increased plasma sodium (Na) concentration in females of the mid-concentration group, which was considered to be a chance finding, because a concentration-response relationship was absent.

Since statistically significant differences were observed in animals of the main groups, clinical chemistry parameters were also examined in animals of the recovery groups. No exposurerelated changes were observed at the end of the recovery period. A few statistically significant differences in females of the high-concentration group when compared to concurrent controls (slightly lower ALP activity, slightly lower average plasma albumin concentration and – consequently – a decreased albumin/globulin ratio) were considered chance findings, since corresponding changes were not observed at the end of the exposure phase.

PLEASE REFER TO ATTACHED TABLES - 'Clinical chemistry (main and recovery)'

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Organ weight data obtained at necropsy of animals of the main groups at the end of the exposure period showed a statistically significant increase in relative weight of the testes and adrenals in males of the high-concentration group, and a decrease in absolute and relative weight of the thymus in females of the high-concentration group. Thymus weight also seemed decreased in males of the high-concentration group, but statistical significance was not reached. A few statistically significant changes in organ weight of females of the low-concentration group (decreased absolute spleen weight, decreased absolute and relative thymus weight) were considered chance findings, because a concentration-response relationship was absent.

No exposure-related changes were observed in the weight of selected organs (seminal vesicles, testes, epididymides, and thymus) of animals sacrificed at the end of the recovery period.

PLEASE REFER TO ATTACHED TABLES - 'Absolute organ weights (main and recovery)' 'Relative organ weights (main and recovery)'

Gross pathological findings:

no effects observed

Description (incidence and severity):

At necropsy, no exposure-related macroscopic changes were observed in the animals of the main and recovery groups. The few gross changes observed represented background pathology in rats of this strain and age and occurred only incidentally or at random incidence in the different groups.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Microscopic evaluation revealed an exposure-related, concentration-dependent increase of degeneration of the respiratory and olfactory epithelium in the nasal tissues of animals of the mid- and high-concentration groups sacrificed at the end of the exposure period. In the highconcentration group, all animals showed minimal to mild degeneration of the respiratory epithelium in the rostral parts of the nose (level 2) and mild to moderate degeneration of the olfactory epithelium in the dorsocaudal parts of the nose (levels 3-6). The incidence and severity of these lesions was lower in animals of the mid-concentration group, with minimal to mild degeneration of the respiratory epithelium observed at level 2 in 4/10 animals, and minimal to mild degeneration of the olfactory epithelium observed at levels 3 (10/10 animals), 4 (9/10 animals) and 5 (2/10 animals). No exposure-related nasal pathology was observed in animals of the low-concentration group.

The other organs and tissues did not reveal any exposure-related histopathological changes. The histopathological changes observed were about equally distributed amongst the different treatment groups or occurred in one or a few animals only. They are common findings in rats of this strain and age or occurred as individual chance findings. Therefore, they were not considered to be related to the exposure.

Because of the histopathological changes observed in animals of the main groups at the end of the exposure period, microscopic examination of the nasal tissues was extended to animals of the recovery groups (control and high-concentration). Substantial – but not complete – recovery of the nasal lesions was observed at the end of the 4-week exposure-free recovery period. Minimal to mild degeneration of the olfactory epithelium was still observed in 7/10 animals at level 3 and in 1/10 animals at level 4 of the nasal tissues of animals of the high-concentration group. No exposure-related changes were observed in the respiratory epithelium – or in other
parts – of the nasal tissues of animals of the recovery groups.

PLEASE REFER TO THE ATTACHED TABLES - 'Microscopic observations (main and recovery)'

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Bronchoalveolar lavage and measurements
Analysis of BAL parameters at the end of the exposure period revealed an increase in the absolute number of macrophages in BAL fluid of exposed males (and consequently a trend towards increased total / viable cell numbers), which reached the level of statistical significance in males of the high-concentration group. These findings were not associated with any changes in the percentage distribution of white blood cells (BAL fluid normally contains ~100% macrophages in healthy animals). No exposure-related changes in cell differentials were observed in female animals. Investigation of biochemical parameters revealed a slight, but statistically significant increase in gamma-glutamyltransferase (GGT) activity in BAL fluid of females of the mid- and high-concentration group; no changes were found in male animals at the end of the exposure period.

Analysis of BAL parameters at the end of the recovery period did not show any statistically significant differences between exposed animals an unexposed controls, although a trend towards an increased absolute number of macrophages and total cells was still observed in males of the high-concentration group.

PLEASE REFER TO ATTACHED TABLES - 'Bronchiolalveolar lavage: biochemical determinations (main and recovery)' 'Brochiolar lavage: volume and absolute cell count (main and recovery)' 'Bronchiolar lavage: relative cell count (main and recovery)'

Key result

Dose descriptor:

NOAEC

Effect level:

49.7 ppm (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Remarks on result:

other:

Remarks:

Systemic toxicity

Key result

Dose descriptor:

NOAEC

Effect level:

10 ppm (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Remarks on result:

other:

Remarks:

Local toxicity

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

49.7 ppm

System:

respiratory system: upper respiratory tract

Organ:

nasal cavity

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Estrus cyclicity

Estrus cycle evaluation during the last three weeks of the exposure period did not reveal any exposure-related changes in cycle length or normality of the cycle.

Sperm analysis

Epididymal sperm count

No exposure-related statistically significant differences were observed on epididymal sperm count between the control- and exposure groups.

Epididymal sperm morphology

No exposure-related changes were observed in epididymal sperm morphology. A statistically significantly increased occurrence of ‘small hooks’ in males of the high-concentration recovery group was considered to be a chance finding, because corresponding changes at the end of the exposure period – or any corroborative changes in sperm morphology – were absent.

Epididymal sperm motility

No exposure-related statistically significant differences were observed on epididymal sperm motility. The statistically significantly increased average curvilinear velocity (VCL) in males of the high-concentration recovery group (relative to concurrent controls, but in the same range as controls of the main groups) was considered to be a chance finding, because a similar change was not observed in animals sacrificed at the end of the exposure period (also, a decrease – rather than an increase – in VCL in commonly associated with adverse sperm changes).

Testicular sperm count

There were no statistically significant differences in testicular sperm count parameters between the control- and the high-concentration group.

Analysis of the exposure conditions

Actual concentration

The overall average actual concentrations (± standard deviation) of Dimethylethylamine in the test atmospheres, as determined by total carbon analysis, were 10.0 (± 0.2), 49.7 (± 0.8) and 249.0 (± 4.0) ppm for the low-, mid- and high-concentration groups, respectively. These concentrations were very close to the respective target concentrations of 10, 50 and 250

ppm.

Time to attain chamber equilibration (T95)

The time to reach 95% of the steady state concentration (T95), based on chamber volume and the average total flow, was calculated to be 5.1 – 5.7 minutes.

Generation efficiency

The overall average (± standard deviation) generation efficiency, as calculated from the actual concentration, flows and test material consumption, was 84.0 (± 5.3)%. The efficiency of a vapor test atmosphere generation is commonly close to 100%. The slightly lower (than 100%) efficiency of the current test atmosphere generation was probably related to minor losses of test material due to condensation in the ‘liquid catch’, which was installed after the evaporator to prevent condensation down the line.

Total flow, temperature, relative humidity, oxygen and carbon dioxide concentration

The average (± standard deviation) total flows of test atmosphere were 25.9 (± 0.4), 24.5 (± 0.0), 25.2 (± 0.0) and 27.4 (± 0.2) L/min for the control, low-, mid- and high-concentration groups, respectively.

The average temperature (± standard deviation) was 23.05 (± 0.26), 22.99 (± 0.25), 22.81 (± 0.27) and 23.14 (± 0.30) °C for the control, low-, mid- and high-concentration groups,

respectively (Table 1.3). The average relative humidity during exposure was 48.73 (± 0.92), 49.47 (± 0.90), 46.75 (± 0.80) and 39.02 (± 0.84) % for the control, low-, mid-, and highconcentration groups, respectively.

The oxygen concentration during exposure was in the range of 20.2 – 20.4% (v/v) and the carbon dioxide concentration was in the range of 0.275 – 0.549% (v/v), which was

well within the limits of >19% oxygen and <1% carbon dioxide described in OECD guideline 412.

Conclusions:

Under the conditions of the current study, sub-acute inhalation exposure to Dimethylethylamine at actual concentrations of:
• 249.0 ppm resulted in local toxicity in the upper respiratory tract as characterized by degeneration of the respiratory and olfactory epithelium in the nasal tissues and systemic toxicity, as evidenced by significant decrease in body weight and food consumption;
• 49.7 ppm resulted in no systemic toxicity but presence of adverse local changes in the upper respiratory tract, as characterized by degeneration of the respiratory and olfactory epithelium in the nasal tissues;
• Both in the 49.7 and 249.0 ppm groups, substantial, but not full recovery of the nasal pathology was observed within a 4-week post-exposure recovery period;
• 10 ppm resulted in no systemic or local adverse changes.

Based on these findings, the No-Observed-Adverse-Effect-Concentration (NOAEC) for systemic toxicity upon sub-acute inhalation exposure to Dimethylethylamine in rats was placed at 49.7 ppm, and the NOAEC for local toxicity was placed at 10.0 ppm.

Executive summary:

The toxicity of Dimethylethylamine upon repeated exposure by inhalation was investigated in a sub-acute (28-day) study in Wistar rats. Four main groups of five male and five female rats each were exposed (nose-only) to target concentrations of 0 (control), 10, 50 or 250 ppm for 6 hours/day, 5 days/week over a 28-day period. Animals of the main groups were sacrificed on the day after the last exposure. In addition, two recovery groups, also consisting of five male and five female animals each, were simultaneously exposed with the main study animals to the control or 250 ppm test atmosphere, and were sacrificed after a 28-day recovery period following the last exposure. Endpoints to assess toxicity included clinical and ophthalmoscopic

observations, growth, food consumption, hematology, clinical chemistry and organ weights. Estrus cyclicity was evaluated during the last three weeks of the exposure period, and sperm

analysis was conducted at sacrifice. In addition, the animals were macroscopically examined at necropsy, the right lung lobes were lavaged and used for determination of biochemical markers and cell differentials, and the left lung lobes together with the full respiratory tract and a number of organs and tissues were examined microscopically.

The concentrations to be tested in the sub-acute study were selected on the basis of a 7-day range finding study in which five groups of five male and five female Wistar rats were exposed

to target concentrations of 0, 10, 100, 500 or 1000 ppm for 6 hours/day, 5 days/week. Exposure at 1000 ppm resulted in marked toxicity, as evidenced by clinically observed breathing

abnormalities and general signs of substantial distress, depressed growth and food intake, increased relative weight of several organs (heart, lungs, and in males also testes and adrenals),

and histopathological changes along the entire respiratory tract indicating severe epithelial damage (e.g. ulceration, atrophy, necrosis) in the upper airways and primarily inflammatory

changes in the lower airways. Exposure at 500 ppm resulted in transient clinical abnormalities, growth depression (mainly in males), atrophy of the nasal olfactory epithelium, and

inflammatory changes in the lower airways of several animals. Minimal to mild atrophy of the olfactory epithelium was still observed in animals exposed at 100 ppm. Exposure at 10 ppm did

not result in any exposure-related changes.

In the sub-acute main study, the target concentrations of Dimethylethylamine were accurately achieved as demonstrated by the results of total carbon analysis of the test atmosphere. The

overall average (± standard deviation) actual concentrations were 10.0 (± 0.2), 49.7 (± 0.8) or 249.0 (± 4.0) ppm for the low-, mid- and high-concentration groups, respectively.

Mortality did not occur during the study. Clinical and ophthalmoscopic observations revealed no exposure-related abnormalities.

Body weight data showed a statistically significantly reduced growth in male animals of the high concentration group throughout the exposure period; average body weight was about 10%

below controls at the end of the exposure period. Catch-up growth was observed during the 28- day recovery period. Body weight gain of males of the mid-, and females of the high concentration group was also below control level (-28%) during the first ~2 weeks of the exposure phase (though statistical significance was not reached).

Food consumption was slightly lower than controls in males of the high-concentration group throughout the exposure period (maximally approximately 15%) followed by normal food intake

during the recovery period and in females of the high-concentration group during the first two weeks of the study (maximally approximately 10%).

Estrus cycle evaluation did not reveal any exposure-related abnormalities.

No exposure-related changes were observed on epidydimal and testicular sperm analysis.

Analysis of hematology parameters showed a decrease in absolute number of eosinophils in blood of males of the high-concentration group sacrificed at the end of the exposure period. This

finding was not accompanied by any changes in other white blood cell parameters, by any changes in females, or in males of the recovery groups. No exposure-related changes were

observed in red blood cell and coagulation parameters.

Investigation of clinical chemistry parameters at the end of the exposure period revealed a statistically significant increase in plasma thyroxine (T4) concentration in females of the high concentration group when compared to concurrent controls (but which was within the historical control range, and in line with results obtained in control animals of the recovery group.

Therefore, is was not considered as treatment-related). No further changes in clinical chemistry parameters were observed in animals of the main and recovery groups.

Analysis of cellular parameters in bronchoalveolar lavage (BAL) fluid did not reveal any adverse changes in response to the exposure to the test material. An increase in the absolute number

of macrophages in BAL fluid of males of the high-concentration group was considered to be of no toxicological significance, because changes in the percentage distribution of white blood cells in BAL fluid (which normally contains ~100% macrophages in healthy animals) or any histopathological changes in the lungs were absent. Investigation of biochemical parameters

revealed a slight, but statistically significant increase in gamma-glutamyltransferase (GGT) activity in BAL fluid of females of the mid- and high-concentration main groups, which was fully

reversible within the 28-day recovery period.

Organ weight data obtained at necropsy of animals of the main groups showed an increase in relative (to body weight) weight of the testes (+15%) and adrenals (+28%) in males of the

high-concentration, and a decreased weight of the thymus in males (-28%) and females (-27%) of the high-concentration group. At the end of the recovery period, no exposure-related changes were observed in the weight of selected organs (seminal vesicles, testes, epididymides, and thymus).

Macroscopic examination at scheduled termination revealed no exposure-related gross pathology.

Microscopic examination revealed exposure-related histopathological changes in the nasal tissues of animals of the mid- and high-concentration group, characterized by degeneration of

the respiratory and olfactory epithelium. The incidence and severity of these lesions were evidently concentration-dependent. In the mid-concentration group, animals displayed minimal

to mild degeneration of the respiratory epithelium (in 4/10 animals at level 2 of the nose), and minimal to mild degeneration of the olfactory epithelium at levels 3 (10/10 animals), 4 (9/10

animals) and 5 (2/10 animals) of the nose2. In the high-concentration group, minimal to mild degeneration of the respiratory epithelium was observed in all animals at level 2, and mild to

moderated degeneration of the olfactory epithelium was found in all animals throughout levels 3 to 6 of the nasal tissues. At the end of the 28-day recovery period, minimal to mild

degeneration of the olfactory epithelium was observed at level 3 in 7/10 animals and at level 4 in only one animal of the high-concentration group (changes in the respiratory epithelium were

no longer found), indicating substantial - but not full - recovery of the nasal pathology. No exposure-related microscopic changes were observed in the lower respiratory tract or in any of

the other organs and tissues examined.

Conclusion

Under the conditions of the current study, sub-acute inhalation exposure to Dimethylethylamine at actual concentrations of:

• 249.0 ppm resulted in local toxicity in the upper respiratory tract as characterized by degeneration of the respiratory and olfactory epithelium in the nasal tissues and systemic

toxicity, as evidenced by significant decrease in body weight and food consumption;

• 49.7 ppm resulted in no systemic toxicity but presence of adverse local changes in the upper respiratory tract, as characterized by degeneration of the respiratory and olfactory

epithelium in the nasal tissues;

• Both in the 49.7 and 249.0 ppm groups, substantial, but not full recovery of the nasal pathology was observed within a 4-week post-exposure recovery period;

• 10 ppm resulted in no systemic or local adverse changes.

Based on these findings, the No-Observed-Adverse-Effect-Concentration (NOAEC) for systemic toxicity upon sub-acute inhalation exposure to Dimethylethylamine in rats was placed at 49.7 ppm, and the NOAEC for local toxicity was placed at 10.0 ppm.

Reference 3

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

20 december 2017-17 January 2018

Reliability:

2 (reliable with restrictions)

Reason / purpose for cross-reference:

reference to same study

Guideline:

other:

Version / remarks:

Range finding study

GLP compliance:

yes

Limit test:

yes

Specific details on test material used for the study:

Name1 : Dimethylethylamine
Chemical name 1 : Ethyldimethylamine; N,N-Dimethylethylamine
CAS registry number1 : 598-56-1
Molecular formula1 : C4H11N
Structural formula1 :
Molecular weight1 : 73.13 g/mol
Batch/lot number1 : SAMP160321
Purity1 : 99.41%
Solubility in water1 : completely soluble; exothermic reaction!
Melting point1 : -140 °C
Boiling point1 : 36.3 °C
Vapor pressure 1 : 527 hPa at 20° C
Density 1 : 660 kg/m3 at 20 °C
Viscosity1 : 0.67 mPa.s (OECD Test Guideline 114)
Flash Point1 : -25°C closed cup (ISO 3679)
Quantity received : 6.75 kg
Supplier : Arkema
Approved for use until1 : 20 November 2018
Storage conditions : ambient temperature (15-25°C)
Date of receipt : 23 November 2017
1Characteristics provided by the sponsor
The Triskelion dispense reference number is 17025D.

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

7-day range finding study
27 male and 27 female rats were ordered, and upon arrival on 20 December 2017 they were
taken in their unopened shipping containers to a quarantine room (animal room 5.1.20) and
were checked for overt signs of ill health and anomalies. During the quarantine period,
serological investigation of the microbiological status was conducted in blood samples taken
from four randomly selected animals. On 22 December 2017, the results of the serological
examinations were received and indicated an acceptable microbiological status. The animals
were subsequently released for experimental use, and they were transferred to their definitive
room (animal room 6.0.06) on the same day. The duration of the acclimatization period to the
laboratory conditions prior exposure (period between arrival and the start of the exposure
period) was 21 days.
On 8 January 2018, shortly before initiation of exposure of the rats, they were allocated to the
various groups by computer randomization proportionally to body weight. Cross reference
listings showing animal, group and cage numbers are given in Annex 3.1. The surplus animals
(2 males, 2 females) were kept in reserve to serve as sentinels. These animals were not used
in the present study.

Route of administration:

inhalation: vapour

Type of inhalation exposure:

nose only

Details on inhalation exposure:

The animals were exposed to the test atmosphere in nose-only exposure units, in an illuminated
laboratory room different from the room where the animals were housed. Animals of groups 2,
3, 4 and – for the range finding – 5 were exposed in inhalation chambers consisting of a
cylindrical aluminium column, surrounded by a transparent cylinder (a modification of the
chamber made by ADG Developments Ltd., Codicote, Hitchin, Her ts, SG4 8UB, United
Kingdom). The column had a volume of 46.7 L and consisted of a top assembly with the entrance
of the unit, a mixing section, two rodent tube sections and at the bottom the base assembly
with the exhaust port. Each rodent tube section had 20 ports for animal exposure. Control
animals (group 1) were exposed to clean air in a polypropylene nose-only inhalation chamber
with a volume of 48.2 L (manufactured by P. Groenendijk Kunststoffen BV) which was very
similar in construction to the aluminium chambers described above.
Empty ports were used for test atmosphere sampling (for analysis of the actual concentration)
and measurement of oxygen, carbon dioxide, temperature and relative humidity. The animals
were secured in plastic animal holders (Battelle), positioned radially through the outer cylinder
around the central column. Animals were rotated with respect to their position in the column
every week (main study) or two days (range finding study). The remaining ports were closed.
Only the nose of the rats protruded into the interior of the column. Habituation to the restraint
in the animal holders was not performed because in our experience habituation does not help
to reduce possible stress (Staal et al., 2012).
In our experience, the animal’s body does not exactly fit in the animal holder which always
results in some leakage from the high to the low pressure side. By securing a positive pressure
in the central column and a slightly negative pressure in the outer cylinder, which encloses the
entire animal holder, dilution of test atmosphere by air leaking from the animals’ thorax to the
nose was avoided. The unit was illuminated externally by normal laboratory fluorescent tube lighting. The total air
flow through the unit was at least 1 liter/min for each rat. The air temperature and relative
humidity in the unit were maintained at 22 ± 3°C and between 30 and 70%, as far as possible.

Generation of the test atmosphere

The inhalation equipment was designed to expose the animals to a continuous supply of fresh
test atmosphere. A schematic diagram of the generation and the exposure system is presented
in Figure 1. To generate the test atmospheres, a continuous flow of liquid test material3,
controlled by a peristaltic pump (Minipulse 3, Gilson, Velliers le Bel, France) was allowed to
evaporate in a mass flow controlled (Bronkhorst Hi Tec, Ruurlo, the Netherlands) stream of
compressed dry air, by directing it through a glass evaporator which was kept at a constant
temperature of 48°C by circulating heated water. The resulting single stream of concentrated
vapor was led through a condense trap (not used during the range finding study) and was
subsequently divided for the different groups using mass flow controllers (Bronkhorst Hi Tec)
and mixed with a controlled stream of humidified compressed air via an eductor (Fox Valve
Development Corp., Dover, NJ, USA). The eductors were calibrated by measuring the total air
flow at a range of driving air pressures of the eductors encompassing the driving pressures used
during the study; the driving air pressure was used to monitor the total flow. The resulting test
atmosphere was led to the inlet at the top of the exposure chamber and directed downward
towards the noses of the animals; the atmosphere was exhausted at the bottom. As of 9 April
2018, a PI feedback system was installed to automatically control the feedrate of the peristaltic
pump, based on the measured actual concentration (see paragraph 4.10.1). The feedback
system took into account the proportional (P) and the integrated deviations (I) of the
concentrations from the setpoint.
The exposure chamber for the control animals (group 1) was supplied with a stream of
humidified compressed air only, which was controlled by a reducing valve and measured by
mass view meter (Bronkhorst Hi Tec).
The animals were placed in the exposure unit after stabilization of the test atmospheres (see
paragraph 5.1.2 for T95). Test atmosphere generation and animal exposure were performed in
an illuminated laboratory at room temperature.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The actual concentration of the test material in the test atmospheres was measured by total
carbon analysis (groups 2 and 3 of the main study and group 3 of the range finding study:
Ratfisch RS55T, Munich, Germany; remaining groups: Sick Maihak GMS 810 EuroFID Total
Hydrocarbon Analyzer; Sick Instruments Benelux, Hedel, the Netherlands). Test atmosphere
samples were taken continuously from the exposure chamber at the animals’ breathing zone
and were passed to the total carbon analyzer (TCA) through a sample line.

Group 2 (target concentration 10 ppm)
On 21 and 22 December 2017, concentrations of 7.8, 8.5, 9.2, 9.7, 9.8, 9.8, 9.9, 10.9, 11.5,
19.7 and 21.5 ppm were analyzed to calibrate the TCA used for group 2. The response (Y) of
the analyzer was related to the concentration (X) of the test material as:
Y (%) = 4.481 * X (ppm) – 6.876, with a coefficient of determination (R2) of 0.9887.
Group 3 (target concentration 100 ppm)
On 20 December 2017, concentrations of 73, 79, 109, 111, 124 and 127 ppm were analyzed to
calibrate the TCA used for group 3. The response (Y) of the analyzer was related to the
concentration (X) of the test material as:
Y (%) = 2.305*10-1 * X (ppm) + 1.942*10-1 (R2 = 0.9996).
Group 4 (target concentration 500 ppm)
On 9 January 2018, concentrations of 358, 374, 440, 474, 615 and 727 ppm were analyzed to
calibrate the TCA used for group 4. The response (Y) of the analyzer was related to the
concentration (X) of the test material as:
Y (%) = 1.275*10-1 * X (ppm) – 1.344 (R2 = 0.9996).
Group 5 (target concentration 1000 ppm)
On 19 December 2017, concentrations of 852, 1005, 1079, 1142, 1281 and 1338 ppm were
analyzed to calibrate the TCA used for group 5. The response (Y) of the analyzer was related to
the concentration (X) of the test material as:
Y (%) = 6.217*10-2 * X (ppm) - 2.432*10-1 (R2 = 0.9948).

Duration of treatment / exposure:

The animals were exposed for 6 hours per day, 5 days per
week, over a 7-day study period (5 exposure days in total) and sacrificed on the day after the
last exposure. The date of the first exposure (10 January 2018) was named day 0.

Frequency of treatment:

see above

Dose / conc.:

10 ppm

Dose / conc.:

100 ppm

Dose / conc.:

500 ppm

Dose / conc.:

1 000 ppm

Dose / conc.:

0 ppm

No. of animals per sex per dose:

The range finding study will comprise five test groups of five male and five female rats each,
i.e. a control group exposed to clean air and four groups exposed to different concentrations of
the test material.
Actual concentration
The overall average actual concentrations (± standard deviation) of Dimethylethylamine in the
test atmospheres, as determined by total carbon analysis, were 10.0 (± 0.1), 100.5 (± 0.9),
497.2 (± 6.5) and 998.1 (± 4.3) ppm for the low-, mid-, high- and top-concentration groups,
respectively (Table 1.1 of this annex). These concentrations were very close to the respective
target concentrations of 10, 100, 500 and 1000 ppm.

Control animals:

yes

Observations and examinations performed and frequency:

Clinical observations
Animals were observed daily in the morning hours by cage-side observations and, if necessary,
handled to detect signs of toxicity. The observations included – but were not limited to – the
signs listed in Annex 6. The animals were also observed about halfway through the 6-hour
exposure period, in particular to monitor any breathing abnormalities and restlessness;
observation of other abnormalities was hindered due to the animals’ stay in restraining tubes.
All animals were thoroughly checked again in the afternoon. All abnormalities, signs of ill health,
and reactions to treatment were recorded.
Body weights
In the range finding study, the body weight of each animal was recorded 2 days before the start
of the exposure. These pre-test weights served as a basis for animal allocation (see paragraph
4.5). Subsequently, the animals were weighed prior to the first exposure on day 0, on day 4 of
exposure and on their scheduled sacrifice data in order to calculate the correct organ to body
weight ratios.
Food consumption
Food consumption of the animals was measured per cage by weighing the feeders. The results
are expressed in g per animal per day. Food consumption was measured twice weekly for
animals of the range finding study. For animals of the main study, food consumption was
measured once weekly

Sacrifice and pathology:

Sacrifice, organ weights and macroscopic examination
At the end of the exposure period, the animals were sacrificed in such a sequence that the
average time of sacrifice was approximately the same for each group. The animals were sacrificed by exsanguination from the abdominal aorta
under pentobarbital anesthesia (intraperitoneal injection of sodium pentobarbital) and then
examined grossly for pathological changes.
7-day concentration range finding study
The following organs of all animals were weighed (paired organs together) as soon as possible
after dissection to avoid drying. Relative organ weights (g/kg body weight) were calculated from
the absolute organ weights and the terminal body weight:
- heart
- adrenals
- kidneys
- liver
- spleen
- testes
_ovaries
-lung with trachea
Histopathology
7-day concentration range finding study
For histopathological examination, samples of the following tissues and organs from all animals,
and all gross lesions were preserved in a neutral aqueous phosphate-buffered 4% solution of
formaldehyde (10% solution of Formalin). The lungs (after weighing) were infused with the
fixative under ca. 15 cm water pressure to ensure fixation). Tissues were processed and
examined using the same methods as described below for the relevant tissues examined during
the 28-day study. The carcass containing any remaining tissues was retained in the fixative until
completion of the histopathological examination and then discarded.
- kidneys
- nasal turbinates
- larynx
- trachea
- lungs
- tracheobronchial lymph nodes

All preserved tissues of all animals of the control and top-concentration groups were examined
histopathologically (by light microscopy). Since exposure-related changes were observed in the
nasal tissues (all 6 levels), larynx, trachea and lungs of the top-concentration group,
histopathological examination of these tissues was extended to animals of the intermediate
concentration groups of the range finding study.

Statistics:

Ancova/Anova & Dunnett
Pretreatment body weight, body weight after initiation of treatment, clinical pathology (hematology, clinical chemistry), bronchoalveolar lavage parameters, organ weights, sperm motility
(numerical), sperm count, testicular sperm count.
Food consumption - no statistics were applied (only one cage per sex per group).
Kruskal-Wallis & Wilcoxon: Estrus cyclicity: mean length of the longest cycle, number of complete cycles in the test period; sperm motility (expressed as %), sperm morphology
Chie-squared & Fisher's Exact: Incidences of histopathological changes; Estrus cyclicity: number of acyclic females, number of animals with prolonged estrus period

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

All animals survived until scheduled necropsy. Clinical signs were observed in groups 4 (500
ppm) and 5 (1000 ppm). In group 4, clinical signs were mostly observed in male animals and
were generally transient; after the first exposure on day 0, dyspnea, shallow breathing, hunched
posture and piloerection were noted in about half of the group 4 males. After the second
exposure, fewer clinical observations were noted while after subsequent exposures, only
occasional piloerection was noted in group 4 males. Occasional piloerection was also observed
in female animals of group 4; no further clinical abnormalities were noted.
In group 5 animals, all male animals and most female animals displayed dyspnea, sniffing,
irregular respiration, hunched posture, ataxia, blepharospasm and piloerection and hypoactivity.
Annex 11: Results of the range finding study
Triskelion B.V. Report | V21124 | Final
February 2019 139/397
Mouth breathing was observed in 4/5 males and 1/5 females. Most animals displayed increased
salivation, while in some animals grunting, chewing movement and shallow breathing was noted.
These clinical signs persisted throughout the study, were exposure-related, and indicative of severe toxicity.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Concentration-related changes in body weight were observed in males, which increased in
severity from slightly reduced growth in males of the mid-concentration, to substantial body
weight loss in males of the top-concentration group (16% average loss of body weight between
days 0 and 7). In females, a statistically significantly reduced growth was only observed in
animals of the top-concentration group.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

The changes in body weight were accompanied by a decreased food intake in males of the
high- and top-concentration and in females of the top-concentration group.

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

The relative (to body weight) weights of the lungs and heart were statistically significantly
increased in males and females of the top-concentration group when compared to unexposed
controls. In male animals, relative testes weight was also increased at the high- and topconcentration,
and relative weight of the adrenals was increased and relative liver weight was decreased at the top-concentration level.
Statistically significant changes were also observed in absolute organs weights (e.g. decreased weight of the kidneys, liver and spleen in males and of the spleen and ovaries in female of the top-concentration group), but the interpretation of significance of these findings is difficult, because they may be – at least partly – related to the lower body weight at necropsy.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

At necropsy, the lungs of 4/5 top-concentration males and some single animals in the other
treatment groups showed red spots in the lungs that probably were related to treatment. A small
thymus was found in 2/5 males and 2/5 females of the top-concentration group.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Microscopic evaluation revealed exposure-related histopathological changes in the nose, the
larynx, the trachea/bronchi and the lungs.
Nose:
In the mid-concentration animals, the treatment-related histopathological changes were
confined to minimal to mild atrophy of the olfactory epithelium in levels 3, 4 and 5, mainly in
the dorsal meatus.
In the high-concentration group, olfactory epithelial atrophy was also observed in most animals.
In addition, inflammatory changes were seen in several animals, in levels 1 to 4, occasionally
accompanied by necrosis or ulceration.
The top-concentration animals showed severe histopathological changes: level 2 was most
affected with the highest score grades for (granulocytic) inflammation, ulceration and necrosis.
The damaged areas were mainly the nasal septum, the conchae and the lateral walls of the nasal
cavity. In severe cases of necrosis, parts of the conchae and/or nasal septum were completely
gone. High score grades for inflammation and ulceration were also observed in levels 3 and 4,
while slightly lower grades were scored in levels 5 and 6. The remains of the damaged epithelium
showed large areas with squamous metaplasia. In the necrotic areas sometimes colonies of
(most likely) opportunistic bacteria were seen. In many cases, the nasal cavity contained
inflammatory exudate (a mix of fluid, cellular debris and granulocytic inflammatory cells). Since
the exact origin of this exudate could not be established, it was not mentioned in the table at a
specific level of the nose.
Larynx:
The main exposure-related finding in the larynx was minimal to mild granulocytic inflammation,
mild squamous metaplasia and minimal to mild ulceration. This was seen in 8/10, 8/10 and 4/10
top-concentration animals, respectively. Minimal to mild granulocytic inflammation was also
present in 4/10 high-concentration animals.
Trachea/bronchi:
The main treatment-related finding in the trachea/bronchi was minimal to mild granulocytic
inflammation in 9/10 top-concentration animals and in 3/10 high-concentration animals.
Lungs:
Histopathological changes that were considered to be related to the exposure were: alveolar
hemorrhages (4/10 top-concentration animals), increased bronchus associated lymphoid tissue
(4/10 top-concentration animals), granulocytic inflammation of bronchi (8/10 top-concentration
animals) and a granulomatous inflammation (2/10 top-concentration animals).
The other histopathological changes observed were about equally distributed amongst the
different treatment groups or occurred in one or a few animals only. They are common findings
in rats of this strain and age or occurred as individual chance findings. Therefore, they were not
considered to be related to treatment.

Histopathological findings: neoplastic:

not examined

Dose descriptor:

NOEC

Remarks:

local nasal irriation

Effect level:

10 ppm

Basis for effect level:

histopathology: non-neoplastic

Dose descriptor:

NOAEC

Remarks:

systemic toxicity

Effect level:

100 ppm

Basis for effect level:

body weight and weight gain

clinical signs

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Critical effects observed:

yes

Lowest effective dose / conc.:

100 ppm

System:

respiratory system: upper respiratory tract

Organ:

nasal cavity

Treatment related:

yes

Conclusions:

The target concentration for the main study were selected at 10, 50, and 250 ppm. Sub-acute exposure to Dimethylethylamine at 250 ppm is anticipated to result in clear respiratory toxicity (without causing lethality or severe distress), while at 10 ppm exposure-related adverse changes are no longer expected.

Executive summary:

Under the conditions of this 7-day concentration range-finding study, inhalation exposure to Dimethylethylamine at target concentrations of 10, 100, 500 and 1000 ppm resulted in the following treatment-related changes:

- At 1000 ppm, marked toxicity was observed; in nearly all animals, dyspnea, sniffing, irregular respiration, hunched posture, ataxia, blepharospasm, piloerection and hypoactivity were observed. Body weight of males exposed to the top concentration were 22% lower than controls (on day 7); for females the reduction in body weight was 15%. Relative weight of the heart and lungs was increased in male and female animals. When assessing histopathology, severe olfactory epithelial atrophy was observed in the nasal tissues. Milder effects were observed in the larynx, trachea/bronchi and lungs, mainly of inflammatory nature.

- At 500 ppm, transient clinical abnormalities (dyspnea, shallow breathing, hunched posture and piloerection) were observed in males especially. Minimal to mild atrophy of the nasal olfactory epithelium was observed in most animals when assessing nose tissue levels 3 and 4 (out of the 6 levels assessed).

- At 100 ppm, minimal atrophy of the nasal olfactory epithelium was still observed in most animals, with 2 cases of mild atrophy observed at a single level of the nose in 2 animals.

- At 10 ppm, no exposure-related toxicity was seen. Thus, the No-Observed-Adverse-Effect-Concentration (NOAEC) in this range finding study was placed at 10 ppm (target concentration).

Based on these observations, the target concentration for the main study were selected at 10, 50, and 250 ppm. Sub-acute exposure to Dimethylethylamine at 250 ppm is anticipated to result in clear respiratory toxicity (without causing lethality or severe distress), while at 10 ppm exposure-related adverse changes are no longer expected.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

318 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:

08 November 2018 - 01 July 2019
report finalized 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.29 (Sub-Chronic Inhalation Toxicity:90-Day Study)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: SAMP160321
- Expiration date of the lot/batch: 17 April 2019


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: In a refrigerator (2 to 8°C), desiccated, in the dark.

Species:

rat

Strain:

Wistar

Details on species / strain selection:

The rat was chosen as the test species because it is accepted as a predictor of toxic change in man and the requirement for a rodent species by regulatory agencies. The Crl:WI(Han) strain was used because of the historical control data available at this laboratory.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Ltd.
- Females (if applicable) nulliparous and non-pregnant: yes]
- Age at study initiation: 53 to 59 days
- Weight at study initiation: Males :222 to 318 g, Females: 143 to 203 g
- Fasting period before study: N/A
- Housing: Cages-Polycarbonate body with a stainless steel mesh lid, changed at appropriate intervals. The cages constituting each group were blocked together by sex on separate batteries. Five of the same sex per cage (main study and recovery), unless reduced by mortality or isolation. Wood based bedding which was changed at appropriate intervals each week.
- Diet (e.g. ad libitum): Teklad 2014C Diet. Non-restricted (removed overnight before blood sampling for hematology or blood chemistry and during the period exposure).
- Water (e.g. ad libitum): Potable water from the public supply via polycarbonate bottles with sipper tubes. Bottles were changed at appropriate intervals. Non-restricted (except during exposure).
- Acclimation period: 11 days before commencement of treatment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24ºC
- Humidity (%): 40-70%.
- Air changes (per hr): Filtered fresh air which was passed to atmosphere and not recirculated
- Photoperiod (hrs dark / hrs light): Artificial lighting, 12 hours light : 12 hours dark.

IN-LIFE DATES (Main study): From: 08 November 2018 To: 18 to 19 February 2019
IN-LIFE DATES (Recovery study): From: 08 November 2018 To: 01 April 2019

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

air

Details on inhalation exposure:

Route: Inhalation - snout only exposure.
Control (Group 1): Air only.
Duration of daily exposure: 6 hours. Weeks 1 to 12: 5 days each week; Week 13: 7 days.
Training for dosing: The animals on study were acclimated to the method of restraint, over a 3 day period immediately preceding the first test item exposure.

Exposure System:
Flow through nose-only chamber
Aluminum alloy construction comprising a base unit, three animal exposure sections, a top section and a pre-chamber

Animal Restraint:
Plastic nose-only restraint tube

Atmosphere Generation:
Glass sintered vaporizer
The test item was supplied to the generator, via a feed line, from a syringe driven at a constant rate by a syringe pump

Inlet Airflow:
From in-house compressed air system – breathing quality
Generator flow: 10-60 L/minute

Extract Airflow:
Drawn by in-house vacuum system
Filtered locally
Extract flow: 40-160 L/minute

Airflow Monitoring:
High quality tapered tube flowmeters - calibrated daily
In-line flowmeters monitored continuously

System Containment:
Systems housed in separate ventilated cabinets.

Administration:
Test group animals (Groups 2 – 4) were exposed to an atmosphere containing dimethylethylamine.
Group 1 animals were exposed to compressed air only
Animals were exposed on five days each week for 13 weeks. Additional animal exposures were conducted on Week/Day 13.6, 13.7 and 14.1 to cover end of study
investigations.
Duration of exposure was 6 hours each day
Exposures commenced on 19 November 2018
Different exposure levels were achieved by varying the concentration of test item in the exposure systems, whilst keeping the duration of exposure constant
The animals on study were acclimatized to the method of restraint for three consecutive days preceding their first exposure
System operating conditions were amended at the discretion of the Study Director to maintain achieved atmosphere concentrations close to target.

Concentration:
Atmosphere samples collected as follows:
Collection media: Dreschel head and solvent trap (bubbler)
Sample solvent: Methanol
Sample flow: 2.0 L/minute
Sample volume: Measured by wet-type gas meter
Sample frequency: 1 sample from Group 1/day (taken at approximately 180 minutes into exposure)
Minimum of 3 samples from Group 2, 3 and 4/day (taken at approximately 60, 180 and 300 minutes during exposure)
Sample location: Animal exposure port
Sample analysis: Chemical
During preliminary characterization trials an assessment was made of the percentage breakthrough of test item through the sample collection media; this was achieved by setting up two bubblers in series and collecting a sample of test atmosphere. The acceptable breakthrough limit to the second solvent trap is = 10%. The percentage break through the sample collection media was less than 10%, therefore one bubbler was used on study to collect chamber atmosphere samples.

Chamber air temperature was measured throughout exposure using an electronic thermometer probe placed in the breathing zone of the animals via an unused exposure port. Chamber air temperature was monitored continuously and recorded at 60-minute intervals.

Chamber relative humidity was measured throughout exposure using an electronic hygrometer probe placed in the breathing zone of the animals via an unused exposure port.
Chamber relative humidity was monitored continuously and recorded at 60-minute intervals.

The mean achieved atmosphere concentrations were 103, 100 and 106% of target for Groups 2, 3 and 4, respectively. Initially the inter and intra exposure variation was higher than anticipated. Bubbles were observed to be forming in the syringes and feed lines containing the test item. This was attributed to the test item expanding as it warmed following refrigerated storage and was
remedied by allowing the test item to warm to ambient temperature prior to generation. Subsequently the test item was observed to be vaporizing in the feed lines with an inversely
proportional relationship to target atmosphere concentration. This was considered to be a consequence of the very low feed rates required to achieve the target atmosphere concentrations and was remedied by reducing the diameter of the feed lines.

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

13 Weeks.

Frequency of treatment:

Dimethylethylamine was administered to Wistar Han rats by snout-only inhalation exposure for 6 hours per day (5 days per week) for 13 weeks.

Dose / conc.:

0 ppm

Dose / conc.:

10 ppm

Dose / conc.:

30 ppm

Dose / conc.:

100 ppm

No. of animals per sex per dose:

10 animals, per sex, per dose.

Control animals:

yes

Details on study design:

The target doses used in this study (0, 10, 30 and 100 ppm) were selected in conjunction with the Sponsor.
In a previous 28-day inhalation study in rats with Dimethylethylamine at 10, 50 or 250 ppm, degeneration of respiratory and olfactory epithelium in the nose was evident at 50 or 250 ppm with additional effects evident at 250 ppm relating to body weight and food consumption. For this study, a high exposure level of 100 ppm was selected and local effects were expected in the nose but it was considered these would be tolerated. Intermediate and low exposure levels of 30 or 10 ppm were selected to assess any relationship to exposure level.

The rat was chosen as the test species because it is accepted as a predictor of toxic change in man and the requirement for a rodent species by regulatory agencies. The Crl:WI(Han) strain was used because of the historical control data available at this laboratory.

Please see study design section under material and methods section below.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
Cages were inspected daily for evidence of animal ill-health amongst the occupants. Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment.

DETAILED CLINICAL OBSERVATIONS: Yes
Signs Associated with Dosing
Daily during the first four weeks of treatment on exposure days and weekly thereafter, detailed observations were recorded at the following times in relation to dose administration:
Pre-exposure observation
As each animal was returned to its home cage
As late as possible in the working day

In addition observations were made in the treatment period, on days without exposures during Weeks 1 to 4, at the following times during the day:
Early in the working day (equivalent to pre-exposure observation)
As late as possible in the working day

Observations during exposure is severely restricted due to tube restraint.

Detailed Physical Examination and Arena Observations
Before treatment commenced and during each week of treatment and recovery, detailed physical examination and arena observations were performed on each animal. On each occasion, the examinations were performed at approximately the same time of day (before dosing during the treatment period), by an observer unaware of the experimental group identities.
After removal from the home cage, animals were assessed for physical condition and behavior during handling and after being placed in a standard arena. Any deviation from normal was recorded with respect to the nature and, where appropriate, degree of severity. Particular attention was paid to possible signs of neurotoxicity, such as convulsions, tremor and abnormalities of gait or behavior.
Findings were either reported as "present" or assigned a severity grade - slight, moderate or marked.

BODY WEIGHT: Yes
The weight of each animal was recorded twice weekly from one week before treatment commenced, on the day that treatment commenced (Day 1) and during Weeks 1 to 4. Weekly body weights were recorded during Weeks 5 to 13, during recovery and on the day of necropsy.

FOOD EFFICIENCY: Not specified

FOOD CONSUMPTION
The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded for the week before treatment started and for each week throughout the study.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Not specified

OPHTHALMOSCOPIC EXAMINATION: Yes
The eyes of all animals were examined by means of a binocular indirect ophthalmoscope during pretreatment and Week 13.

HAEMATOLOGY: Yes
Blood samples were collected after overnight withdrawal of food duing week 13 from all animals.
Animals were held under light general anesthesia induced by isoflurane. Blood samples (nominally 0.5 mL) were withdrawn from the sublingual vein, collected into tubes containing EDTA anticoagulant and examined for the following characteristics using a Bayer Advia 120 analyzer:
Hematocrit (Hct)*
Hemoglobin concentration (Hb)
Erythrocyte count (RBC)
Absolute reticulocyte count (Retic)
Mean cell hemoglobin (MCH)*
Mean cell hemoglobin concentration (MCHC)*
Mean cell volume (MCV)
Red cell distribution width (RDW)
Total leucocyte count (WBC)
Differential leucocyte count:
Neutrophils (N)
Lymphocytes (L)
Eosinophils (E)
Basophils (B)
Monocytes (M)
Large unstained cells (LUC)
Platelet count (Plt)

*Derived values calculated in ClinAxys

Blood film (prepared for all samples) - Romanowsky stain, examined for abnormalities by light microscopy, in the case of flags from the Advia 120 analyzer. Confirmation or a written description from the blood film was made where appropriate. Additional blood samples (nominally 0.5 mL) were taken into tubes containing citrate anticoagulant and examined using a Stago STA Compact Max analyzer and appropriate reagent in respect of:
Prothrombin time (PT) - using IL PT Fibrinogen reagent. Activated partial thromboplastin time (APTT) - using IL APTT reagent.

BLOOD CHEMISTRY: Yes
Blood samples were collected after overnight withdrawal of food duing week 13 from all animals.
Animals were held under light general anesthesia induced by isoflurane. Blood samples (nominally 0.7 mL) were withdrawn from the sublingual vein and collected into tubes containing lithium heparin as anticoagulant. After separation, the plasma was examined using a Roche P Modular Analyzer in respect of:
Alkaline phosphatase (ALP)
Alanine aminotransferase (ALT)
Aspartate aminotransferase (AST)
Total bilirubin (Bili)
Urea*
Blood urea nitrogen (BUN)
Creatinine (Creat)
Glucose (Gluc)
Total cholesterol (Chol)
Triglycerides (Trig)
Sodium (Na)
Potassium (K)
Chloride (Cl)
Calcium (Ca)
Inorganic phosphorus (Phos)
Total protein (Total Prot)
Albumin (Alb)

*Numerically equivalent to blood urea nitrogen (BUN)

Albumin/globulin ratio (A/G Ratio) was calculated from total protein concentration and analyzed albumin concentration.

URINALYSIS: Not specified

NEUROBEHAVIOURAL EXAMINATION: Yes
Sensory reactivity and grip strength assessments were performed (on non-dosing days) on all main study animals in Groups 2 and 3 and all recovery phase animals during Week 12 of treatment. Animals were tested by an observer who was unaware of the treatment group to which each animal belonged. Before the start of observations, cage labels showing the treatment group were replaced by labels stating only the study, animal and cage numbers. Animals were not necessarily all tested on the same day, but the numbers of animals and the times of testing were balanced across the groups on each day of testing.
The following measurements, reflexes and responses were recorded:

Approach response
A blunt probe was brought towards the animal’s head until it was close to the animal’s nose (but not touching the whiskers).
Pinna reflex
The inside of one ear was touched lightly with a nylon filament and the reaction recorded.
Auditory startle reflex
The animal’s response to a sudden sharp noise was assessed.
Tail pinch response
The animal’s tail was pinched sharply with forceps approximately one third from the tip and the response graded.
Grip strength
Forelimb and hindlimb grip strength was measured using Mecmesin Basic Force Gauges. Three trials were performed.

Motor Activity
During Week 12 of treatment (on non-dosing days), the motor activity of all main study animals in Groups 2 and 3 and all recovery phase animals was measured using a Rodent Activity Monitoring System (Version 2.0.6), with hardware supplied by Pearson Technical Services and software developed and maintained by Envigo.
Animals were tested individually in clear polycarbonate cages and motor activity was measured by counting infra-red beam breaks over ten 6-minute intervals (one hour total). Ten beams were set at two height levels (five low and five high) to detect cage floor and rearing activity respectively. Animals were not necessarily all tested on the same day, but the numbers of animals and the times of testing were balanced across the groups on each day of testing.


IMMUNOLOGY: Not specified

OTHER:
Estrous Cycles – Vaginal Smears: Dry smears were taken For 14 days during Weeks 12 and 13 of treatment and during the recovery phase, using cotton swabs.

Mortality: A viability check was performed near the start and end of each working day. Animals were isolated or killed for reasons of animal welfare where necessary.
A complete necropsy was performed in all cases.

All observations regarding Thyroid hormone analysis please see materials and methods section below.

Sacrifice and pathology:

All main study and recovery animals were subject to a detailed necropsy. After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative. The retained tissues were checked before disposal of the carcass.

Other examinations:

Bone Marrow
Bone marrow smears were prepared immediately following death, on completion of the scheduled treatment or recovery periods and from animals killed prematurely during the study

Bronchoalveolar Lavage (BAL)
The right lung was used for bronchoalveolar lavage sampling and the left lung was processed for histology and light microscopy.

Sperm Analysis
Immediately after scheduled sacrifice of each male and collection of blood and bone marrow, the left vas deferens, epididymis and testis were removed and the epididymis and testis were weighed.
The following tests were performed:
Sperm motility – all groups
Sperm morphology – Groups 1 and 4
Sperm count – all groups
Homogenisation-resistant spermatid count – all groups

Stage-dependent Evaluation of Spermatogenesis
Stage dependent evaluation of spermatogenesis was conducted on sections of testes from all animals of Groups 1 (Control) and 4 (106 ppm) sacrificed on completion of the scheduled treatment period prepared and stained using the PAS method. A qualitative examination of spermatogenic stages was made for normal progression of the stages of the spermatogenic cycle, cell associations, and proportions expected to be present during normal spermatogenesis.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related clinical signs or dosing observations during the 13 weeks of treatment or during the 6 week recovery period.
Signs associated with the administration procedure included wet fur and/or red staining of the head, nose and eyes on return to home cage, in which the majority had resolved by end of working day. These signs were seen in animals from all groups including control, therefore are considered to be due to the method and duration of restraint and are commonly seen on inhalation studies of this study design. There were no test-item related effects observed during the physical examination and arena observations.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

There was one unscheduled death. A Group 2 male, number 20, died under anaesthetic during blood sample collection for hematology and blood chemistry during Week 13. The reason for death is unknown as the animal was considered normal prior to induction of anaesthesia and no macroscopic abnormalities were seen at necropsy.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

After thirteen weeks of treatment, group mean body weight gain was statistically lower than control for both sexes exposed to 106 ppm (0.77X and 0.79X control, males and females respectively).
There were no test-item related effects on body weight gain for either sex exposed to 10.3 or 29.9 ppm.
After 6 weeks of recovery, group mean body weight gain was higher than control for males previously exposed to 106 ppm (1.36X control).

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There were no test item-related effects on food consumption after 13 weeks of treatment or 6 weeks of recovery.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There was a higher incidence of superficial opacities in males that received 106 ppm after 13 weeks of treatment, evaluation of animals in groups that received 29.9 or 10.3 ppm did not reveal a similar effect. This finding is considered incidental in absence of a similar effect in females or animals in the lower exposure levels.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related effects on haematology.
All differences from control were minor, lacked exposure relationship or were inconsistent between the sexes. Therefore, these were considered to be due to individual variation and unrelated to treatment.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related effects on blood chemistry.
All differences from control were minor, lacked exposure relationship or were inconsistent between the sexes. Therefore, these were considered to be due to individual variation and unrelated to treatment.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related effects.
Group mean hindlimb grip strength was higher than control for all treated groups (not exposure related), however all were within the range of the historical data therefore this was considered incidental. A small number of differences attained statistical significance, however these were isolated and are attributed to normal variation.

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related effects.
After 13 weeks of treatment mean adjusted ovary weights were lower than control for females exposed to 106 ppm (0.86X), however there was no exposure-related effect evident and statistical significance was not achieved.
All other differences from control were minor, lacked exposure relationship or were inconsistent between the sexes. Therefore, these were considered to be due to individual variation and unrelated to treatment.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Animals Killed After 13 Weeks of Treatment: The macroscopic examination performed after 13 weeks of treatment revealed no intergroup differences of note. The incidence and distribution of all findings were considered to be unrelated to treatment.

Animals Killed After 6 Weeks of Recovery: The macroscopic examination performed after 13 weeks of treatment and 6 weeks of recovery revealed no intergroup differences of note.
The incidence and distribution of all findings were considered to be unrelated to treatment.

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Animals Killed After 13 Weeks of Treatment: Changes related to treatment with the test item were seen in the nose/turbinates. Degeneration/atrophy of the olfactory epithelium mainly affecting the dorsal parts of the nasal vestibules was observed in animals receiving 29.9 or 106 ppm and was associated with loss of axon bundles in the sub adjacent lamina propria. Incidence and severity of these changes showed an exposure level response.

Animals Killed After 6 Weeks of Recovery: Minimal degeneration/atrophy of the olfactory epithelium associated with loss of axon bundles in the sub adjacent lamina propria was observed in animals previously exposed to 106 ppm. These changes were mainly distributed in the dorsal part of the nasal cavities.

Histopathological findings: neoplastic:

no effects observed

Details on results:

Estrus cycle: When compared with control, estrus cycles in females exposed to 106 ppm during Weeks 12 and 13 showed a higher number of irregular cycles, 5 versus 1; there were also two individuals exposed to 106 ppm that either had extended estrus or were determined to be acyclic. During the recovery period, there was one control female with an irregular cycle and extended estrus for two females previously exposed to 106 ppm.

T3/T4 analysis: There were no test item-related effects. All samples taken from all groups, including control, at termination and from control and animals previously exposed to 106 ppm at the end of the recovery phase showed T3 and T4 concentrations were consistent among groups.

Thyroid Stimulating Hormone analysis: Individual serum TSH concentrations were found to be variable. Group mean TSH concentrations were lower for males exposed to 10.3 ppm when compared with control. Group mean TSH concentrations for males exposed to 29.9 or 106 ppm were similar to control. There was a slight increase in TSH concentrations for males previously exposed to 106 ppm when compared with control. Females showed an increase in TSH concentrations with increasing concentration of Dimethylethylamine when compared with control. There was a slight decrease in TSH concentrations for females previously exposed to 106 ppm when compared with control.
Given the high degree of variability, lack of exposure related response in males and inconsistency between the sexes, it is considered the observed changes are likely to be a result of biological variation rather than a test-item related effect.
For information regarding Thyroid hormones measurements please see the attached full study report

Sperm Analysis: No adverse effects on sperm motility, testicular spermatid numbers, cauda epididymal sperm numbers or sperm morphology were observed following treatment with Dimethylethylamine compared with control.

Bronchoalveolar Lavage (BAL): There were no test item-related effects.
Group mean cell counts were variable when compared with control, however individual values for test animals were within the control range and therefore all differences were attributed to normal biological variation.

Total Protein and Lactate Dehydrogenase: Although group mean data may suggest lower total protein and lactate dehydrogenase concentrations in treated males and higher total protein and lactate dehydrogenase concentrations in females, there was a large degree of variation and overlap in individual data when comparing test data with control, so there is no convincing test item-related effect.
After 6 weeks of recovery, higher group mean total protein (up to 1.82X control) and lactate dehydrogenase concentrations (up to 1.64X control) were observed in both sexes exposed to 106 ppm when compared with control.

Dose descriptor:

NOAEC

Remarks:

nasal local effects

Effect level:

10.3 ppm (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Remarks on result:

other:

Remarks:

30 mg/m3

Dose descriptor:

NOAEC

Remarks:

systemic toxicity

Effect level:

>= 106 ppm (analytical)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Remarks:

318 mg/m3

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

106 ppm (analytical)

System:

respiratory system: upper respiratory tract

Organ:

nasal cavity

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

The test item, Dimethylethylamine (DMEA), was administered by snout-only inhalation administration to Wistar Han rats, for 6 hours a day, 5 days a week, for 12 weeks and for 7 days in Week 13 at achieved exposure levels of 10.3, 29.9 or 106 mg/L and was clinically well tolerated, recovery was assessed during a 6 week off-dose period. There were no test item-related deaths or effects on clinical signs, food consumption, sensory reactivity and grip strength or motor activity. There were also no effects on ophthalmoscopy, haematology, blood chemistry, thyroid hormone levels, sperm motility, bronchoalveolar lavage, organ weights or macroscopic pathology.

 

After 13 weeks of exposure to DMEA, test item-related histopathological changes were evident in the nasal turbinates of animals exposed to 29.9 or 106 ppm and consisted of minimal to moderatedegeneration/atrophy of the olfactory epithelium mainly affecting the dorsal parts of the nasal vestibules and was associated with loss of axon bundles in the sub adjacent lamina propria and was considered adverse at 106 ppm. This finding was only present at minimal severity in a proportion animals exposed to 29.9 ppm, 2 out of 10 males and 4 out of 10 females, compared with all animals being affected up to moderate severity at 106 ppm. After 6 weeks of recovery this finding was observed in 8 out of 10 males and 4 out of 10 females previously exposed to 106 ppm and the severity was reduced compared with animals killed after 13 weeks of exposure to DMEA, only achieving slight severity in 3 males, indicating that partial recovery had occurred following 6 weeks without exposure to the test item. 

 

Statistically Reduced body weight gain evident in both sexes exposed to 106 ppm at the end of the treatment phase was not accompanied by reduced food consumption. Body weight gain returned to similar values, or exceeded those, seen in control during the recovery period; therefore, in the absence of any histopathological correlate the reduced bodyweight gain is considered to be non-adverse.

 

A higher incidence of irregular estrus cycles, extended estrus or acyclic animals were apparent for females exposed to 106 ppm when compared with control; extended estrus was still evident for females previously exposed to 106 ppm during the recovery period. Irregular and extended cycles were also observed in the control group, albeit at a lower incidence; however in the absence of any relationship to exposure and the absence of any findings correlating with these observations, they are considered to be non-adverse.

Conclusions:

The test item, Dimethylethylamine (DMEA), was administered by snout-only inhalation administration to Wistar Han rats, for 6 hours a day, 5 days a week, for 12 weeks and for 7 days in Week 13 at achieved exposure levels of 10.3, 29.9 or 106 ppm and was clinically well tolerated, recovery was assessed during a 6 week off-dose period. There were no test item-related deaths or effects on clinical signs, food consumption, sensory reactivity and grip strength or motor activity. There were also no effects on ophthalmoscopy, haematology, blood chemistry, thyroid hormone levels, sperm motility, estrous cycle, bronchoalveolar lavage, organ weights or macroscopic pathology.
Test item-related changes were evident in the nasal turbinates of animals exposed to 29.9 or 106 ppm and consisted of minimal to moderate degeneration/atrophy of the olfactory epithelium mainly affecting the dorsal parts of the nasal vestibules and was associated with loss of axon bundles in the sub adjacent lamina propria. There was evidence of partial recovery after 6 weeks without exposure to DMEA; however, based on the incidence and severity these findings were considered adverse.
Reduced body weight gain evident at the end of the treatment period for both sexes exposed to 106 ppm resolved following 6 weeks of recovery.
Based on the findings in this study a No Observed Adverse Effects Concentration (NOAEC) for the local toxicity is considered to be 10.3 ppm based on the minimal to moderate nasal degeneration/atrophy of the olfactory epithelium recorded at 29.9 or 106 ppm and the NOAEC for systemic toxicity is considered to be 106 ppm.

Executive summary:

Four main groups of 10 male and 10 female Wistar Han rats each were exposed (nose-only) to target concentrations of 0 (control), 10, 30 or 100 ppm for 6 hours/day, 5 days/week over a 13-week period. Animals of the main groups were sacrificed on the day after the last exposure. In addition, two recovery groups, also consisting of 10 male and 10 female animals each, were simultaneously exposed with the main study animals to the control or 100 ppm test atmosphere, and were sacrificed after a 6-week recovery period following the last exposure.Animals received the air control, or the test item, Dimethylethylamine by inhalation for 13 weeks. Recovery animals were similarly treated for 13 weeks followed by a 6 week off dose period. During the study, clinical condition, detailed physical and arena observations, sensory reactivity, grip strength, motor activity, estrous cycle, body weight, food consumption, ophthalmoscopy, hematology (peripheral blood), blood chemistry, thyroid hormone (T3 and T4), thyroid hormone (TSH), organ weight, sperm analysis, bronchoalveolar lavage, macropathology and histopathology investigations were undertaken.

The mean achieved atmosphere concentrations were 10.3, 29.9 and 106 ppm (103, 100 and 106% of target) for Groups 2, 3 and 4, respectively.

Statistically significant test item-related reduced body weight gain was evident for both sexes exposed to 106 ppm (0.77X and 0.79X control, males and females respectively).  After 6 weeks of recovery, group mean body weight gain was higher than control for males previously exposed to 106 ppm (1.36X control).

Histopathological changes were evident in the nasal turbinates in animals that received 29.9 or 106 ppm.  Minimal to moderate degeneration/atrophy of the olfactory epithelium mainly affecting the dorsal parts of the nasal vestibules was observed in animals receiving 29.9 or 106 ppm and was associated with loss of axon bundles in the sub adjacent lamina propria.  Incidence and severity of these changes showed an exposure level response.  There was evidence of partial recovery following 6 weeks without exposure to DMEA.  No histopathological changes were evident in animals that received 10.3 ppm.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

30 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

Justification for classification or non-classification

According to CLP and GHS criteria, no classification is warranted for specific target organ toxicity after repeated exposure.