Registration Dossier

Toxicological information

Repeated dose toxicity: inhalation

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Administrative data

Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
08 November 2018 - 01 July 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.29 (Sub-Chronic Inhalation Toxicity:90-Day Study)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
Test item: Dimethylethylamine
Test item identity (including alternative names): DMEA
CAS number: 598-56-1
EC number: 209-940-8
Intended use: Industrial chemical
Appearance: Colorless liquid
Storage conditions: In a refrigerator (2 to 8°C), desiccated, in the dark.
Supplier: Sponsor
Batch number: SAMP160321
Stability/expiry date: 17 April 2019
Purity: 99.33%
Supplier’s responsibilities: Characterization of the test item and the documentation of the methods of synthesis, fabrication or derivation and stability.
Archive sample: A 0.5 g representative sample of the test item. This sample was placed in a well closed glass container and stored in the archives under the same conditions as the bulk material.
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.

Test animals

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 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

Administration / exposure

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.
Doses / concentrationsopen allclose all
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.

Examinations

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.



Results and discussion

Results of examinations

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 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.



Effect levels

open allclose all
Dose descriptor:
NOAEC
Remarks:
nasal local effects
Effect level:
29.9 ppm (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Remarks on result:
other:
Remarks:
89.7 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

Target system / organ toxicity

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

Any other information on results incl. tables

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. Considering the reduction in incidence and severity seen at 106 ppm after 6 weeks of recovery, it is highly likely the minimal findings seen in a small number of animals following exposure to 29.9 ppm at the end of the treatment phase would have resolved once exposure to DMEA had stopped, and therefore the findings at 29.9 ppm are considered non-adverse.

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.

Applicant's summary and conclusion

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 mg/L and was clinically well tolerated, recovery was assessed during a 6 week off-dose period.
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, the incidence and severity of findings in animals exposed to 106 ppm was considered adverse.
Based on the findings in this study a No Observed Adverse Effects Concentration (NOAEC) is considered to be 29.9 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.

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.

Body weight gainwas lower than control 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. 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 after 6 weeks without exposure to DMEA; however, the incidence and severity of findings in animals exposed to 106 ppm was considered adverse.

Based on the findings in this study a No Observed Adverse Effects Concentration (NOAEC) for systemic toxicity was considered to be equal or higher than 106 ppm and the NOAEC for local (nasal) effect to be 29.9 ppm.