Ethyldiisopropylamine

Endpoint summary

Description of key information

A 28 days inhalation study is available for EDIPA (Covance, 2021)

Two additionnal supportive 28 days inhalation GLP and OECD 412 studies are available for the analogues DMIPA and DMEA. In these three 28 days inhalation studies, the lead effect was local toxicity with no specific systemic toxicity as indicated in the robust study summaries.

No 90 days inhalation toxicity study is available for EDIPA, however a 90 days toxicity by inhlation study is available for DMEA (Beebe, 2021). This study is used as read across to fulfill the 90 subchronic endpoint (see read across justification document).

A two weeks by oral route was performed with EDIPA as preliminary study to OECD 414 in rats.

1) 7-Days inhalation study EDIPA(Rogers, 2021):

The test item, N,N-Diisopropylethylamine (EDIPA), was administered by nose-only inhalation administration to Wistar Han rats, for up to 6 hours a day, for up to 5 days a week, for 1 week at achieved exposure levels of 9.78, 49.7, 100, 156 or 231 ppm resulted in test item-related deaths at 156 or 231 ppm, which resulted in cessation of exposures at these levels after four days or one day respectively, however, exposure to EDIPA was generally well tolerated at the lower exposure levels, 100 ppm or below. 

Histopathological changes related to treatment with EDIPA were seen in the nose (inflammation, vacuolation/degeneration of the olfactory, respiratory and glandular epithelium and ulceration of the respiratory epithelium), larynx (inflammation, vacuolation/degeneration of the respiratory and glandular epithelium, and ulceration of the respiratory epithelium) and trachea (inflammation and vacuolation/degeneration of the respiratory epithelium) in decedents exposed to 156 or 231 ppm.

In animals killed after 7 days (9.78, 49.7 or 100ppm), 4 days (156 ppm) or 1 day (231 ppm) of treatment, changes were seen in the nose (inflammation and vacuolation/degeneration of the olfactory epithelium) in animals exposed to 100, 156 or 231 ppm, and in the trachea in (vacuolation/degeneration of the respiratory epithelium) in animals exposed to 231 ppm.
All these changes were more focal and less severe than in decedent animals.

 

Exposure to EDIPA at 156 or 231 ppm resulted in marked clinical signs in the majority of the animals and contributed to the deterioration in the clinical condition of some individuals which necessitated early termination, it is therefore considered these exposure levels are unsuitable for selection for the following 4-week inhalation study with EDIPA.  Body weight loss in males and reduced body weight gain in females exposed to 100 ppm during the treatment period together with the degenerative histopathological changes in the nose seen at this exposure level also precludes its selection for use in the 4-week inhalation study with EDIPA.

 

Based on the results of this study exposure levels below 100 ppm should be considered for selection in the 4-week inhalation study with EDIPA.

2) 28 days inhalation study EDIPA (Rogers, 2021):

The test item N,N-Diisopropylethylamine (EDIPA) was administered by snout-only inhalation administration to Wistar Han rats, for 6 hours a day, 5 days a week, for 4 weeks at achieved exposure levels of 10.8, 37.3 and 77.8 ppm and was clinically well tolerated, recovery was assessed during a 4 week off-dose period.

There were no test item-related decedents on the study with minimal effects on clinical signs, body weight, and organ weights with recovery seen after 4 weeks without exposure. No effects were seen on food consumption, ophthalmoscopy, hematology and blood chemistry, bronchoalveolar lavage, sperm analysis or macroscopic pathology and the variations seen in the estrous cycles were questionable due to the lack of relevant historical control data, and the absence of correlation with any relevant changes in organ weights or microscopic findings in the ovaries and uterus as well as comparable estrous profiles observed between treated groups and controls at the end of recovery period.

Test-item related changes were evident in the nasal turbinates of animals exposed to 37.3 or 77.8 ppm and consisted of minimal to slight vacuolation/atrophy of the respiratory epithelium. Minimal to slight vacuolation/atrophy of the olfactory epithelium was also seen in the majority of animals exposed to 77.8 ppm. There was full recovery after 4 weeks without exposure to EDIPA in the respiratory epithelium and near complete recovery in the olfactory epithelium and therefore these findings were considered to be non-adverse.

The No Observed Adverse Effect Concentration for systemic and local toxicity is considered to be 77.8 ppm based on the non-adverse histopathology findings in the nasal turbinates showing recovery after 4 weeks without exposure and the effects seen on clinical signs, body weight or organ weights which recovered at the end.

3) 90-day toxicity study by inhalation with analogue DMEA (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.

 

Two weeks oral toxicity study with EDIPA (Covance, 2021)

A two weeks oral stuy was performed with EDIPA as a preliminary study to set the dose-levels for the range finding embryofetal developmental study in rats by oral route.

The purpose of this study was to assess the systemic toxic potential of Ethyldiisopropylamine, in a 14 day oral gavage study in Crl:CD(SD) rats and to aid in the selection of suitable dose levels for future repeat dose reprotoxicity studies by oral route.

Three groups, each comprising two female Crl:CD(SD) rats, received Ethyldiisopropylamine at doses of 25, 75 or 150 mg/kg/day. A similarly constituted control group received the vehicle, 1% w/v methylcellulose plus 0.1% Tween 80, at the same volume dose as control groups.

During the study clinical condition, body weight, food consumption and macropathology investigations were undertaken.

Results

From Day 1 to 3 of study, females that received 150 mg/kg/day showed signs comprised of elevated gait, decreased activity, piloerection, closed and/or partially closed eyelids, hunched posture and chromodacryorrhea. Subsequently, this group was entirely prematurely killed on the grounds of animal welfare on Day 3 of study. Macroscopic examination of these females revealed no abnormalities.

Females that received 75 mg/kg/day showed signs of a lesser severity and comprised partially closed eyelids from Day 1 to 13 of treatment and for 1 female, unsteady gait on Day 1 of treatment only.

There were no signs recorded for females receiving 25 mg/kg/day.

Body weight gain from Day 1 to 15 of study was not clearly affected by treatment at 25 or 75 mg/kg/day.

There was a suggestion of slightly reduced food intake from Day 1 to 3 of treatment for females that received 75 mg/kg/day. Food intake was not clearly affected by treatment for females that received 25 mg/kg/day.

There were no macroscopic findings after 14 days of treatment for females that received 25 or 75 mg/kg/day.

Considering the adverse clinical signs observed at 150 mg/kg/day and clinical signs observed at 75 mg/kg/day, it was concluded that the selection of 100 mg/kg/day is a suitable high dose for a subsequent preliminary study for effects on embryo-fetal development (Covance Study No. 8434891).

Key value for chemical safety assessment

Toxic effect type:

concentration-driven

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

Experimental start date (Animal arrival) 19 February 2020
Experimental completion date (Necropsy) 11 March 2020

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

equivalent or similar to guideline

Guideline:

other: The Acute oral toxicity of Ethyldiisopropylamine (EDIPA) was evaluated in rats according to a protocol similar to the OECD 423 guideline (Acute Oral Toxicity - Acute Toxic Class Method, 1997)

GLP compliance:

no

Remarks:

The study was not designed to meet any particular regulatory requirements. No claim for compliance with Good Laboratory Practice was made, although the work performed generally followed Good Laboratory Practice principles.

Limit test:

no

Species:

rat

Strain:

Wistar

Details on species / strain selection:

The rat (virgin) 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:CD (SD) rat strain was used because it is accepted by regulatory agencies and because of the historical control data available at this laboratory

Sex:

female

Details on test animals or test system and environmental conditions:

Strain/Species Crl:CD(SD) rat.

Supplier Charles River (UK) Ltd.

Number of animals 10 females.

Spare animals were removed from the study room after treatment commenced.

Duration of acclimatization Seven days before commencement of treatment.

Age of animals at start of treatment 72 days.

Weight range of animals at the start of treatment 206 to 266 g

Allocation and Identification
Allocation Randomly allocated on arrival.

Using the sequence of cages in the battery, one animal at a time was placed in each cage with the procedure being repeated until each cage held the appropriate number of animals.

Identification of animals Each animal was assigned a number and identified uniquely within the study by a microchip inserted shortly after arrival.

Identification of cages Each cage label was color-coded according to group and was numbered uniquely with cage and study number, as well as the identity of the occupants.

Animal Replacement
Before the start of treatment checks upon health and body weight were performed. All
animals were considered acceptable for use upon the study and so no replacements were
performed.

Animal Care and Husbandry
Environmental Control
Animal facility Limited access - to minimize entry of external biological and chemical agents and to minimize the transference of such agents between rooms.

Air supply Filtered fresh air which was passed to atmosphere and not recirculated.

Temperature and relative humidity Monitored and maintained within the range of 20-24¿C and 40-70%.

There were no deviations from these ranges.

Lighting Artificial lighting, 12 hours light: 12 hours dark.

Electricity supply Public supply with automatic stand-by generators.

Animal Accommodation
Cages Polycarbonate body with a stainless steel mesh lid, changed at appropriate intervals.

Cage distribution The cages constituting each group were dispersed in batteries so that possible environmental influences arising from their spatial distribution were equilibrated, as far as was practicable.

Number of animals per cage Up to two of the same sex, unless reduced by mortality.

Bedding Wood based bedding which was changed at appropriate intervals each week.

Environmental Enrichment
Aspen gnawing material Provided to each cage throughout the study and replaced when necessary.

Plastic shelter Provided to each cage throughout the study and replaced when necessary.

Diet Supply
Diet SDS VRF1 Certified, pelleted diet.

Availability Non-restricted

Water Supply
Supply Potable water from the public supply via polycarbonate bottles with sipper tubes. Bottles were changed at appropriate intervals.

Availability Non-restricted.

Supplier Certificates of Analysis
Certificates of analysis for the diet were scrutinized and approved before any batch of diet was released for use. Certificates of analysis are routinely provided by the water supplier.
Certificates of analysis were also received from the suppliers of the wood based bedding and Aspen gnawing material.

No specific contaminants were known that may have interfered with or prejudiced the outcome of the study and therefore no special assays were performed.

Route of administration:

oral: gavage

Details on route of administration:

The oral gavage route of administration was chosen to simulate the conditions of possible human exposure.

Vehicle:

methylcellulose

Remarks:

1% w/v methylcellulose plus 0.1% Tween 80

Details on oral exposure:

Method of preparation
The required amount of test item was ground in a mortar using a pestle and mixed with some vehicle to form a paste. Further amounts of vehicle were gradually added and mixed to produce a smooth, pourable suspension. The suspension was quantitatively transferred and diluted to volume and finally mixed using a high-shear homogenizer.

A series of suspensions at the required concentrations were prepared by dilution of individual weightings of the test item.

The pH of formulations were adjusted with aqueous HCl to pH 8.0

Frequency of preparation Daily.

Storage of formulation Refrigerated (2 to 8°C) for up to 24 hours and ambient (15 to 25°C) for two hours.

Test item accounting Detailed records of compound usage were maintained. The amount of test item necessary to prepare the formulations and the amount actually used were determined on each occasion. The difference between these amounts was checked before the formulations were dispensed.

Formulation Analysis
Stability and homogeneity The homogeneity and stability of formulations in the concentration range of 5 to 200 mg/mL during ambient and refrigerated storage were confirmed as part of another study, Covance Study No. LB21BK.

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

14 days

Frequency of treatment:

Once daily

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Control only

Dose / conc.:

25 mg/kg bw/day (nominal)

Dose / conc.:

75 mg/kg bw/day (nominal)

Dose / conc.:

150 mg/kg bw/day (nominal)

No. of animals per sex per dose:

2 females

Control animals:

yes, concurrent vehicle

Details on study design:

Purpose
The purpose of this study was to assess the systemic toxic potential of Ethyldiisopropylamine, an industrial chemical, in a 14 day oral gavage study in Crl:CD(SD) rats and to aid in the selection of suitable dose levels for future repeat dose studies.

Rationale for Dose Level Selection
The doses used in this study (0, 25, 75 and 150 mg/kg/day) were selected in conjunction with the Sponsor.

The Acute oral toxicity of Ethyldiisopropylamine (EDIPA) was evaluated in rats according to a protocol similar to the OECD 423 guideline (Acute Oral Toxicity - Acute Toxic Class Method, 1997) (ECHA registration dossier). Groups of three female Wistar rats were given a single oral dose of EDIPA using dose steps of 2000, 500 and 200 mg/kg bw, with three males also receiving 200 mg/kg bw. Following treatment, rats were observed daily for clinical sings and weighed weekly. A gross necropsy examination was performed at the time of euthanasia (Day 14). 3/3 females died within one hour of receiving EDIPA at 2000 mg/kg bw. Animals showed slight hyperemia of the glandular stomach and moderate erythema of the small intestine. 1/3 females died within one hour of receiving EDIPA at 500 mg/kg bw, the remaining two females died within three hours. Animals showed slight thickening of the fore stomach and glandular stomach walls. No death was observed at the dose of 200 mg/kg bw. Clinical symptoms were observed (poor general state, dysponea, apathy, staggering, tremor, twitching and piloerection) at 500 mg/kg bw up to hour two and at 200 mg/kg bw in all males up to Day 2 and all females between three and five hours post administration.

Observations and examinations performed and frequency:

Serial Observations
Clinical Observations
Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of animal ill-health amongst the occupants. Any deviation from normal was recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as appropriate.

During the acclimatization period, observations of the animals and their cages were recorded at least once per day.

Signs Associated with Dosing

Detailed observations were recorded daily at the following times in relation to dose administration:

• Pre-dose
• Approximately 30 minutes after dosing#
• One to two hours after dosing
• Three to four hours after dosing#
• As late as possible in the working day

# - Additional dose observations were observed.

Clinical Signs
A detailed physical examination was performed on each animal to monitor general health, for three days prior to the start of treatment and daily throughout the study.

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.

Body Weight
The animals were weighted daily for three days prior to the start of treatment, on the day that treatment commenced (Day 1), daily throughout the study and before necropsy.

Food Consumption
The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded for three days prior to the start of treatment and daily throughout the study.

Water Consumption
Fluid intake was assessed by daily visual observation. No effect was observed and consequently quantitative measurements were not performed.

Sacrifice and pathology:

Necropsy
All 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 and abnormalities retained and fixed. All animals, thoracic and abdominal cavities opened, cranial cavity opened only if observations indicate possible neurotoxic action. Special attention to be paid to stomach tissue and the required tissue samples preserved in appropriate fixative.

The retained tissues were checked before disposal of the carcass.

Schedule Animals were killed following 14 days of treatment.

Sequence To allow satisfactory inter-group comparison.

Fixation
Tissues were routinely preserved in 10% Neutral Buffered Formalin.

Statistics:

No statistical analysis was performed on this study.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Between Days 1 to 3 of treatment, female’s No. 7 and 8 that received 150 mg/kg/day showed signs associated with the administration of dose comprising elevated gait, decreased activity, piloerection, closed and/or partially closed eyelids, hunched posture and chromodacryorrhea. Given the persistence of these observations, female’s No. 7 and 8 were prematurely euthanized without being dosed on Day 3 of treatment on animal welfare grounds. From Day 1 to 3 of treatment, female No. 7 had lost a total 8g body weight and female No. 8 had lost a total 12g body weight. Furthermore, food consumption for these animals was markedly reduced when compared to the Control group and 25 or 75 mg/kg/day groups. At the macroscopic examination, there were no abnormalities detected in either female No. 7 or 8.

Between Days 1 to 13 of treatment, female’s No. 5 and 6 receiving 75 mg/kg/day showed signs at the 1 to 2 hour and late in the working day post-dose observation comprising partially closed eyelids. Furthermore, female No. 5 was observed to have unsteady gait on Day 1 of treatment only.

There were no signs recorded for females receiving 25 mg/kg/day.

PLEASE REFER TO THE ATTACHED TABLE: "CLINICAL SIGNS - GROUP DISTRIBUTION OF OBSERVATIONS"

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

150 mg/kg/day : From Day 1 to 3 of treatment, female No. 7 had lost a total 8g body weight and female No. 8 had lost a total 12g body weight.

From Day 1 to 15 of study, there was no clear treatment related effect on body weight change for females receiving 25 or 75 mg/kg/day.

PLEASE REFER TO THE ATTACHED TABLE: "BODY WEIGHT - GROUP MEAN VALUES"

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

150 mg/kg/day: Female No. 7 and female No. 8 - food consumption for these animals was markedly reduced when compared to the Control group and 25 or 75 mg/kg/day groups.

There was a suggestion of a slight reduction in food consumption for females receiving 75 mg/kg/day on Days 1 to 3 of treatment, from Days 3 to 15 of study, there was no clear treatment related effect on food consumption.

There was no clear treatment related effect on food consumption for females receiving 25 mg/kg/day on Days 1 to 15 of study.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no macroscopic abnormalities detected at scheduled termination on Day 15 of study.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Dose descriptor:

other: Considering the adverse clinical signs observed at 150 mg/kg/day and clinical signs observed at 75 mg/kg/day, it was concluded that the selection of 100 mg/kg/day is a suitable high dose for a subsequent preliminary study for effects on embryo-fetal devel

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Critical effects observed:

no

Conclusions:

Considering the adverse clinical signs observed at 150 mg/kg/day and clinical signs observed at 75 mg/kg/day, it was concluded that the selection of 100 mg/kg/day is a suitable high dose for a subsequent preliminary study for effects on embryo-fetal development (Covance Study No. 8434891).

Executive summary:

SUMMARY

The purpose of this study was to assess the systemic toxic potential of Ethyldiisopropylamine, an industrial chemical, in a 14 day oral gavage study in Crl:CD(SD) rats and to aid in the selection of suitable dose levels for future repeat dose studies.

Three groups, each comprising two female Crl:CD(SD) rats, received Ethyldiisopropylamine at doses of 25, 75 or 150 mg/kg/day. A similarly constituted control group received the vehicle, 1% w/v methylcellulose plus 0.1% Tween 80, at the same volume dose as control groups.

 

During the study clinical condition, body weight, food consumption and macropathology investigations were undertaken.

Results

From Day 1 to 3 of study, females that received 150 mg/kg/day showed signs comprised of elevated gait, decreased activity, piloerection, closed and/or partially closed eyelids, hunched posture and chromodacryorrhea. Subsequently, this group was entirely prematurely killed on the grounds of animal welfare on Day 3 of study. Macroscopic examination of these females revealed no abnormalities.

Females that received 75 mg/kg/day showed signs of a lesser severity and comprised partially closed eyelids from Day 1 to 13 of treatment and for 1 female, unsteady gait on Day 1 of treatment only.

There were no signs recorded for females receiving 25 mg/kg/day.

Body weight gain from Day 1 to 15 of study was not clearly affected by treatment at 25 or 75 mg/kg/day.

There was a suggestion of slightly reduced food intake from Day 1 to 3 of treatment for females that received 75 mg/kg/day. Food intake was not clearly affected by treatment for females that received 25 mg/kg/day.

There were no macroscopic findings after 14 days of treatment for females that received 25 or 75 mg/kg/day.

Conclusion

Considering the adverse clinical signs observed at 150 mg/kg/day and clinical signs observed at 75 mg/kg/day, it was concluded that the selection of 100 mg/kg/day is a suitable high dose for a subsequent preliminary study for effects on embryo-fetal development (Covance Study No. 8434891).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LOAEL

75 mg/kg bw/day

Study duration:

subacute

Species:

rat

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

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:

no adverse effect observed

Dose descriptor:

NOAEC

318 mg/m³

Study duration:

subchronic

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:

no 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

Additional information

 

 

 

Justification for classification or non-classification

No classification for repeated dose toxicity is warranted according to REGULATION (EC) No 1272-2008 and Annex VI of Commission Directive 2001/59/EC.