4-morpholinecarbaldehyde

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24.06.2016 - 07.04.2017

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3465 (90-Day Inhalation Toxicity)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

4-morpholinecarbaldehyde

EC Number:

224-518-3

EC Name:

4-morpholinecarbaldehyde

Cas Number:

4394-85-8

Molecular formula:

C5H9NO2

IUPAC Name:

morpholine-4-carbaldehyde

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 92076288Q0
- Expiration date of the lot/batch: 21 Jan 2018
- Purity: 99,707%


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: the stability of the test substance under storage conditions over the test period was guaranteed by the sponsor.
- Storage conditions: Room temperature
- Physical state / appearance: Liquid / colorless, clear

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

The rat is the most frequently used laboratory animal, and there is extensive experience with this species. The rat is also proposed as a suitable test animal by OECD and the EPA. The Wistar strain is selected because a huge amount of historical control data are available for this strain.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH; Sandhofer Weg 7, 97633 Sulzfeld
- Age at study initiation: supply: 7 weeks; start of pre-exposure 8 weeks; start of exposure 9 weeks
- Housing:Type of cage / No. of animals per cage: Typ 2000P: ca. 2065 cm2 (polysulfone cages) supplied by TECNIPLAST, Germany / up to 5 animals
Dust-free wooden bedding
- Diet: Kliba laboratory diet, mouse/rat maintenance “GLP”, 12 mm pellets, Provimi Kliba SA, Kaiseraugst, Basel Switzerland
- Water: ad libitum
- Acclimation period: During the acclimatization period the animals are accustomed to the surroundings of the study and to the diet.

DETAILS OF FOOD AND WATER QUALITY:

ENVIRONMENTAL CONDITIONS
- Temperature 20 - 24°C
- Humidity 30-70%
- Air changes 15 changes per hr
- Photoperiod:06.00 a.m. - 06.00 p.m. light, 06.00 p.m. - 06.00 a.m. dark

Administration / exposure

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

air

Mass median aerodynamic diameter (MMAD):

>= 1.9 - <= 3.11 µm

Details on inhalation exposure:

During exposure of the animals, exposure mixtures (inhalation atmospheres: test substance in air) will be generated continuously in such a way that they are as homogeneous and of as constant a composition as possible.

Equipment:
Piston metering pumps KP 2000 (DESAGA; SARSTED AG & Co, Nürnbrecht, Germany)
Two-component atomizers (stainless steel, Model 970; Düsen-Schlick GmbH, Untersiemau/Coburg, Germany)

Generation technique:
For each concentration, a respective constant amount of the test substance will be supplied to a two-componentatomizer by means of a metering pump and sprayed with compressed air. The so generated aerosol will be mixed with conditioned air and passed into the inhalation system.

Exposure systems
The following exposure systems with the specific technical conditions will be used: Nose-only inhalation systems:
The test atmospheres are passed into the aerodynamic exposure apparatuses with the supply air. The rats are restrained in exposure tubes, their snouts projecting into the inhalation chamber to inhale the atmosphere.
The exhaust air system connected to the exposure systems is adjusted in such a way that the amount of exhaust air is lower than the supply air (positive pressure). Thus the test atmosphere is not diluted with laboratory air in the breathing zones of the animals.

Measurement and recording of technical conditions in the exposure systems
In general, the technical parameters will be measured and recorded as follows:
The air flow rates of supply and exhaust air, relative humidities and temperatures in the inhalation systems will be measured continuously by an automated measuring system and will be monitored against preset limits and partially regulated.
All these parameters are recorded continuously by an computerized data acquisition and control system BaseLab (BASF SE, Ludwigshafen, Germany) as analog signals (between 0 or 4 and 20 mA), converted into digital data every 10 seconds, transferred to a personal computer and displayed on the screen. The computer (Baselab-Software, BASF SE, Ludwigshafen, Germany) checks the incoming values against the preset threshold values, gives warnings if violations of these values occur and record the start and the end of the violation. Daily protocols are prepared from the values measured every 10 seconds using Microsoft Excel. lf values above or below the preset limits occur for longer than 5 minutes, values will be printed and documented in the printed daily records. The digital data and the printed daily records are considered as raw data. The systems and software are validated in house.
The pump rate of the dosing pumps are read and recorded once per exposure. The atomizer pressure is measured continuously by manometers and recorded once per exposure.

Analysis of the inhalation atmosphere
The analytical determination of the concentrations of the inhalation atmospheres will be performed in the Inhalation Laboratory and in the Laboratory for Chemical Analysis of Experimental Toxicology and Ecology of BASF SE. The results are summarized in the final report.

Nominal concentration
The nominal concentration of the inhalation atmospheres will be calculated from the amounts of test substance dosed and air-flow per unit time.

Particle size analysis by means of cascade impactor
To determine particle size distribution, the cascade impactor is assembled with metal collecting discs. Metal collecting discs are eluted individually to determine the amounts ot test substance deposited behind the various impactor stages. The test substance will be determined using the gas chromatographic method mentioned above. The amount of material adsorbed in the sampling probe and on the walls ot the impactor (wall losses) will also be determined quantitatively.
The distribution ot masses deposited on the stages ot the meta! collecting disc are used to calculate MMAD and GSD. The evaluations ot the particle size distribution follow the recommendation ot DIN 66141 and DIN 66161.
Because low liquid aerosol concentration is expected to be low in test group 1, cascade impactor measurement will be performed once weekly in test groups 2 and 3 during the first four weeks. lt comparable data of particle size distribution can be provided by APS during this time period, cascade impactor measurement will be stopped after four weeks. Particle size measurements will be performed by APS once weekly per concentration. lt the data of these two devices differ significantly, APS measurement will be stopped, cascade impactor measurement will be continued once weekly per concentration.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The aerosol generation effectiveness was as expected for these high concentrations (46.2 64.9 %).
Real time surveillance of the inhalation atmospheres with scattered light photometers generally proved the constancy of each concentration throughout the daily exposures. Examples of recorder protocols are depicted in the figure below (original protocols are archived with the raw data). Short and transient variations occurred in all concentration groups on very few days. Due to their extremely short duration, they were not considered relevant. Details were archived with the raw data.
The air flows were constantly maintained in the desired range. An air change of about 67 times per hour can be calculated by dividing the supply air flow through the volume of each inhalation system.
The daily mean relative humidities in the inhalation systems ranged between 45.4 and 71.1 %.
Mean temperatures in the inhalation systems ranged between 20.6 and 23.9°C. All these values were within the range suggested by the respective testing guidelines.

Cascade impactor measurements showed MMADs between 1.90 and 3.28 μm with GSDs between 1.85 to 3.51. APS measurements showed generally larger MMAD than those of the cascade impactor measurements.

Duration of treatment / exposure:

6 hours; 65 exposures

Frequency of treatment:

on each workday

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/m³ air

Remarks:

Control

Doses / concentrations 2

Dose / conc.:

100 mg/m³ air (nominal)

Remarks:

Mean 105.7 +- 20.7 SD
MMAD not measured

Doses / concentrations 3

Dose / conc.:

300 mg/m³ air (nominal)

Remarks:

Mean 309.1 +- 44.8 SD
2.36 - 3.07 µm MMAD

Doses / concentrations 4

Dose / conc.:

1 000 mg/m³ air (nominal)

Remarks:

Mean 1035.1 +- 106.7 SD
1.90 - 3.11 µm MMAD

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Examinations

Observations and examinations performed and frequency:

Mortality
A check for moribund or dead animals will be carried out twice per day on working days and once per day on weekends and holidays.

Symptoms
A clinical observation will be performed on each animal at least three times on exposure days and once a day during pre-exposure and post exposure observation days. Signs and findings are recorded for each animal. During exposure only a group wise examination is possible.

Body weight
The animals will be weighed prior to the pre-exposure period, at the start of the exposure period (day 0) and twice weekly thereafter until one day prior to gross necropsy.
The body weight change of the respective week will be calculated as the difference of Friday to the previous Monday.

Food consumption
Food consumption will be determined weekly (e.g. Monday-Friday) and calculated as mean food consumption in grams per animal and day.

Ophthalmology
Before the beginning of administration, the eyes of all animals will be examined with an ophthalmoscope (HEINE OPTOTECHNIK, Herrsching, Germany) after administration of a mydriatic (Mydrum, Firma Chauvin ankerpharm GmbH, Rudolstadt, Germany). Against the end of the exposure period, the eyes of all animals of the control and high concentration group will be examined. The eyes of the animals of the other animals will be examined only if there is a striking discrepancy between the high concentration group and the control group.

Detailed clinical observations
All animals will be subjected to detailed clinical observations outside their cages once before the beginning of the administration period (day 0) and subsequently once on study day 42 and once on study day 84, generally in the morning. For observation, the animals will therefore be removed from their cages and placed in a standard arena (50 x 37.5 x 25 cm). The scope of examinations and the scoring of the findings that are observed will be based on the current index of findings in PDS ToxData® software and includes but is not limited to the following parameters listed:
Abnormal behavior in handling, Fur, Skin, Posture, Salivation, Respiration, Activity/ arousal level. Tremors, Convulsions, Abnormal movements, Gait abnormalities, Lacrimation, Palpebral closure, Exophthalmos, Assessment of the feces discharged during the examination (appearance/ consistency), Assessment of the urine discharged during the examination, Pupil size.

Functional observational battery (FOB)
The functional observational battery (FOB) will be carried out in the first 5 male and 5 female animals for each test group on study day 77. On the day of FOB the examined animals as weil as the remaining animals of each test group are not exposed to test substance. The examinations will generally start in the morning.
At least one hour before the start of the FOB the animals will be transferred to single animal polycarbonate cages (floor area about 800 cm2). Drinking water will be provided ad libitum, but no food will be offered during the measurements.
The FOB will start with passive observations without disturbing the animals, followed by removal from the home cage, open field observations in a standard arena and sensory motor tests as weil as reflex tests. The findings will be ranked according to the degree of severity, if applicable. The observations will be performed at random.

Horne cage observation
The animals will be observed in the rack for a short period (about 10-30 seconds) in their cages with the lids closed; during this period disturbing influences (touching of the cage and loud noises) should be avoided. Besides other abnormalities, particularly the following parameters will be observed:
Posture, Tremors, Convulsions, Abnormal movements, Gait abnormalities, Other findings

Open field observation
For observation, the animals will be removed from their cages by the investigator and placed in a standard arena (50 x 50 x 25 cm). Besides other abnormalities, the following parameters listed will be assessed:
Behavior on removal from the cage, Fur, Skin, Salivation, Nasal discharge, Lacrimation, Eyes/pupil size, Posture, Palpebral closure, Respiration, Tremors, Convulsions, Abnormal movements/stereotypes, Gait, Activity/arousal level, Feces excreted within 2 minutes, Urine excreted within 2 minutes, Rearing within 2 minutes, Other findings

Sensory motor tests / Reflex tests
The animals will be removed from the open field and will be subjected to the sensory motor and reflex tests listed below.
Reaction to an object being moved towards the face (Approach response), Touch sensitivity (Touch response), ision (Visual placing response), Pupillary reflex, Pinna reflex, Audition (Auditory startle response), Coordiantion of movements (Righting response), Behavior during handling, Vocalization, Pain perception (Tail pinch)
Other findings, Grip strength of forelimbs and hindlimbs, Landing foot-splay test


Measurement of motor activity (MA)
Motor activity (MA) will be measured from 14:00 h onwards on the same day as the FOB will be performed (in a randomized sequence on each examination day). The MA will be carried out in 5 male and 5 female animals. On the day of MA the examined animals as well as the remaining animals of each test group are not exposed.
The examinations will be performed using the TSE Labmaster System supplied by TSE Systems GmbH, Bad Homburg, Germany. For this purpose, the animals will be placed in new clean polycarbonate cages with a small amount of bedding for the duration of the measurement. Eighteen beams will be allocated per cage. The number of beam interrupts will be counted over 12 intervals for 5 minutes per interval. The sequence in which the animals will be placed in the cages will be selected at random. On account of the time needed to place the animals in the cages, the starting time will be "staggered" for each animal. The measurementperiod will begin when the 1st beam will be interrupted and will finish exactly 1 hour later. No food or water will be offered to the animals during these measurements and the measurement room will be darkened after the transfer of the last animal. The program requires a file name for the measured data to be stored. This name consists of the reference number and a serial number.

Estrous cycle determination
Vaginal smears for cycle determination will be prepared in the morning and evaluated according to the timetable for at least 3 weeks. The samples will be disposed after examination.

Sacrifice and pathology:

Organ weights:

anesthetized animals, adrenal glands, brain, cauda epididymis, epididymides, heart, kidneys, liver, ovaries, prostate, seminal vesicles incl. coagulating glands, spleen, testes, thymus, thyroid glands, uterus

Organ / tissue fixation
all gross lesions, adrenal glands, aorta, bone marrow (femur), brain, cecum, cervix, coagulating gland, colon, duodenum, epididymis, left (modified Davidson’s solution), esophagus, extraorbital lacrimal glands, eyes with optic nerve (modified Davidson’s solution), femur with knee joint, harderian glands, heart, ileum, jejunum, kidneys, larynx, liver, lungs, lymph nodes, mammary gland (male and female), nose (nasal cavity), ovaries, pancreas, parathyroid glands, pharynx, pituitary gland, prostate, rectum, salivary glands (mandibular and sublingual glands), sciatic nerve, seminal vesicles, skeletal muscle, skin, spinal cord (cervical, thoracic and lumbar cord), spleen, sternum with marrow, stomach (forestomach and glandular stomach), teeth, testis, left (modified Davidson’s solution), thymus, thyroid glands, tongue, trachea, urinary bladder, uterus, ureter, urethra

Other examinations:

Haematology:
leukocyte count (WBC), erythrocyte count (RBC), hemoglobin (HCT), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelet count (PLT), differential blood count, reticulocytes (RET), prothrombin time (Hepato Quick´s test) (HQT), preparation of blood smears

Clinical chemistry:
alanine aminotransferase (ALT), aspartate aminotransferase (AST), Alkaline phosphate (ALP), γ-Glutamyltransverase (GGT), sodium (Na), potassium (K), Chloride (Cl), inorganic phosphate (INP), calcium (Ca), urea (UREA), creatinine; glucose (GLUC), total bilirubin (TBIL), total protein (TPROT), albumin (ALB), globulins (GLOB), triglycerides (TRIG), cholesterol (CHOL).

Sperm parameters
lmmediately after necropsy and organ weight determination the right testis and cauda epididymis will be taken from all male animals.

Sperm motility (cauda epididymis)
Sperm morphology (cauda epididymis)
Sperm head count (cauda epididymis)
Sperm head count (testis)

Sperm motility examinations will be carried out immediately after necropsy, in a randomized sequence. For sperm head count, the right testis and right cauda epididymis will be deep frozen at -20°C until evaluation. At first, sperm head count and sperm morphology will be evaluated in controls and the high dose group, only. lf alterations occur the other test groups will also be examined

Statistics:

Body weights, body weight change , Food consumption: DUNNET
Feces, rearing, grip strength fore and hindlimbs, foot-splay test, motor activity, estrous cycle; clinical pathology parameters: KRUSKAL-WALLIS and WILCOXON Test

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

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

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Immunological findings:

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test group 3 (1000 mg/m³):
• Minimal to moderate degeneration of the olfactory epithelium in five males and five
females
• Minimal to slight inflammatory cell infiltrates in the nasal cavity in two males and one
female
• Slight to severe increase of mucous cells in the ventral nasal location in six males and
four females
Test group 2 (300 mg/m³):
• Minimal to moderate degeneration of the olfactory epithelium in two males and five
females
• Slight to severe increase of mucous cells in the ventral nasal location in one male and
four females
Test group 1 (100 mg/m³):
• Minimal degeneration of the olfactory epithelium in one male

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Effect levels

1

Key result

Dose descriptor:

NOAEC

Remarks:

systemic toxicity

Effect level:

ca. 1 000 mg/m³ air (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: systemic toxicity

2

Key result

Dose descriptor:

LOAEC

Remarks:

Local effects

Effect level:

ca. 100 mg/m³ air (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: local effects

Target system / organ toxicity

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

100 mg/m³ air (nominal)

System:

other: olfactory epithelium

Organ:

other: olfactory epithelium

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

no

Applicant's summary and conclusion

Executive summary:

Inhalation exposure to N-formylmorpholine for 90 days (65 exposures) caused degeneration of olfactory epithelia in the nasal cavity levels III and IV, which was occasionally accompanied by inflammatory cell infiltrates and increased mucous cells. These effects were considered treatment-related and adverse and showed concentration-response relationship. At the lowest concentration of 100 mg/m³, minimal olfactory epithelia degeneration was still observed in one male rat. Thus, a No Observed Adverse Effect Concentration (NOAEC) could not be established for local effect in nasal cavity. The Low Observed Adverse Effect Concentration (LOAEC) was 100 mg/m³ for local effect on the respiratory tract under the current study condition.

No systemic effects were observed. Thus, the NOAEC for systemic effects was 1000 mg/m³ under the current study condition.