4-morpholinecarbaldehyde

Endpoint summary

Description of key information

oral (OECD 407): NOAEL = 1000 mg/kgbw [BASF, 1998]

inhalation (OECD 413): NOAECsystemic= 1000 mg/m3 & LOAEClocal= 100 mg/m3[BASF, 2016]

No Observed Adverse Effect Concentration (NOAEC) could not be established due to local effects in nasal cavity

Key value for chemical safety assessment

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:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

yes

Remarks:

the weight of prostate and seminal vesicles with coagulating glands as a whole was not determinded

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Dr. Karl Thomae GmbH
- Age at study initiation: 49 days old
- Weight at study initiation: males mean ca. 236 g; females mean ca. 172 g
- Housing: individually in type DK III stainless steel wire mesh cages
- Diet: ad libitum, ground Kliba maintenance diet rat/mouse/hamster
- Water: ad libitum from water bottles
- Acclimation period: 8 (males) or 9 (females) days


ENVIRONMENTAL CONDITIONS
- Temperature: 20-24 °C
- Humidity: 30-70 %
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

doubly distilled

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test substance was administered as a solution in doubly distilled water. The test substance was weighed in depending on the dose group, then doubly distilled water was filled up to the desired volume. The solutions were mixed by shaking. The mixtures were prepared at least once a week.

VEHICLE
- Concentration in vehicle: 0.5, 2.0 and 10.0 g/100 mL
- Amount of vehicle: 10 ml/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The analyses were carried out at the Analytical Department of BASF Aktiengesellschaft. The stability of the test substance in water
was tested over a period of 10 days at room temperature. Concentration analyses were performed in samples of all concentrations at the start of the administration period. As the test substance was administered as a solution, no homogeneity analyses were conducted.

Duration of treatment / exposure:

28 days

Frequency of treatment:

once daily

Remarks:

Doses / Concentrations:
0, 50, 200 and 1000 mg/kg bw/d
Basis:

No. of animals per sex per dose:

5 animals per sex per dose plus another 5 animals per sex in the control and high dose groups (recovery)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
In a preceding test study, N-formylmorpholin was administered to groups of 3 male and female Wistar rats by gavage at dose levels of 0, 300, or 1000 mg/kg bw/d for 2 weeks. No substancerelated findings were observed. On this basis, the dose levels (0, 50, 200 and 1000 mg/kg bw/d) were selected for the present study.

- Rationale for selecting satellite groups:
Animals of the highest dose and control group underwent a recovery period of two weeks in order to prove reversibility and/or delayed onset of possible effects.

- Post-exposure recovery period in satellite groups:
2 weeks

Positive control:

none

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS / DETAILED CLINICAL OBSERVATIONS:
- The animals were examined for evident signs of toxicity or mortality twice a day (in the morning and in the late afternoon) from Mondays to Fridays and once a day (in the morning) on Saturdays, Sundays and public holidays. Additionally, further clinical examinations were carried out daily just before treatment, less than 1 hour after treatment, between 3 and 4 hours after treatment, as well as once a day during the recovery period.

BODY WEIGHT:
- Time schedule for examinations: The body weight was determined before the start of the administration period in order to randomize the animals. During the administration period and the recovery period the body weight was determined on day 0 (start of administration period) and thereafter at weekly intervals. The difference between the body weight on the respective day of weighing and the body weight on day 0 was calculated as body weight change.

FOOD CONSUMPTION:
- Food consumption was determined weekly over a period of 7 days and calculated as mean food consumption in grams per animal and day.

FOOD EFFICIENCY:
- body weight gain in g/food consumption in g/per unit time x 100 calculated as time-weighted averages

WATER CONSUMPTION:
- Water consumption was observed daily by visual inspection of the water bottles for any overt changes in volume.

HAEMATOLOGY:
- Time schedule for collection of blood: day 24 (males) and 26 (females) for all animals and day 40 (females, recovery) and 41 (males, recovery)
- Anaesthetic used for blood collection: no
- Animals fasted: no
- How many animals: all animals
- Parameters examined: leukocytes, erythrocytes, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelets, differential blood count, prothrombin time (Hepato Quick's test)


CLINICAL CHEMISTRY:
- Time schedule for collection of blood: day 24 (males) and 26 (females) for all animals and day 40 (females, recovery) and 41 (males, recovery)
- Animals fasted: yes. withdrawal of food and water during the collection period
- How many animals: all animals
- Parameters examined: alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, serum-y-glutamyltransferase, sodium, potassium, chloride, inorganic phosphate, calcium, urea, creatinine, glucose, total bilirubin, total protein, albumin, globulins, triglycerides, cholesterol, magnesium

URINALYSIS:
- Time schedule for collection of urine: day 23 (all animals) and day 36 (females, recovery) and 37 (males, recovery)
- Metabolism cages used for collection of urine: yes
- Animals fasted: no
- Parameters examined: volume, color, turbidity, nitrite, pH, protein, glucose, ketones, urobilinogen, bilirubin, blood, specific gravity, sediment

NEUROBEHAVIOURAL EXAMINATION:
- Time schedule for examinations: Detailed clinical examinations (open field observations) were carried out in all animals prior to the test substance administration (day -1), and on day 7, 14, 21, and in all animals of recovery groups on day 35, each time from about 2.00 p.m. onwards.
Functional observational batteries and motor activity measurements were carried out in all animals on the day of the last administration (day 28), and in all animals of recovery groups on day 42. Functional observational batteries were performed each time from about 2.00 p.m . and motor activity measurements were conducted each time from about 11.00 a.m . (main groups) and 12.30 p.m. (recovery groups) onwards.
- Dose groups that were examined: all animals
- Battery of functions tested: sensory activity / grip strength / motor activity

Sacrifice and pathology:

GROSS PATHOLOGY: Yes as well as organ weights

HISTOPATHOLOGY: Yes: all gross lesions, brain, pituitary gland, thyroid glands with parathyroid glands, thymus, trachea, lungs, heart, liver, spleen, kidneys, adrenal glands, testes/ovaries, uterus/vagina, accessory genital organs (epididymides, prostate, seminal vesicle), stomach (glandular and non-glandular), duodenum, jejunum, ileum, cecum, colon, rectum, urinary bladder, mandibular and mesenteric lymph nodes, sciatic nerve, bone marrow (femur), eyes, spinal cord (cervical, thoracic and lumbar cord)

Other examinations:

Organ weights:
The weight of the anesthetized animals as well as the weights of liver, kidneys, adrenal glands, testes, epididymides, ovaries, brain, thymus, heart, and spleen from all animals sacrificed at scheduled dates were determined .

Statistics:

Means and standard deviations of each test group were calculated for several parameters.
Further statistical analyses were performed but are too large in number to be described here.

Clinical signs:

no effects observed

Description (incidence and severity):

No animal died during the study period and no substance-related findings were obtained.

Mortality:

no mortality observed

Description (incidence):

No animal died during the study period and no substance-related findings were obtained.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

No statistically significant or biologically relevant deviations of body weights were observed. Concerning body weight change data, statistically significantly increased values were seen in males of the high dose recovery group on days 21 (+10.8%), day 28 (+6.3%) and day 42 (+10.1%). These slight deviations were most probably due to the (incidentally) increased food consumption in these animals, but not related to treatment.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Food consumption was statistically significantly increased in high dose males (recovery group) on day 21 (+10.1%) and in high dose females (recovery group) on day 35 (+15.0%). Due to either the single occurrence or the fact that the effect occurred during even in the recovery period, this was assessed as being incidental and not related to treatment.

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

Food efficiency was statistically significantly decreased in high dose males (main group) on day 28 and statistically significantly increased in high dose females (main group) on day 21. Both findings were assessed as being incidental in nature.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

At the end of the study significant decreases in white blood cell counts were observed in the peripheral blood of all treated males of the main groups. These findings were not considered to be test substance-related since there was no dose-response relationship and all leukocyte values of the treated animals were within the limits of historical control data. Moreover, statistically significantly decreased white blood cells were observed in the males only. It is very likely that the statistically significant decreases in leukocytes were only due to the high value of the control group. Thus, the lowered white blood cell counts were regarded to be incidental in nature and were not related to treatment. The other hematological and clotting examinations revealed no treatment-related changes.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

At the end of the administration period slightly increased alanine aminotransferase activities were found in the sera of the high dose males and females of the main and recovery groups. After cessation of the test compound administration these findings returned to normal. There are no treatment-related changes in the other clinical chemistry parameters measured.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment-related changes in the urinalytical parameters measured.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Detailed clinical observations (open field observations): No substance-related effects were observed.
Functional observational battery: No substance-related effects were observed
Home cage observations: No substance-related effects were observed.

Open field observations : No abnormalities were obtained in any of the main test groups when observing the animals in the open field. Rearing was statistically significantly decreased in the high dose males (recovery group) on day 28. As no such effect was seen in the main group, this was assessed as being incidental.
Sensorimotor tests/Reflexes: All findings were within the biological range of variation. No treatment-related effect was observed.
Quantitative observations (Grip strength, Landing foot-splay test): Grip strength hindlimbs was statistically significantly decreased in low dose females on day 28. Due to the lack of a dose-response relationship, this was assessed as being incidental. All other findings were within the biological range of variation. No treatment-related effect was observed.
Motor activity measurement: Regarding the overall motor activity no statistically significant or biologically relevant deviation was seen in any of the treatment groups. The comparison of the single intervals with the control group resulted in a statistically significantly higher value in high dose females (recovery group) on day 28 (interval 11). This was, however clearly incidental in nature and not related to treatment.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

The mean absolute weight of the spleen was significantly decreased in males of the low and high dose groups, whereas it was significantly increased in females of the low dose group. The other mean absolute weight parameters of the animals of the main groups did not show significant differences when compared with the concurrent control group. Due to a slightly decreased mean terminal body weight, the mean weight of the kidneys was significantly increased in males of the high dose group. This was not regarded to be treatment related. The mean weight of the spleen was significantly increased in females of the low dose group. The other mean relative weight parameters (when related to terminal body weight) of the animals of the main groups did not show significant differences when compared with the concurrent control group.

Recovery groups:
Due to a slight increase of the mean terminal body weight (+ 2.6%), the mean weight of the liver was also slightly (+ 9.2%) significantly increased in males of the high dose recovery group. This was not regarded to be treatment related. The other mean absolute weight parameters of the animals of the recovery groups did not show significant differences when compared with the concurrent control group. The mean relative weight parameters (when related to terminal body weight) of the animals of the recovery groups did not show significant differences when compared with the concurrent control group.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

The only gross lesions noted were in the glandular stomach of a low dose male (erosion/ulcer) and in the kidneys of a low dose female (pelvic dilation).

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

There were no remarkable microscopic findings noted in any of the organs investigated. All microscopic findings recorded were either single observations, or they occurred in control animals only, or they were recorded at comparable incidence and graded severity in control and high dose males and/or females. There was no indication of a morphologic affection of the organs of the central or peripheral nervous system, the reproductive system, and the immune system.

Recovery groups:
Histopathology correlated the gross lesions in the glandular stomach with an incidental focus of cystic malformation. The focus in the liver turned out as tension lipidosis, a spontaneous observation caused by the traction of the ligamentum hepato-colicum at its insertion site on the surface of the liver. The few other microscopic findings noted in the liver were also of spontaneous origin and unrelated to treatment.

Description (incidence and severity):

There were additional statistically significant intergroup differences in the results of clinical pathology testing. These deviations were marginal, incidental or inconsistent, when compared with the other sex, or lacked a dose-response relationship. Accordingly, these findings were considered to be of no toxicological significance.

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Critical effects observed:

not specified

Chemical analyses of dose formulations revealed that the test item was stable in the vehicle over a period of 10 days when kept at room temperature. The mean analytical concentration values were 100 -105 % of the nominal concentraions.

Executive summary:

The oral administration of the test item by gavage to rats at the high dose level of 1000 mg/kg bw/day for 4 weeks resulted in slight increases in alanine aminotransferase activity in the sera indicating mild hepatocellular damage. No test substance-related changes were seen in the low and mid dose animals (50 and 200 mg/kg bw/day). The increases in liver enzyme activities in the sera observed at the end of the administration period were reversible during the course of the treatment-free recovery period. The no observed adverse effect level (NOAEL) under the conditions of this study was 1000 mg/kg bw/day.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

GLP and Guideline study

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:

24.06.2016 - 07.04.2017

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3465 (90-Day Inhalation Toxicity)

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: 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

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

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

Dose / conc.:

0 mg/m³ air

Remarks:

Control

Dose / conc.:

100 mg/m³ air (nominal)

Remarks:

Mean 105.7 +- 20.7 SD
MMAD not measured

Dose / conc.:

300 mg/m³ air (nominal)

Remarks:

Mean 309.1 +- 44.8 SD
2.36 - 3.07 µm MMAD

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

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

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

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

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

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

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

1 000 mg/m³

Study duration:

subchronic

Species:

rat

Quality of whole database:

GLP and Guideline study

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:

24.06.2016 - 07.04.2017

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3465 (90-Day Inhalation Toxicity)

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: 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

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

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

Dose / conc.:

0 mg/m³ air

Remarks:

Control

Dose / conc.:

100 mg/m³ air (nominal)

Remarks:

Mean 105.7 +- 20.7 SD
MMAD not measured

Dose / conc.:

300 mg/m³ air (nominal)

Remarks:

Mean 309.1 +- 44.8 SD
2.36 - 3.07 µm MMAD

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

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

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

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

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

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

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.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LOAEC

100 mg/m³

Study duration:

subchronic

Species:

rat

Quality of whole database:

GLP and Guideline study

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

Oral

In the key subacute repeated dose toxicity study (BASF AG, 1998; 3850173/97029) the test item was administered to male and female Wistar rats by gavage for 4 weeks at nominal doses of 0 (vehicle control), 50, 200 and 1000 mg/kg bw/day. The analytically confirmed concentrations were 0 .5, 2 .1 and 10 .0 g/100 mL, respectively. The vehicle used was doubly distilled water, and the administration volume was 10 mL/kg. Control and high dose group consisted of each 10 animals per sex; low and mid dose group consisted of each 5 animals per sex. Five animals per sex of all groups were treated for 4 weeks and sacrificed thereafter (main groups); the remaining 5 animals per sex of control and high dose group were maintained for another 14-days without administration of the test substance (recovery groups).

The following substance-related findings were obtained:

 

High dose groups; main study and recovery (1000 mg/kg bw/day)

 - slight increase in alanine aminotransferase activity in the sera in both sexes

 Mid and low dose group (50 mg/kg bw/day and 200 mg/kg bw/day)

 - no treatment-related changes

 

Thus, the oral administration of the test item by gavage to rats at a dose level of 1000 mg/kg bw/day for 4 weeks resulted in slight increases in alanine aminotransferase activity in the sera potentially indicating mild hepatocellular damage. No test substance-related changes were seen in the low and mid dose animals (50 and 200 mg/kg bw/day). The increases in liver enzyme activity in the sera observed at the end of the administration period were reversible during the course of the treatment-free recovery period. In the study report, the authors concluded that the NOAEL under the conditions of this study was 200 mg/kg bw/d due to the slight increase in alanine aminotransferase activity in the sera at the high dose level of 1000 mg/kg bw/d.

However, scientifically reasons are given to adapt and correct this value: The slight increase in alanine aminotransferase activity in the sera is an isolated finding and variations of other liver enzyme activities in the sera were missing. Moreover, a statistically significant increase was only observed in the recovery-group. Effects were reversible during the course of the treatment-free recovery period. The increase of enzyme activity in the sera was not dose dependent and had no relevant correlate with regards to histopathology.

 

Therefore, the NOAEL is considered to be 1000 mg/kg bw/d, the highest dose tested.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint: 

The key study was selected. 

Inhalation

NFM has recently been investigated in a 3 months- repeated dose toxicity study (OECD 413) in Wistar rats. According to a 14 days dose-range finder, the doses to be tested are 0, 100, 300 and 1000 mg/m³ of NFM-vapour / aerosol.

Please find results from the dose-range finder below indicating that no Hazard is to be expected up to the high dose:

DRF

The following findings were noted:

  

Test group 3 (1000 mg/m³):

No findings were noted in organ weights, gross pathology or histopathology

Test group 2 (300 mg/m³):

Statistically significant increased relative weights of the epididymis(+30%) and the lungs (+16%)

Test group 1 (100 mg/m³):

No findings were noted in organ weights, gross pathology or histopathology

  

As there was no dose response – relationship or histopathological correlate for the increased relative weights of the lungs in test group 2 males, this was regarded as incidental. The increased relative weights of the epididymides were due to a low mean control weight as one control animal (no 3) showed severe oligospermia in the epididymis and diffuse tubular degeneration in the testis.

No treatment – related findings were noted in male Wistar rats after 14 days of exposure.

3 months (90-day study)

To evaluate the toxicity profile of N-formylmorpholine after inhalation exposure, groups of ten male and ten female Wistar rats per test group were exposed nose-only to dynamic atmosphere of N-formylmorpholine for 6 hours per day on 5 consecutive days per week for 3 months (90-day study). The target concentrations were 100, 300 and 1000 mg/m³. A concurrent control group was exposed to conditioned air. For adaptation to the experimental conditions all animals were kept in glass restraining tubes identical to those used in the main study and were exposed nose-only to fresh air on three days before start of the exposure period.

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

 

Conclusion:

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 effect was 1000 mg/m³ under the current study condition.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint: 

The key study was selected. 

Justification for selection of repeated dose toxicity via inhalation route - systemic effects endpoint: 

The key study was selected. 

Justification for classification or non-classification

The test item did not induce adverse effects indicative of serious damage at dose levels relevant for classification and labelling when tested for oral and inhalative repeated dose toxicity. Thus, the test item is not subjected to classification and labelling for repeated dose toxicity according to Regulation 1272/2008/EC.