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Diss Factsheets

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

NOAEL = 1000 mg/kg bw/d (BASF, 2012)

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2011-08-02 to 2012-09-11
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD Guideline study according to GLP
Qualifier:
according to guideline
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS

- Strain: Crl:WI(Han)
- Source: Charles River Laboratories, Germany
- Age at study initiation: 11-13 weeks old
- Weight at study initiation: Males: 311.9 g - 341.1 g; Females: 206.7 g - 229.0 g
- females were nulliparous and non-pregnant at the beginning of the study
- male and female animals were derived from different litters, necessary to rule out the possibility of sibling mating


- Housing: During the study period, the rats were housed individually in Makrolon type M III cages supplied by Becker & Co., Castrop-Rauxel, Germany (floor area of about 800 cm²), with the following exceptions:

• During overnight matings, male and female mating partners were housed together in Makrolon type M III cages.
• Pregnant animals and their litters were housed together until PND 4.

Pregnant females were provided with nesting material (cellulose wadding) toward the end of gestation.

- Diet: ground Kliba maintenance diet rat/mouse "GLP" meal (Provimi Kliba SA, Kaiseraugst, Switzerland), ad libitum
- Water: drinking water from bottles, ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature: 20 - 24 °C
- Humidity: 30 - 70 %
- Air changes: 15 per hour
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES:
From: 08 Aug 2011 To: 05 Sept 2011 (males), 26 Sept 2011 and 04 Oct 2011 (females)
Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test substance preparations in drinking water were prepared at the beginning of the administration period and thereafter in intervals, which took into account the analytical results of the stability verification.

For the preparation of the administration solutions the test substance was weighed in a graduated measuring flask depending on the dose group, topped up with drinking water and subsequently thoroughly mixed by shaken until completely dissolved.

- Amount of vehicle: 10 mL/kg bw based on the most recent individual body weight
Details on mating procedure:
In general, each of the male and female animals was mated overnight in a 1 : 1 ratio for a maximum of 2 weeks. Throughout the mating period, each female animal was paired with a predetermined male animal from the same dose group.

Mating was accomplished by placing the female in the cage of the male mating partner from about 16.00 h until 7.00 - 9.00 h of the following morning. Deviations from the specified times were possible on weekends and public holidays and were reported in the raw data.

A vaginal smear was prepared after each mating and was examined for the presence of sperm. If sperm was detected, pairing of the animals was discontinued. The day on which sperm were detected was denoted "gestation day (GD) 0" and the following day "gestation day (GD) 1".
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The analyses were carried out at the Analytical Chemistry Laboratory of Experimental Toxicology and Ecology of BASF SE, Ludwigshafen, Germany.

Analytical verifications of the stability of the test substance in drinking water for a period of 7 days at room temperature were carried out prior to the start of the study.

Given that N-formylmorpholin is completely miscible with drinking water, solutions were considered to be homogenous without further analysis.

Samples of the aqueous test substance solutions were sent to the analytical laboratory once at the beginning of the study for verification of the concentrations
Duration of treatment / exposure:
After the acclimatization period, the test substance was administered to the parental animals orally by gavage, once daily at approximately the same time in the mornings. The duration of treatment covered a 2 week premating period and mating in both sexes (mating pairs were from the same dose group) as well as entire gestation and 4 days of lactation period in females up to one day prior to the day of schedule sacrifice of the animals. Females in labor were not treated during parturition. The male animals were treated for a total of 28 days.
Frequency of treatment:
once daily
Remarks:
Doses / Concentrations:
0, 100, 300 and 1000 mg/kg bw/day
Basis:
nominal conc.
No. of animals per sex per dose:
10 rats
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The animals were tested upto the limit dose level for this study type.
Positive control:
None
Parental animals: Observations and examinations:
MORTALITY

A check for moribund or dead animals was made twice daily on working days or once daily (Saturday, Sunday or on public holidays). If animals were in a moribund state, they were sacrificed and necropsied. The examinations of these animals were carried out according to the methods established at the pathology laboratory.


CAGE SIDE OBSERVATIONS / CLINICAL OBSERVATIONS:
A cageside examination was conducted at least once daily for any signs of morbidity, pertinent behavioral changes and signs of overt toxicity. Abnormalities and changes were documented daily for each animal.

The parturition and lactation behavior of the dams was generally evaluated in the mornings in combination with the daily clinical inspection of the dams. Only particular findings (e.g. inability to deliver) were documented on an individual dam basis.

On weekdays (except public holidays) the parturition behavior of the dams was inspected in the afternoons in addition to the evaluations in the mornings.

The day of parturition was considered the 24-hour period from about 15.00 h of one day until about 15.00 h of the following day.


BODY WEIGHT:
In general, the body weight of the male and female parental animals was determined once a week at the same time of the day (in the morning) until sacrifice.

The body weight change of the animals was calculated from these results.

The following exceptions are notable for the female animals:

• During the mating period the parental females were weighed on the day of positive evidence of sperm (GD 0) and on GD 7, 14 and 20.

• Females with litter were weighed on the day of parturition (PND 0) and on PND 4.

• Females without litter, waiting for necropsy, were weighed weekly

FOOD CONSUMPTION:
Generally, food consumption was determined once a week (in a period of 6-7 days) for male and female parental animals, with the following exceptions:

• Food consumption was not determined during the mating period (male and female F0 animals).
• Food consumption of the F0 females with evidence of sperm was determined for gestation days (GD) 0-7, 7-14 and 14-20.
• Food consumption of F0 females, which gave birth to a litter was determined for PND 1-4.

Food consumption was not determined in females without litter during lactation period.
Oestrous cyclicity (parental animals):
Not examined
Sperm parameters (parental animals):
Not examined
Litter observations:
PARAMETERS EXAMINED
The live pups were examined daily for clinical symptoms (including gross-morphological findings) during the clinical inspection of the dams.
The pups were weighed on the day after birth (PND 1) and on PND 4. Pups' body weight change was calculated from these results. The individual weights were always determined at about the same time of the day (in the morning).



GROSS EXAMINATION OF DEAD PUPS:
Please refer to "Postmortem examinations (Offspring)".
Postmortem examinations (parental animals):
Necropsy
All parental animals were sacrificed by decapitation under isoflurane anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology, special attention being given to the reproductive organs.

Organ weights:
The following weights were determined in animals sacrificed on schedule:

1. Anesthetized animals > terminal body weight (all animals)
2. Epididymides (males)
3. Testes (males)

Organ/tissue fixation:
The following organs or tissues were fixed in 4 % buffered formaldehyde solution or in modified Davidson’s solution:

1. All gross lesions
2. Cervix
3. Coagulating gland
4. Epididymides (modified Davidson’s solution)
5. Ovaries (modified Davidson’s solution)
6. Oviducts
7. Prostate gland
8. Seminal vesicles
9. Testes (modified Davidson’s solution)
10. Vagina
11. Uterus

Histological assessment:
Fixation was followed by histotechnical processing (HE staining) and examination by light microscopy.
The testes and epididymides of all control and high dose males and the ovaries of all control and high dose females were examined for histopathological findings.
Postmortem examinations (offspring):
All pups with scheduled sacrifice on PND 4 were sacrificed under isoflurane anesthesia with CO2. All pups were examined externally and eviscerated; their organs were assessed macroscopically.

All stillborn pups and all pups that died before PND 4 were examined externally, eviscerated and their organs were assessed macroscopically.

All pups without notable findings or abnormalities were discarded after their macroscopic evaluation.
Animals with notable findings or abnormalities were evaluated on a case-by-case basis, depending on the type of finding noted.
Statistics:
For the clinical examinations, Dunnett's test, Fisher's exact test, and the Wilcoxon test were used. The Kruskal-Wallis test followed by the Wilcoxon test was used to analyze the pathology data.
Reproductive indices:
For the males, the mating and fertility indices were calculated.
For the females, the mating, fertility, gestation, live birth indices as well as the postimplantation loss were calculated.
Offspring viability indices:
Live birth index and post implantation loss were determined.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No clinical signs or changes of general behavior, which may be attributed to the test substance, were detected in any male or parental animals at dose levels of 100, 300 or 1000 mg/kg bw/d during the different study periods.
One sperm positive female the control group (No. 102), two females of low dose group (Nos. 115 and 120 – 100 mg/kg bw/d), one female of the mid dose group (No. 125 – 300 mg/kg bw/d) and two females of the high dose group (Nos. 132 and 134 – 1000 mg/kg bw/d) did not deliver F1 pups.
In one female of the mid dose group (No. 126 – 300 mg/kg bw/d) pups were palpable in its abdomen after delivery on PND 0 and 1. Afterwards it was without abnormal findings again until scheduled sacrifice.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
There were no test substance-related or spontaneous mortalities in any of the groups.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Mean body weights and mean body weight gain of the F0 males in all test groups (100, 300 and 1000 mg/kg bw/d) were comparable to the concurrent control throughout the entire study period.
Mean body weight and mean body weight gain of the F0 females in all test groups (100, 300 and 1000 mg/kg bw/d) were comparable to the concurrent control throughout the entire premating, gestation and lactation periods.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption of the male and female F0 generation parental animals in all test substance-treated groups (100, 300 and 1000 mg/kg bw/d) was comparable to the concurrent control group during the entire study period, covering premating, gestation and lactation.
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
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
All findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.
Histopathological findings: neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
All findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.
Other effects:
not examined
Reproductive function: oestrous cycle:
effects observed, non-treatment-related
Description (incidence and severity):
The female mating index calculated after the mating period for F1 litter was 100% in all test groups.
The mean duration until sperm was detected (GD 0) varied between 2 and 3 days without any relation to dosing.
All sperm positive rats delivered pups or had implants in utero with the following exception:
• control female No. 102 (mated with male No. 2) did not become pregnant.
• low-dose females Nos. 115 and 120 (mated with males Nos. 15 and 20) did not become pregnant.
• mid-dose female No. 125 (mated with male No. 25) did not become pregnant.
• high-dose females Nos. 132 and 134 (mated with males Nos. 32 and 34) did not become pregnant.

The fertility index varied between 80% (low and high dose group [100 and 1000 mg/kg bw/d]) and 90% (control and high dose group [1000 mg/kg bw/d]). These values reflect the normal range of biological variation inherent in the strain of rats used for this study.

The non-pregnant female rat of the control group (No. 102) exhibited a cyst in the right oviduct as well as a dilated uterus and vagina. High-dose female No. 132 (1000 mg/kg bw/d) had a dilated uterus at gross necropsy. None of the other non-pregnant females in test groups 1-3 (Nos. 115, 120, 125 and 134) had any relevant gross lesions.
The mean duration of gestation was similar in all test groups (i.e. 22 days).
The gestation index was 100% in all test groups.
Implantation was not affected by the treatment since the mean number of implantation sites was comparable between all test substance-treated groups and the controls, taking normal biological variation into account (11.5 / 11.0 / 12.7 and 10.1 implants/dam in all test groups (0, 100, 300 and 1000 mg/kg, respectively). There were no statistically significant differences in post-implantation loss between the groups (4.9 % / 9.2 % / 10.5 % / 9.6 % respectively), and the mean number of F1 pups delivered per dam remained unaffected (12.1 / 12.4 / 13.2 and 11.6 pups/dam at 0, 100, 300 and 1000 mg/kg bw/d, respectively).
The rate of liveborn pups was also not affected by the test substance, as indicated by live birth indices of 95.8 % (mid dose group), 98.9 % (high dose group) and 100 % (control and low dose group). Moreover, the number of stillborn pups was comparable between the groups.

Thus, N-Formylmorpholin did not adversely affect reproduction and delivery of the F0 generation parental females.
Reproductive function: sperm measures:
effects observed, non-treatment-related
Description (incidence and severity):
For all F0 parental males, which were placed with females to generate F1 pups, copulation was confirmed. Thus, the male mating index was 100 % in all groups including the controls.
Fertility was proven for most of the F0 parental males within the scheduled mating interval for F1 litter.
One sperm positive female of the control group (No. 102), two females of the low dose group (Nos. 115 and 120 – 100 mg/kg bw/d), one female of mid dose group (No. 125 – 300 mg/kg bw/d) and two females of high dose group (Nos. 132 and 134 – 1000 mg/kg bw/d) did not deliver F1 pups.
Thus, the male fertility index ranged between 80 % (low and high dose group) and 90 % (control and mid dose group ) without showing any relation to dosing. This reflects the normal range of biological variation inherent in the strain of rats used for this study.
The apparently infertile male rats of test groups 1-3 (Nos. 15, 20, 25, 32 and 34 – 100, 300 or 1000 mg/kg bw/d) did not show relevant gross lesions. However, a degeneration was recorded in the testes of control male rat No. 2.
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: There were no test item related signs of systemic toxicity and no adverse effects on reproduction/fertility up to and including the high dose level of 1000 mg/kg bw/d
Clinical signs:
no effects observed
Description (incidence and severity):
There were no test substance related adverse clinical signs observed in any of the F1 generation pups of the different test groups.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Description (incidence and severity):
The viability index indicating pup mortality during lactation (PND 0-4) varied between 99.2 % (mid and high dose) and 100 % (control and low dose).

The mean number of delivered F1 pups per dam and the rates of liveborn and stillborn F1 pups were evenly distributed about the groups. The respective values reflect the normal range of biological variation inherent in the strain used in this study.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Mean pup body weight and pup body weight change of all test substance-treated groups were statistically comparable to the concurrent control group.
One male and 3 female runts were seen in the control group. Two male runts were seen in test group 2 (300 mg/kg bw/d); 3 male and 7 female runts were seen in test high dose group (1000 mg/kg bw/d).
Description (incidence and severity):
The sex distribution and sex ratios of live F1 pups on the day of birth and PND 4 did not show substantial differences between the control and the test substance-treated groups; slight differences were regarded to be spontaneous in nature.
Description (incidence and severity):
PUP NECROPSY OBSERVATIONS:
One F1 pup of test group 3 (1000 mg/kg bw/d) showed post mortem autolysis at gross necropsy. No association to treatment is assumed.
PUP NUMBER AND STATUS AT DELIVERY:
The mean number of delivered F1 pups per dam and the rates of liveborn and stillborn F1 pups were evenly distributed about the groups. The respective values reflect the normal range of biological variation inherent in the strain used in this study.

PUP VIABILITY/MORTALITY:
The viability index indicating pup mortality during lactation (PND 0-4) varied between 99.2 % (mid and high dose) and 100 % (control and low dose).

SEX RATIO:
The sex distribution and sex ratios of live F1 pups on the day of birth and PND 4 did not show substantial differences between the control and the test substance-treated groups; slight differences were regarded to be spontaneous in nature.

PUP CLINICAL OBSERVATIONS:
There were no test substance related adverse clinical signs observed in any of the F1 generation pups of the different test groups.

PUP BODY WEIGHT DATA:
Mean pup body weight and pup body weight change of all test substance-treated groups were statistically comparable to the concurrent control group.
One male and 3 female runts were seen in the control group. Two male runts were seen in test group 2 (300 mg/kg bw/d); 3 male and 7 female runts were seen in test high dose group (1000 mg/kg bw/d).

PUP NECROPSY OBSERVATIONS:
One F1 pup of test group 3 (1000 mg/kg bw/d) showed post mortem autolysis at gross necropsy. No association to treatment is assumed.



Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no developmental differences in the F1 generation were detected up to and including the high dose level of 1000 mg/kg bw/d
Reproductive effects observed:
not specified

Dose formulation analysis demonstrated adequate stability ot the test substance in drinking water when kept over a period of 7 days at room temperature. All determined concentrations were in range between 90 and 110 % of the nominal concentrations, i.e. were regarded to correspond to the ecpected values.

Executive summary:

Generally, no toxicologically-relevant adverse effects of N-formylmorpholin were noted in parental animals up to a dose of 1000 mg/kg bw/d. However, a significant reduction in the absolute and relative weights of the epididymides was observed in parental male animals receiving 1000 mg/kg bw/day. As neither a pathological change nor a decline in fertility was detected, this effect was judged non-adverse. Thus the NOAEL (no observed adverse effect level) for reproductive toxicity in both genders is 1000 mg/kg bw/d. However, due to the decline in absolute and relative epididymides weights at this dose, the NOEL (no observed effect level) for reproductive toxicity in males is 300 mg/kg bw/d. No developmental differences in the F1 generation were detected during this study.

Therefore, the NOAEL for developmental toxicity is 1000 mg/kg bw/d, the highest dose tested.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The GLP compliant key study is of high quality (Klimisch score = 1).
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

In an OECD Guideline 421 reproduction/developmental toxicity screening test under GLP the test item N-formylmorpholin was given once daily as an aqueous solution to groups of 10 male and 10 female Wistar rats (F0 animals) by gavage at doses of 100, 300 or 1000 mg/kg bw/d (BASF SE, 2012; 80R0173/97R001). Control animals were dosed daily with the vehicle only (drinking water). The duration of treatment covered a 2 week premating period and mating in both sexes (mating pairs were from the same dose group) as well as entire gestation and 4 days of lactation period in females up to one day prior to the day of schedule sacrifice of the animals.

The parents' and the pups' state of health was checked each day, and parental animals were examined for their mating and reproductive performances.

F0 animals were mated 13 days after the beginning of treatment to produce a litter (F1 generation pups). As soon as sperm was detected in the vaginal smear, mating was discontinued. F0 animals were examined for their reproductive performance including determinations of the number of implantations and the calculation of the post implantation loss in all F0 females. Food consumption of the F0 parents was determined regularly once weekly before and after the mating period, as well as in dams during gestation (days 0 - 7, 7 - 14, 14 - 20) and lactation (days 1 - 4). In general, the body weights of F0 animals were determined once a week. However, during gestation and lactation, F0 females were weighed on gestation days (GD) 0, 7, 14 and 20, and on postnatal days (PND) 0 and 4. The pups were sexed and examined for macroscopically evident changes on PND 0. They were weighed on PND 1 and PND 4 and their viability was recorded. At necropsy on PND 4, all pups were sacrificed under isoflurane anesthesia with CO2 and examined macroscopically for external and visceral findings at necropsy. All F0 parental animals were sacrificed by decapitation under isoflurane anesthesia, and were assessed by gross pathology. Weights of selected organs were recorded and a histopathological examination was performed.

 

Generally, no toxicologically-relevant adverse effects of N-formylmorpholin were noted in parental animals up to a dose of 1000 mg/kg bw/d. However, a significant reduction in the absolute and relative weights of the epididymides was observed in parental male animals receiving 1000 mg/kg bw/d. As neither a pathological change nor a decline in fertility was detected, this effect was judged non-adverse.

Inhalation exposure to N-formylmorpholine for 90 days (65 exposures) did not cause systemic effects. Thus, the NOAEC for systemic effect was 1000 mg/m³. Investigation on the estrous cycle data revealed regular cycles in the females of all test groups including the control. Concerning motility of the sperms and the incidence of abnormal sperms in the cauda epididymidis as well as sperm head counts in the testis and in the cauda epididymidis no treatment-related effects were observed. Multifocal degeneration in the testes corresponded to the macroscopically observed reduced size of the testes. There was no dose response-relationship (1/7/5/5), and the severity was less in test group 3 (1000 mg/m³) compared to test group 2 (300 mg/m³). It is known that in nose-only expopsed rats a higher incidence of testicular findings can occurs (Lee at al. 1993), which is demonstrated als by inhouse historical control data her up to 8 out of 10 animals revealed testicular degeneration. Therefore, an incidental finding might be possible, especially with unchanged sperm parameters in clinical pathology. 

Thus the NOAEL (no observed adverse effect level) for reproductive toxicity in both genders is 1000 mg/kg bw/d.

However, due to the decline in absolute and relative epididymides weights at this dose, the NOEL (no observed effect level) for reproductive toxicity in males is 300 mg/kg bw/d.

 

No developmental differences in the F1 generation were detected during this study.

Therefore, the NOAEL for developmental toxicity is 1000 mg/kg bw/d, the highest dose tested.


Short description of key information:
The NOAEL (no observed adverse effect level) for reproductive toxicity in both sexes is 1000 mg/kg bw/d, the highest dose tested.

Justification for selection of Effect on fertility via oral route:
The key study was selected. (only one OECD 421 study available)

Effects on developmental toxicity

Description of key information

In conclusion, the no observed adverse effect level (NOAEL) for maternal toxicity is 300 mg/kg body weight/day based on temporary body weight loss and decreased (net) body weight gain in animals treated with 1000 mg/kg body weight/day N-formylmorpholine.

The no observed adverse effect level (NOAEL) for prenatal developmental toxicity is 300 mg/kg body weight/day, based on slightly reduced fetal weights and subsequent marginal delays of ossification. The fetal findings were minor and considered to be secondary to the body weight effects in the dams. Therefore, the test item does not cause selective prenatal developmental toxicity.

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Age at study initiation: About 10-13 weeks old
- Weight at study initiation: no data
- Fasting period before study: no
- Housing: Makrolon cages type M III, No. of animals per cage: 1,
- Diet: Ground Kliba maintenance diet mouse/rat “GLP”, Provimi Kliba SA, Kaiseraugst, Switzerland; ad libitum
- Water: Drinking water ad libitum
- Acclimation period: From GD 0 (day of supply) to the beginning of administration (GD 6), the animals were accustomed to the environmental conditions and to the diet.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): humidity 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 hours light (06:00 h -18:00 h) , 12 hours darkness (18:00 h - 06:00 h)


IN-LIFE DATES:
From: xxx To: xxx
Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

For the test substance preparation, the specific amounts of test substance were weighed, topped up with drinking water in a graduated flask and intensely mixed with a magnetic stirrer until it was completely dissolved.The test substance preparations were prepared at intervals which guaranteed that the test substance concentrations in the vehicle remained stable.
Storage conditions of the preparations: Room temperature


VEHICLE

- Amount of vehicle (if gavage): 10 mL/kg; the body weight determined most recently was used to calculate the administration volume

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The analytical investigations of the test substance preparations were carried out at the Analytical Chemistry Laboratory of Experimental Toxicology and Ecology of BASF SE, Ludwigshafen, Germany, as a part of this study.
The stability of the test substance in drinking water at room temperature over a period of 10 days had been verified prior to the start of the study in a similar batch (Project No.: 97L00390).
Details on mating procedure:
- Impreganation procedure: purchased time pregnant
- Verfication of of same strain and source of both sexes: yes

The animals paired by the breeder (time-mated animals) were supplied at noon on the day of evidence of mating; this day is referred to as GD 0 and the following day as GD 1.
Duration of treatment / exposure:
days 6 - 19 of gestation
Frequency of treatment:
Once daily (GD 6-19)
Duration of test:
21 days
Remarks:
0,100, 300, 1000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
25 females per dose
Control animals:
yes, concurrent vehicle
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Cage side examination were conducted at least once daily for any signs of morbidity, pertinent behavioral changes and/or signs of overt toxicity. If such signs occurred, the animals were examined several times daily. Abnormalities and changes were be documented for each animal (GD 0 through 20).

BODY WEIGHT: Yes
- Body weights were recorded on GD 0, 1, 3, 6, 8, 10, 13, 15, 17, 19 and 20.

FOOD CONSUMPTION: Yes
- Food consumption was recorded for GD 0-1, 1-3, 3-6, 6-8, 8-10, 10-13, 13-15, 15-17, 17-19 and 19-20.

POST-MORTEM EXAMINATIONS: Yes
- On GD 20, the surviving dams were anesthetized with isoflurane, sacrificed by cervical dislocation in a randomized sequence and examined. The fetuses were removed from the uterus, which had been opened before and examined.

Moribund animals and animals that died intercurrently were examined if possible (with the exception of the uterus weight).

- Examinations:

- After the dams were sacrificed, the following examinations or the following weight determinations and counts were carried out:

- Gross-pathological examination
- Weight of the unopened uterus
- Number of corpora lutea
- Number of implantations (differentiated according to live and dead fetuses and early or late resorptions)
- Early resorptions according to SALEWSKI in animals that do not appear to be pregnant & animals with single-horn pregnancy
- Site of implantations in the uterus
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: Site of implantations in the uterus
Fetal examinations:
After the fetuses were removed from the uterus, the following examinations or weight determinations were carried out:

- Weight of each fetus
- Sex
- Weight of the placentas
- External examinations: yes (all per litter)
- Soft tissue examinations: yes (all per litter)
- Skeletal examinations: yes (all per litter)
- Head examinations: no

Gross-pathological examination of the fetuses after dissection from the uterus (including abnormalities of the fetal membranes, placentas, amniotic fluid and umbilical cord); then all fetuses were sacrificed by subcutaneous injection of pentobarbital.
About half of the fetuses of each dam were skinned, fixed in ethyl alcohol and, after fixation, stained according to a modified method (KIMMEL and TRAMMELL ) to show the skeleton and the cartilage. After the skeletons/cartilage had been examined, these fetuses were archived individually.
The other half of the fetuses of each dam were fixed in Harrison’s fluid. After fixation, the soft tissues of these fetuses were examined according to a modified microdissection method (BARROW and TAYLOR ). After the examination, these fetuses were discarded.

Statistics:
Means and standard deviations were calculated. In addition, the following statistical analyses were carried out:

DUNNETT’s test:
Food consumption, body weight, body weight change, corrected body weight gain, carcass weight, weight of the unopened uterus, weight of the placentas and fetuses, corpora lutea, implantations, pre and postimplantation losses, resorptions and live fetuses

FISHER's exact test:
Number of pregnant animals at the end of the study, mortality rate (of the dams) and number of litters with fetal findings

WILCOXON test:
Proportion of fetuses with findings per litter
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Test group 3 (1000 mg/kg bw/d):
DAMS
• Body weight loss on GD 6-8
• Reduced corrected body weight gain (10% below control)
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Test group 3 (1000 mg/kg bw/d):
DAMS
• Reduced food consumption on GD 6-8 (11% below control)
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Changes in number of pregnant:
no effects observed
Other effects:
not examined
Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
body weight and weight gain
Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
Test group 3 (1000 mg/kg bw/d):

FETUSES
• Slightly reduced fetal body weights (-6%)
• Marginally higher incidence of two incompletely ossified bones (skull, sternebra), without structural changes

The mean fetal body weights of all viable fetuses in test group 3 (1000 mg/kg bw/d) were statistically significantly reduced (about -6% in comparison to the corresponding control value). If the sexes were considered separately, the weights were 8% lower in male and 9% lower in female fetuses.
The mean fetal weights of test groups 1 and 2 (100 and 300 mg/kg bw/d) were not influenced by the test substance and did not show any biologically relevant differences in comparison to the control group.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Description (incidence and severity):
The sex distribution of the fetuses in test groups 1-3 (100, 300 and 1000 mg/kg bw/d) was comparable to the control fetuses. Any observable differences were without biological relevance.
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
no effects observed
External malformations:
no effects observed
Skeletal malformations:
no effects observed
Visceral malformations:
no effects observed
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test substance-related and/or biologically relevant differences between test groups 0, 1, 2 and 3 (0, 100, 300 and 1000 mg/kg bw/d) in conception rate, in the mean number of corpora lutea and implantation sites or in the values calculated for the pre- and the postimplantation losses, the number of resorptions and viable fetuses. All observed differences are considered to reflect the normal range of fluctuations for animals of this strain and
age.

Two soft tissue variations, i.e. dilated renal pelvis and dilated ureter, were detected in all test groups including the controls. These are common findings in this rat strain and there is no doe-related increase of incidence. Thus, these variants are not considered to be biologically relevant.

For all test groups, skeletal variations of different bone structures were observed, with or without effects on corresponding cartilages. The observed skeletal variations were related to several parts of fetal skeletons and appeared without a relation to dosing.
The overall incidences of skeletal variations were comparable to the historical control data
Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes
other: please see below
Abnormalities:
not specified
Developmental effects observed:
not specified
Conclusions:
In conclusion, the no observed adverse effect level (NOAEL) for maternal toxicity is 300 mg/kg body weight/day based on temporary body weight loss and decreased (net) body weight gain in animals treated with 1000 mg/kg body weight/day N-formylmorpholin.
The no observed adverse effect level (NOAEL) for prenatal developmental toxicity is 300 mg/kg body weight/day, based on slightly reduced fetal weights and subsequent marginal delays of ossification. The fetal findings were minor and considered to be secondary to the body weight effects in the dams. Therefore, the test item does not cause selective prenatal developmental toxicity.
Executive summary:

In a prenatal developmental toxicity study the test compound N-formylmorpholine was administered to pregnant Wistar rats daily by gavage from implantation to one day prior to the expected day of parturition (GD 6-19) to evaluate its potential maternal and prenatal developmental toxicity.Analyses confirmed the correctness of the prepared concentrations and the stability of the test substance in the vehicle.

Generally, clinical observations revealed no toxicologically relevant difference between the dams receiving 100 or 300 mg/kg bw/d N-formylmorpholine and controls. The high-dose of the test substance (1000 mg/kg bw/d) produced marginal adverse effects in gestating female Wistar rats. Food consumption and body weight gain were significantly reduced shortly after onset of treatment (gestation days 6-8), but recovered afterwards. Corrected (net) body weight gain was also reduced (10% below control) in these animals, though not statistically significant. No differences of toxicological relevance were noted between the control and the treatmentgroups (100, 300 or 1000 mg/kg bw/d) for any reproductive parameters. This included conception rate, mean number of corpora lutea, mean number of implantations and pre- and postimplantation loss. Similarly, no influence of the test compound on sex distribution was noted at any dose. The mean fetal weights in the high-dose group (1000 mg/kg bw/d) were marginally, but statistically significantly reduced. These weight reductions in offspring go along with a body weight loss during early pregnancy and were in the same order of magnitude or less than a consistently reduced net body weight gain in the dams. Another consequence might be a marginal developmental delay as shown by an increased incidence of two incompletely ossified structures (skull and sternebra), both with no morphological change in the underlying anatomy. A number of feed restriction studies in rats have been conducted to evaluate the potential effects of maternal decreased body weight gain on embryo–fetal development, and were recently reviewed by Beyer et al. (2011). The outcome of these studies offers supporting evidence for the hypothesis that reduced maternal food intake, regardless of the cause, will result in maternal undernutrition which is well known to decrease fetal growth, with associated skeletal ossification delays.

In the present study, the marginally decreased fetal body weight and ossification delay occurred secondary to decreased net body weight gain in the dams at a very high dose (1000 mg/kg bw/d), with no other adverse fetal findings. It is therefore not considered evidence of a selective prenatal developmental toxicity.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
300 mg/kg bw/day
Study duration:
subacute
Species:
rat
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

In a prenatal developmental toxicity study the test compound N-formylmorpholine was administered to pregnant Wistar rats daily by gavage from implantation to one day prior to the expected day of parturition (GD 6-19) to evaluate its potential maternal and prenatal developmental toxicity. Analyses confirmed the correctness of the prepared concentrations and the stability of the test substance in the vehicle. Generally, clinical observations revealed no toxicologically relevant difference between the dams receiving 100 or 300 mg/kg bw/d N-formylmorpholine and controls.

The high-dose of the test substance (1000 mg/kg bw/d) produced marginal adverse effects in gestating female Wistar rats. Food consumption and body weight gain were significantly reduced shortly after onset of treatment (gestation days 6-8), but recovered afterwards. Corrected (net) body weight gain was also reduced (10% below control) in these animals, though not statistically significant.

No differences of toxicological relevance were noted between the control and the treatmentgroups (100, 300 or 1000 mg/kg bw/d) for any reproductive parameters. This included conception rate, mean number of corpora lutea, mean number of implantations and pre- and postimplantation loss. Similarly, no influence of the test compound on sex distribution was noted at any dose.

The mean fetal weights in the high-dose group (1000 mg/kg bw/d) were marginally, but statistically significantly reduced. These weight reductions in offspring go along with a body weight loss during early pregnancy and were in the same order of magnitude or less than a consistently reduced net body weight gain in the dams. Another consequence might be a marginal developmental delay as shown by an increased incidence of two incompletely ossified structures (skull and sternebra), both with no morphological change in the underlying anatomy. A number of feed restriction studies in rats have been conducted to evaluate the potential effects of maternal decreased body weight gain on embryo–fetal development, and were recently reviewed by Beyer et al. (2011). The outcome of these studies offers supporting evidence for the hypothesis that reduced maternal food intake, regardless of the cause, will result in maternal undernutrition which is well known to decrease fetal growth, with associated

skeletal ossification delays.

In the present study, the marginally decreased fetal body weight and ossification delay occurred secondary to decreased net body weight gain in the dams at a very high dose (1000 mg/kg bw/d), with no other adverse fetal findings. It is therefore not considered evidence of a selective prenatal developmental toxicity.

Justification for classification or non-classification

Based on the results obtained in the available OECD 421 and the OECD 414, classification for reproductive toxicity is not warranted according to Regulation (EC) No 1272/2008.

Additional information