4-morpholinecarbaldehyde

Developmental toxicity / teratogenicity

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

4-morpholinecarbaldehyde

EC Number:

224-518-3

EC Name:

4-morpholinecarbaldehyde

Cas Number:

4394-85-8

Molecular formula:

C5H9NO2

IUPAC Name:

morpholine-4-carbaldehyde

Test material form:

other: liquid

Details on test material:

- Name of test substance: N-Formylmorpholin
- Batch No.: O 2891
- CAS No.: 4394-85-8
- Purity: min. 99.5 %
- Homogeneity: Given (visually)
- Expiry date: 16 May 2013
- Physical state/appearance: Liquid / colorless, clear
- Storage conditions: Room temperature

Test animals

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

Administration / exposure

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

Doses / concentrations

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

Examinations

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

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

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

Maternal developmental toxicity

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

Effect levels (maternal animals)

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

Results (fetuses)

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

Effect levels (fetuses)

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

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Applicant's summary and conclusion

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.