4-morpholinecarbaldehyde

Toxicity to reproduction

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

4-morpholinecarbaldehyde

EC Number:

224-518-3

EC Name:

4-morpholinecarbaldehyde

Cas Number:

4394-85-8

Molecular formula:

C5H9NO2

IUPAC Name:

morpholine-4-carbaldehyde

Test material form:

other: liquid

Details on test material:

- Name of test substance: N-Formylmorpholin
- Test substance No.: 97/173-1
- Batch identification: O 2891
- Purity: 99.96 %
- Homogeneity: Given (visually)
- Storage stability:(stable under storage conditions)
- Expiry date: 16 May 2013

ADDITIONAL TEST SUBSTANCE INFORMATION

- Physical state/Appearance: Liquid / colorless, clear
- Storage conditions: Room temperature

Test animals

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)

Administration / exposure

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

Doses / concentrations

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

Examinations

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.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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 / performance (P0)

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.

Effect levels (P0)

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

Results: F1 generation

General toxicity (F1)

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.

Details on results (F1)

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.

Effect levels (F1)

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

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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.