4-(1-oxo-2-propenyl)-morpholine

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 2000-08-23 to 2001-03-02

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 2000-08-23 to 2001-03-02

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

1996

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.6200 (neurotoxicity screening battery)

Version / remarks:

1998

Deviations:

no

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

Lot No.: 0600605-P
Purity: 99.50%

Species:

rat

Strain:

other: CD

Details on species / strain selection:

The rat was chosen because of its acceptance as a predictor of toxic change in man and the requirement for a rodent species by regulatory agencies. The CD strain was used because of the historical control data available in this laboratory for the general toxicity, neurobehavioral and reproductive parameters recorded in this study.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Limited
- Females (if applicable) nulliparous and non-pregnant: not specified
- Age at study initiation: 30 ± 1 days
- Weight at study initiation: Males: 119 to 185 g; Females: 131 to 174 g
- Housing: housed (TR18 cages) in groups of five of the same sex, unless reduced by mortality or isolation. TR18 cages were obtained from Arrowmight Biosciences, Hereford and consisted of stainless steel bodies with lids and floors of stainless steel grid. These cages were suspended in batteries over trays lined with absorbent paper that was changed at appropriate intervals.
- Diet: An expanded rodent diet, Rat and Mouse No. 1 Maintenance Diet, ad libitum
- Water: Water, taken from the public supply was freely available via polyethylene or polycarbonate bottles fitted with sipper tubes.
- Acclimation period: 9 days

DETAILS OF FOOD AND WATER QUALITY:
Both diet suppliers provided a Certificate of Analysis with every batch of diet. Both diets contained no added antibiotic or other chemotherapeutic or prophylactic agent.
The quality of the water supply is governed by regulations published by the Department of the Environment. Certificates of analysis were routinely received from the supplier.
No contaminants were reasonably expected to be present in water, diet or bedding at levels known to be capable of interfering with the progress or outcome of this study. No other specific contaminants that were likely to have been present in the diet or water were analysed, as none that may have interfered with or prejudiced the outcome of the study was known.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-24°C
- Humidity (%): 51-67%
- Air changes (per hr): Each animal room was kept at positive pressure with respect to the outside by its own supply of filtered fresh air which was passed to atmosphere and not re-circulated, providing approximately 15 air changes per hour.
- Photoperiod (hrs dark / hrs light): a 12-hour light: 12-hour dark cycle with the lights on at 06:00 GMT.

IN-LIFE DATES: From: 2000-08-23 To: 2000-12-06

Route of administration:

oral: gavage

Details on route of administration:

Animals received the test substance formulations by oral gavage at a volume-dosage of 10 mL/kg bodyweight. All animals were dosed in ascending dosage order once daily at approximately the same time each day. The volume administered to each animal was calculated from the most recently recorded scheduled bodyweight. Control animals received the vehicle at the same volume dosage over the same treatment period.

Vehicle:

water

Remarks:

reverse osmosis (RO) water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Formulations were prepared by stirring test item with an appropriate amount of reverse osmosis (RO) water until it dissolved. This solution was then diluted to the required volume and magnetically stirred for at least 5 minutes. Formulations were prepared on a weekly basis and any samples taken for analysis were taken from the bulk formulation.

VEHICLE
- Concentration in vehicle: 0.5, 2.0 or 7.5 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The analytical procedure validation, the homogeneity and stability during ambient temperature storage for 3 hours, and refrigerated storage for 10 days were confirmed for test item in aqueous formulations at 0.5 mg/mL and 7.5 mg/mL. Samples of each formulation prepared for administration on one occasion during Weeks 1, 6 and 12 of the study were analysed for test material content and found to be satisfactory.

Duration of treatment / exposure:

13 weeks, except that females of the Reproductive sub-group were administered for two weeks prior to pairing, throughout pairing and gestation until Day 6 of lactation.

Frequency of treatment:

Once daily

Dose / conc.:

5 mg/kg bw/day (actual dose received)

Dose / conc.:

20 mg/kg bw/day (actual dose received)

Dose / conc.:

75 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

Toxicity sub-group: 10 animals per sex per dose
Neurotoxicity sub-group: 5 animals per sex per dose
Reproductive sub-group: 10 females per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
In the 7-day preliminary study it was concluded that 150 mg/kg/day was unsuitable for repeated administration over 28 days due to effects observed on bodyweights and progression of clinical signs. A high dosage of 50 mg/kg/day was subsequently investigated in the 28-day study and was assessed to be the Lowest Observed Adverse Effect Level (LOAEL) with 15 mg/kg/day as the No Observed Adverse Effect Level (NOAEL). In view of the minimal effects observed at 50 mg/kg/day in the 28-day study, it was considered that this might be regarded as too cautious a choice for this study. A high dosage lying between 50 and 150 mg/kg/day was considered more appropriate. 75 mg/kg/day represents a 50% increase from the dosage used in the 28-day study and is exactly half of the dosage considered to be too high in the 7-day study.
The lower dosage represents a four fold interval with the low dosage of 5 mg/kg/day anticipated to be a no-observed effect level equating to the low dosage group (4.5 mg/kg/day) used in the 28-day study.
- Rationale for animal assignment: using a pseudo-random procedure to yield groups with approximately equal mean bodyweights.
- Fasting period before blood sampling for clinical biochemistry: overnight

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals and their cages/cage trays were inspected at least twice daily for evidence of reaction to treatment or ill-health.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: weighed on the day that treatment commenced, weekly thereafter, and at necropsy.

FOOD CONSUMPTION: Yes
- Time schedule: With the exception of Toxicity sub-group males during the period of pairing with Reproductive subgroup females, food consumption was recorded weekly, from the commencement of treatment

OPHTHALMIC EXAMINATION: Yes
- Time schedule for examinations: During Week 13 of treatment, prior to orbital sinus blood sampling
- Dose groups that were examined: all Control and high dosage Toxicity sub-group animals

HAEMATOLOGY AND BLOOD CHEMISTRY: Yes
- Time schedule for collection of blood: During Week 13
- Anaesthetic used for blood collection: Yes (Isoflurane)
- Animals fasted: overnight
- How many animals: all animals in the Toxicity sub-group
- Parameters checked:
Samples using EDTA as an anticoagulant were examined using a Technicon H-l haematology analyser for the following characteristics:
Haematocrit (Hct), Haemoglobin concentration (Hb), Erythrocyte count (RBC), Total and differential leucocyte count (WBC), Platelet count (Plt), Mean cell haemoglobin concentration (MCHC), Mean cell haemoglobin (MCH), Mean cell volume (MCV)
Blood film - Romanowsky stain, examined by light microscopy for abnormal morphology and unusual cell types, including normoblasts.
Using citrate as an anticoagulant, samples were examined for Prothrombin time (PT).
All samples using lithium heparin as an anticoagulant were examined for:
Alkaline phosphatase (Alk. Phos), Alanine amino-transferase (ALT), Aspartate amino-transferase (AST), Gamrna-glutamyl transpeptidase (gGT), Omithine carbamyl transferase (OCT), Glucose (Gluc), Total bilirubin (Bili. Total), Total cholesterol (Chol Total), Creatinine (Creat), Urea, Total protein (Total Prot), Albumin (Alb), Albumin/globulin ratio (AlG Ratio), Sodium (Na), potassium (K) and Chloride (Cl), Calcium concentration (Ca Total), Inorganic phosphorus (Phos)

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations:
Home cage observations: Before commencement of treatment (Week -1) and during Weeks 1, 2, 4, 8 and 12, at approximately the same time of day.
In the hand and standard arena observations: Before commencement of treatment (Week -1) and during Weeks 1, 2, 4, 8 and 12, at approximately the same time of day, after removal from the home cage
Manipulations: Reflexes and responses were assessed before commencement of treatment (Week -1) and during Week 12, at approximately the same time of day.
Motor activity: assessed before commencement of treatment (Week -1) and during Week 12, immediately following the manipulation tests.
- Dose groups that were examined: All Neurotoxicity sub-group animals and 5 males and 5 females from the Toxicity sub-group
- Battery of functions tested: Home cage observations, In the hand and standard arena observations, Manipulations, Motor activity

Sacrifice and pathology:

GROSS PATHOLOGY: Yes.
- Toxicity sub-group animals:
Toxicity sub-group animals were killed, following the 13 week treatment period, by carbon dioxide inhalation and subjected to a detailed necropsy. The necropsy procedure included a review of the history of each animal and a detailed examination of the external features and orifices, the neck and associated tissues and the cranial, thoracic, abdominal and pelvic cavities and their viscera. The requisite organs were weighed after being dissected free of adjacent fat and other contiguous tissue External and cut surfaces of the organs and tissues were examined as appropriate, abnormalities and interactions were noted and the required tissue samples and any abnormalities preserved in appropriate fixative.
- Neurotoxicity sub-group animals:
Following the 13 week treatment period, Neurotoxicity sub-group animals were administered sodium pentobarbitone by intra-peritoneal injection to induce deep anaesthesia. The animals were then killed by exposure of the heart and perfusion of Karnovskis fixative via the aorta. Perfusion fixation was used to ensure the highest order of preservation of nervous tissue.
The necropsy procedure included a review of the history of each animal and a detailed examination of the external features and orifices, the neck and associated tissues and the cranial, thoracic, abdominal and pelvic cavities and their viscera. The brain was weighed. External and cut surfaces of the organs and tissues were examined as appropriate, abnormalities and interactions were noted and the required tissue samples and any abnormalities preserved in appropriate fixative.

HISTOPATHOLOGY: Yes
- Toxicity sub-group animals:
With the exception of the brain, optic nerves, sciatic nerves and spinal cord, organs (including abnormalities) preserved at macroscopic necropsy were processed for all decedents and for animals from the Control and high dosage groups (Groups 3 and 2). Tissue samples were dehydrated, embedded in paraffin wax and sectioned at approximately four to five micron thicknesses. Staining was with haematoxylin and eosin, except testes, which were stained with periodic acid/schiff (PAS). Only one skeletal muscle was sample was processed.
Tissues examined: Adrenals, Epididymides, Femur, Heart, Kidneys, Liver, Lungs, Sternum, Stomach, Thyroid, Uterus
- Neurotoxicity sub-group animals:
Processing of tissue, samples was undertaken for animals from Control and high dosage groups (Groups 3 and 2).
Tissues examined: Brain, Dorsal root ganglia, Dorsal and ventral root fibres, Sciatic nerve, Spinal cord, Eyes and optic nerves (both), Skeletal (gastrocnemius) muscle (right), Sciatic nerve, Tibial nerve

Statistics:

- Bodyweights, Bodyweight changes, Organ weights:
Homogeneity of variance was tested using Bartlett's test (Bartlett, 1937). Whenever this was found to be statistically significant a Behrens-Fisher test (Cochran and Cox, 1957) was used to perform pairwise comparisons, otherwise a Dunnett's test (Dunnett, 1955/64) was used.
- Functional Observation Battery numerical data:
If the data consisted predominantly of one particular value (relative frequency of the mode exceeds 75%), the proportion of animals with different values from the mode was analysed using Fisher's Exact test. Otherwise, Bartlett's test was performed to test for variance heterogeneity between groups. Where significant (1% level) heterogeneity was found, the data were logarithmically transformed and re-tested for heterogeneity. If no statistically significant heterogeneity of variance was detected (with or without logarithmic transformation), a one way analysis of variance was carried out. If the analysis of variance showed evidence (at the 5% level) of differences between the groups, Student's t-test was used to test for differences between treatment groups and the Control group. If heterogeneity was significant and could not be stabilised by logarithmic transformation, the Kruskal-Wallis test on ranks was performed on the untransformed data. If the Kruskal-Wallis test showed evidence (5% level) of differences between the groups, the Wilcoxon Rank-Sum test was used to test for differences between the treatment groups and the Control group.
- Macropathology and micropathology: Fisher's Exact test

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Clinical signs associated with treatment were restricted to post dose salivation for both sexes at 75 mg/kg/day. For Toxicity and Neurotoxicity sub-group animals approximately half the males and the majority of females were affected, although generally only for isolated occasions. The incidence of post dose salivation tended to be higher during the latter half of the treatment period and the lower frequency observed in the Reproductive sub-group females probably reflects their shorter treatment period. Post dose salivation is frequently observed in animals dosed via the oral gavage route and is generally considered to reflect distaste of the dosing formulation.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

There were two unscheduled deaths on the study; while both were among males receiving 20 mg/kg/day, neither death was considered to be related to treatment.
Animal number 52 was noted to have maloccluded teeth during Week 1 of the study. Although the teeth were regularly trimmed to try to prevent this having a detrimental effect on the animal's health, by Week 5 of the study it was considered necessary to kill the animal for humane reasons. It is likely that this condition pre-dated allocation to the study but was not apparent due to the small size of the animal.
Animal number 48 was noted to have Iimited use of a swollen left hindlimb during Week 8. Although the animal was isolated and rehoused in a cage with a solid-bottomed floor, the condition deteriorated and it was considered necessary to kill this animal for humane reasons during Week 11. Necropsy and histopathology confirmed the swelling and inflammation of the left hindpaw and showed atrophy of the muscle in the left hindlimb. No other similar problems were observed in animals on the study including those receiving 75 mg/kg/day and it is considered that this occurrence is coincidental and unrelated to treatment.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

For Toxicity and Neurotoxicity sub-group animals at 75mg/kg/day, combined overall bodyweight gain to Week 13 was lower than Control for both sexes, with differences in bodyweight attaining statistical significance towards the end of the treatment period.
Bodyweight gain of Toxicity and Neurotoxicity sub-group females at 5 and 20 mg/kg/day, although slightly lower than the concurrent Control, was not considered to have been affected by treatment and bodyweight gain of males in the equivalent group showed no intergroup difference.

For Reproductive sub-group females at 75mg/kg/day there was a suggestion of lower gain, compared with Control, during the two week pre-pairing period. Cumulative bodyweight gain was also slightly lower for females during gestation at this dosage but, although bodyweight at Day 1 of lactation was still lower than Control subsequent weight gain to Day 7 of lactation was superior.
Bodyweight gain of Reproductive sub-group females at lower dosages during the pre-pairing period, during gestation and during the first week of lactation was not considered to have been affected by treatment.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

There was no marked effect of treatment on combined food consumption of Toxicity and Neurotoxicity sub-group males and females at any of the dosages investigated, although a lower intake was noted for males at 75 mg/kg/day towards the end of the study when compared with Control values.
Food consumption of Reproductive sub-group females was not obviously affected by treatment during the two week pre-pairing period or during gestation and lactation.

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

At 75 mg/kg/day the efficiency of food utiIisation was generally lower than Control for both sexes, although the effect was more marked in males, particularly in the latter half of the treatment period, and reflected the lower overall bodyweight gain observed at this dosage.
Food conversion efficiency at 5 and 20 mg/kg/day was not obviously affected by treatment.

Ophthalmological findings:

no effects observed

Description (incidence and severity):

Ophthalmic examination during Week 13 of Toxicity sub-group Control animals and those receiving 75 mg/kg/day did not reveal any adverse effects of treatment.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Haematology investigations in Toxicity sub-group males and females during the last week of treatment did not reveal any consistent differences indicative of an obvious adverse effect of treatment, values obtained being essentially similar in all groups.
It was noted for males that white blood cell count was slightly higher than Control, principally due a slight increase in lymphocyte count. These differences were slight and were not considered to be of toxicological significance.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Blood chemistry investigations in Toxicity sub-group males and females during the last week of treatment did not indicate any obvious adverse effect of treatment.
Blood glucose levels were noted to be lower than Control for males at 75 mg/kg/day and for females at all dosages. However intergroup differences were small, showed no clear dosage relationship and were not considered to be of toxicological significance.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Intergroup difference in absolute and bodyweight-relative organ weights of Toxicity sub-group animals did not indicate any adverse effect of treatment. A slight reduction in absolute brain weight, but with a concomitant increase in bodyweight-relative values, was noted for males at 75 mg/kg/day and was considered to reflect the lower bodyweight observed at this dosage.
Other occasional differences (e.g. lower absolute heart and adrenal weights for males at 75 mg/kg/day) were not consistent for both sexes and were not considered to be of toxicological significance.
Brain weights for Neurotoxicity sub-group animals were consistent with that observed for Toxicity sub-group animals, with males at 75 mg/kg/day again showing a slight reduction in absolute brain weight and an increase in bodyweight-relative values.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Macroscopic observations at necropsy of Toxicity and Neurotoxicity sub-group animals following 13 weeks of treatment were unremarkable and did not indicate any obvious adverse effect of treatment.
Macroscopic observations al necropsy of Reproductive sub-group females on Day 7 of lactation were limited to a misshappen liver for one female receiving 75 mg/kg/day.

Neuropathological findings:

no effects observed

Description (incidence and severity):

Home cage, in the hand and arena observations, manipulations and motor activity showed no findings considered to be of neurotoxicological significance.

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Microscopic examination was confined to Toxicity and Neurotoxicity sub-group animals. Findings observed were of a type and severity commonly seen in animals of this age at this laboratory and there were no findings considered to be related to treatment.

Dose descriptor:

NOEL

Effect level:

20 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Remarks on result:

other: for general systemis toxic

Dose descriptor:

NOEL

Effect level:

75 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

neuropathology

Remarks on result:

other: for neurobehavioral toxicity

Dose descriptor:

NOAEL

Effect level:

> 75 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

food efficiency

gross pathology

haematology

histopathology: non-neoplastic

mortality

neuropathology

ophthalmological examination

organ weights and organ / body weight ratios

Critical effects observed:

no

Conclusions:

Other than lower bodyweight gain, particularly towards the end of the treatment period, treatment of test item at 75 mg/kg/day for 13 weeks was generally well tolerated by both male and female rats. While this bodyweight effect precludes 75 mg/kg/day from being classified as an overall no-effect level, this dosage was considered to be a no effect level for neurobehavioral toxicity.
Based on the findings of this study the no-effect-level for general systemic toxic is considered to be 20 mg/kg/day.

Executive summary:

The study was performed to assess the overall toxic potential, including a screen for reproductive effects, of test item when administered daily by oral gavage to the CD rat according to OECD 422 and 408. Animals on the study were distributed to three sub-groups (Toxicity, Neurotoxicity and Reproductive), with dosages of 0 (Control), 5, 20 and 75 mg/kg/day being investigated in each sub-group. The Toxicity sub-group consisted of 10 males and 10 females per group and these animals received 13 weeks of continuous daily treatment with ophthalmic, haematology and blood chemistry investigations being conducted during the final week of treatment. Organ weights were recorded at termination and tissues preserved and processed for microscopic examination. The Neurotoxicity sub-group consisted of 5 males and 5 females per group; these animals received 13 weeks of continuous daily treatment and were subjected to periodic neurobehavioural screening. At necropsy these animals were perfused with fixative to allow a full microscopic examination of the nervous system. The pool of animals assessed for neurobehavioural effects was increased to 10 per sex per group by subjecting 5 males and 5 females from the Toxicity sub-group to the same procedures. The Reproductive sub-group consisted of 10 females per group and animals were given continuous daily treatment until termination. All Control groups received the vehicle alone on the same occasions as the treated animals.

 

There were two deaths among males at 20 mg/kg/day, neither death was considered to be related to treatment.

Clinical signs associated with treatment were restricted to post dose salivation for both sexes at 75 mg/kg/day. This sign was generally observed on isolated occasion with a higher incidence in the latter half of the treatment period for Toxicity and Neurotoxicity sub-group animals.

At 75 mg/kg/day, combined bodyweight gain to Week 13 of Toxicity and Neurotoxicity sub-group animals was lower than Control for both sexes, with differences in bodyweight attaining statistical significance towards the end of the treatment period. For Reproductive sub-group females there was a suggestion of lower gain, compared with Control, during the two week pre-pairing period. Cumulative bodyweight gain was slightly lower for females during gestation and bodyweight at Day 1 of lactation was also lower.

At 5 and 20 mg/kg/day bodyweight gain of Toxicity, Neurotoxicity and Reproductive sub-group animals was unaffected by treatment.

There was no clear adverse effect of treatment observed on food intake at any of the dosages investigated. At 75 mg/kg/day food conversion efficiency was generally lower than Control, particularly for males during the last few weeks of treatment, reflecting the lower overall bodyweight gain observed at this dosage. At 5 and 20 mg/kg/day food conversion efficiency was not obviously affected by treatment.

Ophthalmic examination during Week 13 of Toxicity sub-group Controls and animals receiving 75 mg/kg/day did not reveal any adverse effects of treatment.

Haematology and blood chemistry investigations in Toxicity sub-group animals during the last week of treatment did not indicate any obvious adverse effect of treatment.

Neurobehavioural screening showed no findings considered to be of neurotoxicological significance, at any of the dosages investigated.

Occasional intergroup differences in absolute and bodyweight-relative organ weights of Toxicity subgroup animals were considered to reflect the effect on overall bodyweight and did not indicate any adverse effect of treatment.

Macroscopic necropsy of Toxicity, Neurotoxicity and Reproductive sub-group animals did not reveal any adverse effect of treatment.

Microscopic examination of Toxicity and Neurotoxicity sub-group animals at 75 mg/kg/day did not reveal any changes considered to be related to treatment.

Other than lower bodyweight gain, particularly towards the end of the treatment period, treatment of test item at 75 mg/kg/day for 13 weeks was generally well tolerated by both male and female rats. While this bodyweight effect precludes 75 mg/kg/day from being classified as an overall no-effect level, this dosage was considered to be a no effect level for neurobehavioral toxicity.

Based on the findings of this study the no-effect-level for general systemic toxic is considered to be 20 mg/kg/day.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2001

Report date:

2001

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

1996

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

4-(1-oxo-2-propenyl)-morpholine

EC Number:

418-140-1

EC Name:

4-(1-oxo-2-propenyl)-morpholine

Cas Number:

5117-12-4

Molecular formula:

C7H11NO2

IUPAC Name:

1-(morpholin-4-yl)prop-2-en-1-one

Specific details on test material used for the study:

Lot No.: 0600605-P
Purity: 99.50%

Test animals

Species:

rat

Strain:

other: CD

Details on species / strain selection:

The rat was chosen because of its acceptance as a predictor of toxic change in man and the requirement for a rodent species by regulatory agencies. The CD strain was used because of the historical control data available in this laboratory for the general toxicity, neurobehavioral and reproductive parameters recorded in this study.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Limited
- Females nulliparous and non-pregnant: not specified
- Age at study initiation: (P) Males ca. 7 wks; (F1) ca. 8 wks
- Weight at study initiation: (P) Males: 119-185 g; Females: 217 - 290 g
- Housing: Males housed (TR18 cages) in groups of five of the same sex, unless reduced by mortality or isolation. Females were housed (TR18 cages) in groups of five during the two week pre-pairing treatment period. Females were then paired on a one to one basis with Toxicity sub-group males (RB3 modified cages) for up to 14 days for mating. Following a positive indication of mating, females were individually housed during gestation (RB3 modified cages and RB3 cages) and with their litters during lactation (RB3 cages).
- Diet: Males: An expanded rodent diet, Rat and Mouse No. 1 Maintenance Diet, ad libitum; Females: With the exception of the period of cohabitation, females were given non-restricted access to a pelleted rodent diet, UAR VRF 1 certified (Charles River UK Limited, Margate, Kent, England) formulated as a breeding diet.
- Water: Water, taken from the public supply was freely available via polyethylene or polycarbonate bottles fitted with sipper tubes.
- Acclimation period: Males: 9 days; Females: ca 3 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-24°C
- Humidity (%): 51-67%
- Air changes (per hr): Each animal room was kept at positive pressure with respect to the outside by its own supply of filtered fresh air which was passed to atmosphere and not re-circulated, providing approximately 15 air changes per hour.
- Photoperiod (hrs dark / hrs light): a 12-hour light: 12-hour dark cycle with the lights on at 06:00 GMT.

IN-LIFE DATES: Males: From 2000-08-23 To 2000-12-06; Females: From 2000-08-23 To 2000-11-04

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

reverse osmosis (RO) water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Formulations were prepared by stirring test item with an appropriate amount of reverse osmosis (RO) water until it dissolved. This solution was then diluted to the required volume and magnetically stirred for at least 5 minutes. Formulations were prepared on a weekly basis and any samples taken for analysis were taken from the bulk formulation.

VEHICLE
- Concentration in vehicle: 0.5, 2.0 or 7.5 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg

Details on mating procedure:

- M/F ratio per cage: one-to-one
- Length of cohabitation: up to 2-weeks, all animals mated within the first week of pairing
- Proof of pregnancy: The day on which a sperm positive vaginal smear or at least three copulation plugs were found was designated Day 0 of gestation.
- After successful mating each pregnant female was caged: individually housed during gestation (RB3 modified cages and RB3 cages) and with their litters during lactation (RB3 cages)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The analytical procedure validation, the homogeneity and stability during ambient temperature storage for 3 hours, and refrigerated storage for 10 days were confirmed for test item in aqueous formulations at 0.5 mg/mL and 7.5 mg/mL. Samples of each formulation prepared for administration on one occasion during Weeks 1, 6 and 12 of the study were analysed for test material content and found to be satisfactory.

Duration of treatment / exposure:

Females: two weeks prior to pairing, throughout pairing and gestation until Day 6 of lactation;
Males: 13 weeks, two weeks before commencement of treatment for females

Frequency of treatment:

Once a day

Details on study schedule:

- Age at mating of the mated animals in the study: ca. 10 weeks

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

5 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

20 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

75 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

10 animals per sex per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
In the 7-day preliminary study it was concluded that 150 mg/kg/day was unsuitable for repeated administration over 28 days due to effects observed on bodyweights and progression of clinical signs. A high dosage of 50 mg/kg/day was subsequently investigated in the 28-day study and was assessed to be the Lowest Observed Adverse Effect Level (LOAEL) with 15 mg/kg/day as the No Observed Adverse Effect Level (NOAEL). In view of the minimal effects observed at 50 mg/kg/day in the 28-day study, it was considered that this might be regarded as too cautious a choice for this study. A high dosage lying between 50 and 150 mg/kg/day was considered more appropriate. 75 mg/kg/day represents a 50% increase from the dosage used in the 28-day study and is exactly half of the dosage considered to be too high in the 7-day study.
The lower dosage represents a four fold interval with the low dosage of 5 mg/kg/day anticipated to be a no-observed effect level equating to the low dosage group (4.5 mg/kg/day) used in the 28-day study.
- Rationale for animal assignment: using a pseudo-random procedure to yield groups with approximately equal mean bodyweights.
- Fasting period before blood sampling for clinical biochemistry: overnight

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals and their cages/cage trays were inspected at least twice daily for evidence of reaction to treatment or ill-health.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly.

BODY WEIGHT: Yes
- Time schedule for examinations: Males: weighed on the day that treatment commenced, weekly thereafter, and at necropsy; Females: weighed on the first day of treatment, weekly until pairing and on Days 0, 3, 6, 9, 13, 17 and 20 after mating, and Days 1, 4 and 7 of lactation.

FOOD CONSUMPTION: Yes
- Time schedule: With the exception of the period of mating, food consumption was recorded weekly, from the commencement of treatment

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals were killed, following the 13 week treatment period
- Maternal animals: All surviving animals were killed on Day 7 of lactation

GROSS NECROPSY
- Gross necropsy consisted of :
Females: The number of implantation sites was recorded and the ovaries (with oviducts), uterus and cervix, vagina, pituitary and mammary tissue retained in appropriate fixative.
Males: The necropsy procedure included a review of the history of each animal and a detailed examination of the external features and orifices, the neck and associated tissues and the cranial, thoracic, abdominal and pelvic cavities and their viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
Males: With the exception of the brain, optic nerves, sciatic nerves and spinal cord, organs (including abnormalities) preserved at macroscopic necropsy were processed for all decedents and for animals from the Control and high dosage groups.
Females: restricted to abnormalities observed at macroscopic necropsy

Statistics:

- Bodyweights and Bodyweight changes for males and for females prior to pairing:
Homogeneity of variance was tested using Bartlett's test (Bartlett, 1937). Whenever this was found to be statistically significant a Behrens-Fisher test (Cochran and Cox, 1957) was used to perform pairwise comparisons, otherwise a Dunnett's test (Dunnett, 1955/64) was used.
- Bodyweights and Food consumption during gestation and lactation, Litter size, Survival indices, Sex ratio (percentage male), Offspring bodyweights/changes:
If the data consisted predominantly of one particular value (relative frequency of the mode exceeds 75%), the proportion of animals with different values from the mode was analysed using Fisher's Exact test (Fisher, 1950). Otherwise, Bartlett's test was performed to test for variance heterogeneity between groups. Where significant (1 % level) heterogeneity was found, the data were logarithmically transformed and re-tested for heterogeneity. If no statistically significant heterogeneity of variance was detected (with or without logarithmic transformation); a one way analysis of variance (Snedecor and Cochran, 1967) was carried out. If the analysis of variance showed evidence (at the 5% level) of differences between the groups, Williams' test (Williams' 1971/2) was performed.
If heterogeneity was significant and could not be stabilised by logarithmic transformation, the Kruskal-Wallis test (Kruskal-Wallis, Hollander and Wolfe 1973) on ranks was performed on the untransformed data. If the Kruskal-Wallis test showed evidence (5% level) of differences between the groups, Shirley's test (Shirley 1977) was performed.
- Macropathology and micropathology: Fisher's Exact test

Reproductive indices:

Percentage mating, Conception rate, Fertility index, Gestation length, Gestation index

Offspring viability indices:

Post-implantation survival index, Live birth index, Viability index, Group mean Iitter size, Group mean offspring bodyweight, Sex ratio

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Clinical signs associated with treatment were restricted to post dose salivation for both sexes at 75 mg/kg/day. Approximately half the males and the majority of females were affected, although generally only for isolated occasions. The incidence of post dose salivation tended to be higher during the latter half of the treatment period and the lower frequency observed in the females probably reflects their shorter treatment period. Post dose salivation is frequently observed in animals dosed via the oral gavage route and is generally considered to reflect distaste of the dosing formulation.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

There were two unscheduled deaths on the study; while both were among males receiving 20 mg/kg/day, neither death was considered to be related to treatment.
Animal number 52 was noted to have maloccluded teeth during Week 1 of the study. Although the teeth were regularly trimmed to try to prevent this having a detrimental effect on the animal's health, by Week 5 of the study it was considered necessary to kill the animal for humane reasons. It is likely that this condition pre-dated allocation to the study but was not apparent due to the small size of the animal.
Animal number 48 was noted to have Iimited use of a swollen left hindlimb during Week 8. Althoug h the animal was isolated and rehoused in a cage with a solid-bottomed floor, the condition deteriorated and it was considered necessary to kill this animal for humane reasons during Week 11. Necropsy and histopathology confirmed the swelling and inflammation of the left hindpaw and showed atrophy of the muscle in the left hindlimb. No other similar problems were observed in animals on the study including those receiving 75 mg/kg/day and it is considered that this occurrence is coincidental and unrelated to treatment.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

For males at 75mg/kg/day, combined overall bodyweight gain to Week 13 was lower than Control, with differences in bodyweight attaining statistical significance towards the end of the treatment period. Bodyweight gain of males in the lower groups showed no intergroup difference.
For females at 75mg/kg/day, there was a suggestion of lower gain, compared with Control, during the two week pre-pairing period. Cumulative bodyweight gain was also slightly lower for females during gestation at this dosage but, although bodyweight at Day 1 of lactation was still lower than Control subsequent weight gain to Day 7 of lactation was superior.
Bodyweight gain of females at lower dosages during the pre-pairing period, during gestation and during the first week of lactation was not considered to have been affected by treatment.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Males: There was no marked effect of treatment on combined food consumption at any of the dosages investigated, although a lower intake was noted at 75 mg/kg/day towards the end of the study when compared with Control values.
Females: Food consumption was not obviously affected by treatment during the two week pre-pairing period or during gestation and lactation.

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Males: Macroscopic observations did not indicate any obvious adverse effect of treatment.
Females: limited to a misshappen liver for one female receiving 75 mg/kg/day.

Reproductive function / performance (P0)

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

There were no clear effects of treatment on fertility and reproductive performance. All females successfully mated, gave birth to a live litter and reared young to Day 7 of age.

Details on results (P0)

With the exception of one female at 75 mg/kg/day, all females mated within four days of pairing. AII matings lead to a successful pregnancy with all females successfully producing and rearing a litter to 7 days of age.
There was no clear effect on the duration of pregnancy, although it was noted that 3/10 females at 75 mg/kg/day had a gestation length of 23 days; this is not unusual for this strain of rat even through no similar gestation lengths were recorded in other groups in this particular study.
With the exception of one female at 75 mg/kg/day, all females mated within four days of pairing. AII matings lead to a successful pregnancy with all females successfully producing and rearing a litter to 7 days of age.
There was no clear effect on the duration of pregnancy, although it was noted that 3/10 females at 75 mg/kg/day had a gestation length of 23 days; this is not unusual for this strain of rat even through no similar gestation lengths were recorded in other groups in this particular study.

Effect levels (P0)

Dose descriptor:

NOEL

Effect level:

20 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Target system / organ toxicity (P0)

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Mortality / viability:

no mortality observed

Description (incidence and severity):

Pup survival to Day 7 of age was similarly unaffected by treatment.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

There was no obvious adverse effect of treatment on bodyweight at birth or subsequent bodyweight gain to Day 7 for male or female offspring, values for treated groups being essentially similar to Control.

Details on results (F1)

There was no obvious adverse effect on implantation rate (assessed at necropsy) or litter size at Day 1 of lactation. Subsequent pup survival to Day 7 of age was similarly unaffected by treatment.
Sex ratio at Day 1 and Day 7 was similar for all groups and did not indicate any selective adverse effect of treatment for either sex.
There was no obvious adverse effect of treatment on bodyweight at birth or subsequent bodyweight gain to Day 7 for male or female offspring, values for treated groups being essentially similar to Control.

Effect levels (F1)

1

Dose descriptor:

NOEL

Generation:

F1

Effect level:

75 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

mortality

body weight and weight gain

2

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

> 75 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

body weight and weight gain

Target system / organ toxicity (F1)

Critical effects observed:

no

Overall reproductive toxicity

Reproductive effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

Other than lower bodyweight gain, particularly towards the end of the treatment period, treatment of test item at 75 mg/kg/day for 13 weeks was generally well tolerated by both male and female rats. While this bodyweight effect precludes 75 mg/kg/day from being classified as an overall no-effect level. Based on the findings of this study the no-effect-level for general systemic toxic is considered to be 20 mg/kg/day.
A reproduction developmental toxicity screening test performed during the course of the study did not identify any major cause for concern at dosages of up to 75 mg/kg/day.

Executive summary:

The study was performed to assess the overall toxic potential, including a screen for reproductive effects, of test item when administered daily by oral gavage to the CD rat according to OECD 422 and 408. Animals on the study were distributed to three sub-groups (Toxicity, Neurotoxicity and Reproductive), with dosages of 0 (Control), 5, 20 and 75 mg/kg/day being investigated in each sub-group. The Toxicity sub-group consisted of 10 males and 10 females per group and these animals received 13 weeks of continuous daily treatment. The Reproductive sub-group consisted of 10 females per group and animals were given continuous daily treatment until termination. Following two weeks of treatment these females were paired with Toxicity sub-group males, which had been treated for four weeks, for assessment of fertility and reproductive function. The males were allowed up to 14 days cohabitation after which they returned to the mainstream toxicity study; their females partners were allowed to litter and rear their young until Day 7 of age when parent females and litters were subjected to macroscopic necropsy. All Control groups received the vehicle alone on the same occasions as the treated animals.

 

There were two deaths among males at 20 mg/kg/day, neither death was considered to be related to treatment.

Clinical signs associated with treatment were restricted to post dose salivation for both sexes at 75 mg/kg/day. This sign was generally observed on isolated occasion with a higher incidence in the latter half of the treatment period for Toxicity sub-group animals.

At 75 mg/kg/day, combined bodyweight gain to Week 13 of Toxicity sub-group animals was lower than Control for both sexes, with differences in bodyweight attaining statistical significance towards the end of the treatment period. For Reproductive sub-group females there was a suggestion of lower gain, compared with Control, during the two week pre-pairing period. Cumulative bodyweight gain was slightly lower for females during gestation and bodyweight at Day 1 of lactation was also lower.

At 5 and 20 mg/kg/day bodyweight gain was unaffected by treatment.

There was no clear adverse effect of treatment observed on food intake at any of the dosages investigated.

Macroscopic necropsy did not reveal any adverse effect of treatment.

There were no clear effects of treatment on fertility and reproductive performance. All females successfully mated, gave birth to a live litter and reared young to Day 7 of age. Litter size, pup survival and pup growth were similar in all groups.

Other than lower bodyweight gain, particularly towards the end of the treatment period, treatment of test item at 75 mg/kg/day for 13 weeks was generally well tolerated by both male and female rats. While this bodyweight effect precludes 75 mg/kg/day from being classified as an overall no-effect level. Based on the findings of this study the no-effect-level for general systemic toxic is considered to be 20 mg/kg/day.

A reproduction developmental toxicity screening test performed during the course of the study did not identify any major cause for concern at dosages of up to 75 mg/kg/day.