Registration Dossier

Diss Factsheets

Toxicological information

Developmental toxicity / teratogenicity

Currently viewing:

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
30.10.2007 - 13.02.2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report date:
2009

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Morpholine hydrochloride
EC Number:
233-029-4
EC Name:
Morpholine hydrochloride
Cas Number:
10024-89-2
Molecular formula:
C4H9NO.ClH
IUPAC Name:
morpholine hydrochloride

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 10-12 weeks
- Weight at study initiation: 143.5-197.9 g
- Fasting period before study: no data
- Housing: individually in type M III Makrolon cages supplied by BECKER & CO., Castrop-Rauxel, Germany
- Diet: ad libitum (ground Kliba maintenance diet mouse/rat “GLP”)
- Water: ad libitum (drinking water)
- Acclimation period: no data

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

IN-LIFE DATES: From: 05.11.2007 To: 20.11.2007

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test substance solutions were prepared at the beginning of the administration period and thereafter at intervals of 7 days, which took into account the analytical results of the stability verification. For the preparation of the solutions, appropriate amounts of the test substance were weighed in a beaker, topped up with drinking water and subsequently thoroughly mixed using a magnetic stirrer.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The stability of the test substance solutions over a period of 7 days at room temperature was demonstrated. The results of the analyses of the test substance solutions in tap water confirmed the correctness of the prepared concentrations. The analytical values of the samples corresponded to the expected values within the limits of the analytical method, i.e. were always above 90% and below 110% of the nominal concentrations.
Details on mating procedure:
The animals were paired by the breeder (“time-mated”) and supplied on GD 0 (= detection of vaginal plug/sperm). The animals arrived on the same day (GD 0) at the experimental laboratory. The following day was designated as “GD 1”.
Duration of treatment / exposure:
Test substance was administered on gestation days (GD) 6 through 19.
Frequency of treatment:
The test substance solutions were administered to the animals orally by gavage, once a day, always at approx. the same time in the morning.
A standard dose volume of 10 mL/kg bw was used for each test group. At terminal sacrifice on GD 20, all females had implantation sites.
Doses / concentrationsopen allclose all
Dose / conc.:
75 mg/kg bw/day (nominal)
Dose / conc.:
250 mg/kg bw/day (nominal)
Dose / conc.:
750 mg/kg bw/day (nominal)
No. of animals per sex per dose:
25 female rats
Control animals:
yes, concurrent vehicle

Examinations

Maternal examinations:
MORTALITY: Yes
- Twice a day on working days or once a day on Saturdays, Sundays or on public holidays (GD 0-20)

CAGE SIDE OBSERVATIONS/DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once daily for any signs of morbidity, pertinent behavioral changes and signs of overt toxicity.
- Time schedule: If such signs occurred, the animals were examined several times daily (GD 0-20)

BODY WEIGHT: Yes
- Time schedule for examinations: GD 0, 1, 3, 6, 8, 10, 13, 15, 17, 19 and 20
- Furthermore, the corrected bw gain was calculated after terminal sacrifice (terminal bw on GD 20 minus weight of the unopened uterus minus bw on GD 6).

FOOD CONSUMPTION: Yes
- With the exception of day 0, the consumption of food was determined on the same days as bw

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: Liver, Kidneys, Thyroid glands (with parathyroid glands)
- Cesarean Section: uteri and the ovaries were removed and following data were recorded:
Weight of the unopened uterus, Number of corpora lutea, Number and distribution of implantation sites classified as live fetuses/dead implantations
(cf. "Ovaries and uterine content")

OTHER: CLINICAL PATHOLOGY prior to sacrifice on gestation day 20,
- Blood was taken from the retro-orbital venous plexus from not fasted animals
- Examinations:
- Hematology - WBC, RBC, HGB, HCT, MCV, MCH, MCHC, PLT, Differential blood count, Reticulocytes
- Clinical chemistry - enzymes: ALT, AST, ALP, GGT
- Clinical chemistry - blood chemistry parameter: INP, CA, UREA, CREA, GLUC, TBIL, TPROT, ALB, GLOB TRIG, CHOL
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 and distribution of implantations sites: Yes
- Number of live fetuses: Yes
- Number of dead implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Number of dead fetuses: Yes


Fetal examinations:
- External examinations: Yes
- Each fetus was weighed, sexed, and external tissues and all orifices were examined macroscopically
- Furthermore, the viability of the fetuses and the condition of placentae, umbilical cords, fetal membranes, and fluids were examined.
- Individual placental weights were recorded
- Fetuses were sacrificed: Approximately one half of the fetuses per dam were eviscerated, skinned and placed in ethanol,
the other half was placed in Harrison’s fluid for fixation
- Soft tissue examinations: Yes, fetuses fixed in Harrison’s fluid
- Skeletal examinations: Yes, fetuses fixed in ethanol
- Head examinations: No data
Statistics:
CLINICAL and FETAL EXAMINATIONS:
Food consumption, bw, bw change, corrected bw gain (net maternal bw change), carcass weight, weight of unopened uterus, number of corpora lutea, number of implantations, number of resorptions, number of live fetuses, proportions of preimplantation loss,
proportions of postimplantation, loss, proportions of resorptions, proportion of live fetuses in each litter, litter mean fetal body weight, litter mean placental weight; Female mortality, females pregnant at terminal sacrifice, number of litters with fetal findings; Proportions of fetuses with malformations, variations and/or unclassified observations in each litter.
PATHOLOGY:
Means and standard deviations of each test group were calculated. Weight parameters were evaluated.
Indices:
- Conception rate (in %) [number of pregnant animals/number of fertilized animals x 100]
- Preimplantation loss (in %) [(number of corpora lutea – number of implantations)/number of corpora lutea x 100]
Historical control data:
Yes, parameters of animals of same strain and age available.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
All mid and high-dose dams (250 and 750 mg/kg bw/d) excreted a yellowish discolored urine from GD 7 onwards until terminal sacrifice (GD 20). This urine discoloration mirrors the systemic availability of the test substance rather than being an adverse effect. It is most likely due to the excreted test compound or its metabolite(s). Furthermore 8 high-dose dams showed transient salivation after treatment. Salivation occurred from GD 12 onwards and persisted in the respective females for a few minutes immediately after each administration. After cessation of treatment on GD 19, salivation was no longer observed in these rats. This temporary salivation is considered to be treatment-related. It was likely to be induced by the unpleasend taste of the test substance or by local irritation of the upper digestive tract. It is not considered to be a sign of systemic toxicity. No clinical symptoms were noted in the low-dose group (75 mg/kg bw/d).
Mortality:
no mortality observed
Description (incidence):
There were no substance-related or spontaneous mortalities in any of the groups.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The average body weights were comparable among control and treated groups (0; 75; 250 and 750 mg/kg bw/d) during the entire study. Body weight gain of the high-dose rats was statistically significantly reduced during the initial
phase of treatment (GD 6-8, about 41% below the concurrent control value), which was likely to be a subsequent effect of reduced feed consumption. If calculated for the entire treatment phase (GD 6-19), no reduction in mean body weight gain of these rats was seen. Body weight gain of the low- and mid-dose dams (75 and 250 mg/kg bw/d) was comparable to the concurrent controls. All observable differences in these 2 groups in comparison to the controls are without any biological relevance. This statement includes the statistically significatly increased body weight gain in test group 2 on GD 13-15.
The corrected body weight gain (terminal body weight on GD 20 minus weight of the unopened uterus minus body weight on GD 6) of all test substance-treated groups (75; 250 and 750 mg/kg bw/d) revealed no difference of any biological relevance to the corresponding control group.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
The mean food consumption of the high-dose dams (750 mg/kg bw/d) was statistically significantly reduced (up to about 13% below the concurrent control value) on gestation days 6-10, during the initial phase of exposure. However, on the following days the food consumption of the high-dose rats recovered and became comparable to control. The food consumption of the females of test groups 1 and 2 (75 and 250 mg/kg bw/d) was unaffected and did not show any statistically significant or biologically relevant differences in comparison to the controls.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
On GD 20, the red blood cell counts as well as the hemoglobin and hematocrit values were decreased in the dams of the 250 and 750 mg/kg bw/d dose groups. The relative reticulocyte counts were also increased in these rats.
The relative as well as the absolute eosinophil counts were slightly, but statistically significantly decreased in the dams of the 750 mg/kg bw/d dose group.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
The alanine aminotransferase (ALT) activity was increased in the dams of the 750 mg/kg bw/d dose group. Correspondingly, in the same rats the urea, total bilirubin, and the cholesterol values were increased.
Endocrine findings:
not specified
Description (incidence and severity):
There were no test substance-related effects on the dams concerning uterine, placental and thyroid weights, as well as necropsy observations up to and including a dose of 750 mg/kg bw/day.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Absolute liver weights were slightly, but significantly increased in all treated groups (75; 250; 750 mg/kg bw/d). In the 250 and 750 mg/kg bw/d groups, this increase was correlated to hematological (anemia) findings. Additionally, the 750 mg/kg bw/d group showed clinical pathological findings (increased ALT, urea, bilirubin, cholesterol) indicative of an affection of liver cells and liver cell metabolism. Neither such correlates nor any corroborative gross lesions were noted in the 75 mg/kg bw/d group, thus the rather marginal increase of liver weight in this dose group is not considered as an adverse effect of systemic toxicity.
The mean gravid uterus weights of the animals of all test groups (75; 250 or 750 mg/kg bw/d) were not influenced by the test substance. The differences between these groups and the control group revealed no dose-dependency and were assessed to be without biological relevance. Considering the fluctuations in the mean number of live fetuses/dam, they reflect the normal degree of variation for rats of the strain used in this study.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No gross lesions were noted in the dams of any of the dose groups.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined

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:
not examined
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
The conception rate reached 100% in all test groups including controls (0; 75; 250 and 750 mg/kg bw/d).
Details on maternal toxic effects:
There were no substance-related or spontaneous mortalities in any of the groups. The oral administration of 250 and 750 mg/kg bw/d Morpholine hydrochloride caused a mild, regenerative anemia in the dams, along with increased liver weights. Additionally, transiently reduced mean food consumption and bw gain as well as affection of liver cells and liver cell metabolism was noted at the 750 mg/kg bw/d dose level. The oral administration of Morpholine hydrochloride to the dams at 75, 250 and 750 mg/kg bw/day had no influence on gestational parameters.

Effect levels (maternal animals)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
maternal
Effect level:
75 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
haematology
Key result
Dose descriptor:
NOAEL
Remarks:
developmental
Effect level:
750 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: highest dose tested

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
The mean fetal body weights in test groups 1, 2 and 3 (75; 250 and 750 mg/kg bw/d) were not influenced by the test substance and were comparable to the corresponding control values.
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 (75; 250 and 750 mg/kg bw/d) was comparable to the control fetuses. Observable differences were without biological relevance.
Changes in litter size and weights:
no effects observed
Anogenital distance of all rodent fetuses:
not examined
Changes in postnatal survival:
not examined
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
One sole external malformation (i.e. cleft palate) was recorded for three fetuses from 2 litters in the mid-dose group (250 mg/kg bw/d). Considering the missing doseresponse relationship and the presence of this finding in the historical control
data, these abnormalities were considered to be spontaneous in nature and without a relation to treatment. The incidences of external malformations were comparable to the historical control data. No external variations were observed. No external unclassified observations were seen in any fetuses of any group.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Skeletal malformations were noted in single fetuses of test groups 0, 2 and 3 (0; 250 and 750 mg/kg bw/d). Although some of these findings are not in the historical control data, each of them affected individual fetuses and neither statistically significant differences between the test groups nor a dose-response relationship were observed. The incidences of skeletal malformations were comparable to the historical control data. 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.
Additionally, some isolated cartilage findings without impact on the respective bony structures, which were designated as unclassified cartilage observations, occurred in all groups including the controls. The observed unclassified cartilage findings were related to the skull, the ribs and the sternum and did not show any relation to dosing. The percentages of affected fetuses per litter are within the overall historical control range (mean value 37.4%; range per study: 17.0 – 64.5%) and do not show any relation to dosing. Thus, a toxicological relevance for these findings is not assumed.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Two soft tissue malformations (i.e. short or interrupted spinal cord) were recorded in one fetus each of test groups 0 and 2 (0 and 250 mg/kg bw/d). Although these particular findings are not in the historical control data, they were considered to be spontaneous in nature and without a relation to treatment, because there is no doseresponse relationship. The incidences of soft tissue malformations were comparable to the historical control data. Three soft tissue variations (dilated cerebral ventricle, uni- or bilateral dilation of renal pelvis and ureter) were detected in each group including the controls, without any dose-response relationship. The incidences of soft tissue variations were comparable to the historical control data. No unclassified soft tissue observations were recorded.
Details on embryotoxic / teratogenic effects:
Fetal examinations revealed no influence of the test compound on sex distribution of the fetuses and fetal body weights. Morpholine hydrochloride shows no direct and specific effect on the respective morphological structures. Fetal findings in this study were primarily limited to a slight increase in delayed ossification in the mid- and high-dose groups.These specific skeletal variations mirror common minor effects on fetal morphology which are considered to be transient in nature, being obviously secondary to maternal toxicity. Thus, these findings were regarded to be of no toxicological relevance and are not classified as adverse effects.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
750 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: highest dose tested

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
no

Applicant's summary and conclusion

Categories Display