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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2007-10-30 to 2009-02-13
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Guideline study according to OECD GL 414 (“Prenatal Developmental Toxicity Study”)

Data source

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

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Name of test substance: Morpholine hydrochloride
Test substance No.: 07/0448-1
Batch identification: JB125
Purity: 97 w%
Homogeneity: Homogeneous
Storage stability: It was confirmed by reanalysis that the test substance was stable until Oct 2008
Date of manufacture: 09 Jul 2007
Physical state/Appearance: Solid/white
Storage conditions: Room temperature, under N2 conditions

Test animals

Species:
rat
Strain:
Wistar
Details on test animals 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:


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:
Analyses of the test substance preparations were carried out as a separate study compliance with the Principles of Good Laboratory
Practice at GKA Competence Center Analytics, BASF SE, Ludwigshafen, Germany. As analytical method .....

Samples of the test substance solutions were sent to the analytical laboratory twice during the study period for verification of the concentrations. Analytical verifications of the stability of the test substance in drinking water for a period of at least 7 days at room temperature were carried out before the study was initiated.
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.
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:
Gestational parameters of animals of this strain and age.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Mortality:
no mortality observed
Description (incidence):
There were no substance-related or spontaneous mortalities in any of the groups.
Description (incidence and severity):
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.
Description (incidence and severity):
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.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
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.
Description (incidence and severity):
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.

Maternal developmental toxicity

Details on maternal toxic effects:
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
Dose descriptor:
NOAEL
Effect level:
75 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
haematology
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
750 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: developmental toxicity

Results (fetuses)

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.

Effect levels (fetuses)

Remarks on result:
not determinable due to absence of adverse toxic effects

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

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.

Applicant's summary and conclusion

Executive summary:

In a developmental toxicity study (BASF AG, 2009), Morpholine hydrochloride (97%) was administered as an aqueous solution to 25 time-mated female Wistar rats (Crl:WI[Han]) by gavage at doses of 75; 250 and 750 mg/kg bw/day on gestation days (GD) 6 through 19. The control group, consisting of 25 females, was dosed with the vehicle (drinking water) in parallel. A standard dose volume of 10 mL/kg body weight was used for each test group. At terminal sacrifice on GD 20, all females (25 per group) had implantation sites. The oral administration of 250 and 750 mg/kg bw/d 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 high-dose level. The oral administration of Morpholine hydrochloride to the dams up to 750 mg/kg bw/d had no influence on gestational parameters. Conception rate, mean number of corpora lutea, total implantations, resorptions and live fetuses, fetal sex ratio, and the values calculated for pre- and the postimplantation losses were all unaffected.The maternal NOAEL is 75 mg/kg body weight/day based on statistically significant hematological changes in the dams at 250 and 750 mg/kg bw/day. Fetal examinations revealed no influence of Morpholine hydrochloride 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 events. The developmental NOAEL is 750 mg/kg bw/day. No adverse fetal findings of toxicological relevance were evident at any dose.The developmental toxicity study is classified as acceptable and satisfies the guideline requirement for a developmental toxicity study (OECD 414) in rat.