cis-4-[3-(p-tert-butylphenyl)-2-methylpropyl]-2,6-dimethylmorpholine

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 Feb 2011 - 29 May 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

22 Jan 2001

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

certified by Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht Rheinland-Pfalz (2009)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

cis-4-[3-(p-tert-butylphenyl)-2-methylpropyl]-2,6-dimethylmorpholine

EC Number:

266-719-9

EC Name:

cis-4-[3-(p-tert-butylphenyl)-2-methylpropyl]-2,6-dimethylmorpholine

Cas Number:

67564-91-4

Molecular formula:

C20H33NO

IUPAC Name:

(2R,6S)-4-[3-(4-tert-butylphenyl)-2-methylpropyl]-2,6-dimethylmorpholine

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI[Han]

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: 10-12 weeks
- Weight at study initiation: 144.1-188.2 g
- Fasting period before study: not specified
- Housing: individually
- Diet (e.g. ad libitum): ground Kliba maintenance diet mouse/rat “GLP”, ad libitum
- Water (e.g. ad libitum): water, ad libitum
- Acclimation period: the animals were acclimated to the laboratory conditions between start of the study (beginning of the experimental phase) and first administration (GD 6)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 30-70%
- Air changes (per hr): 15 times per hour
- Photoperiod (hrs dark / hrs light): 12 hours light from 6.00 h to 18.00 h and 12 hours darkness from 18.00 h to 6.00 h

IN-LIFE DATES: From: 15 Feb 2011 To: 09 Mar 2011

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: 1% Carboxymethylcellulose in drinking water and few drops of Cremophor EL

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Justification for use and choice of vehicle (if other than water): not specified
- Concentration in vehicle: 0, 40, 160, 400 mg/100 mL aqueous preparation
- Amount of vehicle (if gavage): 1% Carboxymethylcellulose in drinking water and a few drops Cremophor EL; 10 mL/kg bw of aqueous preparation
- Purity: not specified

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Three samples of the test substance preparations (one from the top, middle and bottom in each case) were taken from the beaker with a magnetic stirrer running and sent to the analytical laboratory at the beginning of administration for verification of the concentrations. The stability of the test substance preparations was demonstrated over a period of 7 days at room temperature. The correctness of the prepared concentrations was shown by HPLC- analysis. The analytical values of the samples corresponded to the expected values within the limits of the analytical method, i.e. were always in the range of 90-110% of the nominal concentrations.

Details on mating procedure:

- Impregnation procedure: purchased timed pregnant
- Proof of pregnancy: supply on gestation day 0 (GD 0), detection of vaginal plug/sperm

Duration of treatment / exposure:

from implantation to one day prior to the expected day of parturition (GD 6 - 19)

Frequency of treatment:

daily

Duration of test:

20 days

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day

Remarks:

control

Doses / concentrations 2

Dose / conc.:

4 mg/kg bw/day

Remarks:

low-dose level

Doses / concentrations 3

Dose / conc.:

16 mg/kg bw/day

Remarks:

mid-dose level

Doses / concentrations 4

Dose / conc.:

40 mg/kg bw/day

Remarks:

high-dose level

No. of animals per sex per dose:

25 females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: As requested by the sponsor
- Fasting period before blood sampling for (rat) dam thyroid hormones: not specified
- Time of day for (rat) dam blood sampling: gestation day 20

Examinations

Maternal examinations:

CAGE SIDE AND CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once daily
- if any signs of morbidity, pertinent behavioral changes and signs of overt toxicity occurred: animals were examined several times daily (GD 0-20)

BODY WEIGHT: Yes
- Time schedule for examinations: on GD 0, 1, 3, 6, 8, 10, 12, 14, 16, 18, and 20.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- With the exception of day 0, the consumption of food was determined on the same days as body weight.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 20
- Organs examined: uteri and ovaries

OTHER: blood samples were obtained by retroorbital venous puncture (Hematology, Clinical chemistry)

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: live/dead fetuses

Blood sampling:

- Plasma: Not specified
- Serum: Yes
- Volume collected: Not specified

Fetal examinations:

- External examinations: Yes: [all per litter ]
- Soft tissue examinations: Yes: [ half per litter ]
- Skeletal examinations: Yes: [ half per litter ]
- Head examinations: No specified
- Anogenital distance of all live rodent pups: yes

Statistics:

- DUNNETT-test (two-sided) for the hypothesis of equal means:
Food consumption ("mean of means"), body weight, body weight change, corrected body weight gain (net maternal body weight 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

- FISHER'S EXACT test (one-sided) for the hypothesis of equal proportions:
Female mortality, females pregnant at terminal sacrifice, number of litters with fetal findings;

- WILCOXON-test (onesided) for the hypothesis of equal medians:
Proportions of fetuses with malformations, variations and/or unclassified observations in each litter;

- KRUSKAL-WALLIS test (two-sided):
Clinical pathology parameters (if the resulting p-value was equal or less than 0.05, a Wilcoxon-test (two-sided) for the equal medians was performed);

- For all: * for p < 0.05; ** for p < 0.01

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;
- postimplantation loss (in %) = ((number of implantations – number of live fetuses) / number of implantations) x 100

Historical control data:

yes

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No test substance-related clinical signs or any disturbances of the general behavior were observed in any dam during the entire study period. The occurrence of transient salivation in one high-dose rat on GD 17-19 was considered to be spontaneous in nature.

Dermal irritation (if dermal study):

not examined

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):

- Body weight (BW) and body weight gain (BWC) of the high-dose rats (40 mg/kg bw/d) were statistically significantly reduced during several days of treatment (BW on GD 10-20, BWC on GD 6-13, up to 7% below the concurrent control value), which was likely to be a subsequent effect of reduced feed consumption. This could also be assumed for mean body weights and body weight gain of the mid-dose rats (16 mg/kg bw/d), which were statistically significantly reduced on GD 10-15 (BW) and GD 6-10 (BWC). If calculated for the entire treatment phase (GD 6-19), mean body weight gain was about 16% (40 mg/kg bw/d) or about 9% (16 mg/kg bw/d) below controls. If calculated for the whole study period (GD 0-20), mean body weight gain was about 12% (high-dose group) or about 9% (mid-dose group) below the concurrent control value. The body weights and the mean body weight gains of the low-dose group (4 mg/kg bw/d) were comparable to the concurrent control values. The slightly, but statistically significantly reduced body weight change value on GD 6-8 was considered not to be test substancerelated. This was also assumed for the statistically significantly increased high-dose body weight change value on GD 15-17.

- The corrected (net) body weight gain was distinctly and statistically significantly lower in the 40 mg/kg bw/d - test group (about 34% below the concurrent control value) and the 16 mg/kg bw/d - test group (about 24% below control). Furthermore, the carcass weight of the high- and mid-dose dams was also statistically significantly reduced in comparison to the control group (about 5% or 4% below controls). These effects are assessed as direct, test substance-related signs of maternal toxicity. The corrected body weight gain of the 4 mg/kg bw/d - test group revealed no difference of any biological relevance to the corresponding control group. Moreover, mean carcass weights remained also unaffected by the treatment.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

The food consumption of the mid- and the high-dose females (16 and 40 mg/kg bw/d) was distinctly reduced during a major part of the treatment period. The values attained statistical significance during GD 6-13 and GD 19-20 in the mid-dose group and for GD 6-15 in the high-dose group. During the entire treatment period (GD 6-19) the total average food consumption of the pregnant rats was about 10% (16 mg/kg bw/d) or about 15% (40 mg/kg bw/d) below controls. The food consumption of the low-dose dams (4 mg/kg bw/d) did not show test substance related impairments.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related changes among hematological parameters were measured. In dams of the 4 mg/kg bw/d - test group absolute neutrophil and eosinophil counts were lower compared to controls, but the values were not dose-dependently decreased and therefore the changes were regarded as incidental and not treatment-related.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Total protein, albumin and globulin as well as calcium levels were decreased in dams of the 16 and 40 mg/kg bw/d - test groups. Cholesterol concentrations were decreased in rats of the 40 mg/kg bw/d test group, only. In dams of the 4 mg/kg bw/d - test group, total bilirubin levels were lower and triglyceride levels were higher compared to controls, but these values were not altered dose-dependently and therefore the changes were regarded as incidental and not treatment-related. Alkaline phosphatase (ALP) activities were lower in dams of the 40 mg/kg bw/d - test group, but the mean was within the historical control range and therefore the alteration was regarded as incidental and not treatmentrelated.

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

The mean gravid uterus weights of the animals of all test groups (4, 16 and 40 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. The mean placental weights were comparable between the dosed groups (4, 16 and 40 mg/kg bw/d) and the corresponding control. Differences observed in comparison to the control were neither statistically significant nor biologically relevant.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No test substance-related or spontaneous findings were observed after sacrifice.

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

Description (incidence and severity):

No differences of toxicological relevance between the control and the treated groups (4, 16
and 40 mg/kg bw/d) were determined for the mean number of implantations, as well as pre- and
postimplantation loss.

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

All animals were supplied pragnant and were still pregnant at terminal sacrifice

Changes in number of pregnant:

no effects observed

Details on maternal toxic effects:

The conception rate reached 100% in all test groups including the control. There were no test substance-related and/or biologically relevant differences between the different test groups in conception rate, in the mean number of corpora lutea and implantation sites or in the values calculated for the pre- and the postimplantation losses, the number of resorptions and viable fetuses. Generally, gestational parameters in the various test groups were within the normal range for animals of this strain and age for historical control data.

Effect levels (maternal animals)

Key result

Dose descriptor:

NOAEL

Effect level:

4 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

body weight and weight gain

clinical biochemistry

food consumption and compound intake

Maternal abnormalities

Abnormalities:

no effects observed

Results (fetuses)

Fetal body weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

The mean fetal weights were not influenced by the test substance and did not show any biologically relevant differences between the test substance-treated groups and the control. However, the fetal body weights of the low-dose group were even increased (attaining statistical significance). Nevertheless, the observable differences between the groups reflect the usual fluctuation for this parameter. All values are well within the historical control range.

Reduction in number of live offspring:

not examined

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The sex distribution of the fetuses in test groups (4, 16 and 40 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 specified

Description (incidence and severity):

The distance between the anus and the base of the genital tubercle was observed to determine the sex of each fetus.

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Description (incidence and severity):

No external malformations and variations were observed.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Three skeletal malformations (i.e. malpositioned and bipartite sternebra, misshapen tuberositas deltoidea and thick humerus) were detected in each group including the controls. Due to the missing dose-response relationship these findings were considered to be spontaneous in nature.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

For one control female fetus multiple visceral malformations were recorded: misshapen heart, malpositioned and small atrium (left), common carotid trunk, fused lung lobes (right) and an absent lung lobe (lobus inferior medialis). This finding was considered to be spontaneous in nature.

Details on embryotoxic / teratogenic effects:

- unclassified external observations: one unclassified external observation was recorded, i.e. placentae fused. The finding occurred in the progeny of one mid-dose dam (16 mg/kg bw/d) and was considered to be spontaneous in nature.

- soft tissue variation: dilated renal pelvis was detected in one female fetus of the mid-dose test group (16 mg/kg bw/d). This finding was considered to be spontaneous in nature.
- skeletal variations: For all test groups, skeletal variations of different bone structures were observed (incomplete ossification of supraoccipital; unchanged cartilage, wavy rib), 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. Incomplete
ossification of the supraoccipital bone with unchanged cartilage is a common and unspecific
finding in this rat strain; this increase likely indicates no more than a slight general
disturbance of ossification processes due to the increase in maternal toxicity. Therefore, this
finding was judged to be a non-adverse, secondary effect of treatment at 40 mg/kg body
weight/day.

- unclassified skeletal observations: 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 sternum and did not show any relation to dosing.

Effect levels (fetuses)

Dose descriptor:

NOAEL

Effect level:

40 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No treatment-related adverse effects observed up to and including the highest dose level of 40 mg/kg bw.

Fetal abnormalities

Abnormalities:

effects observed, non-treatment-related

Localisation:

skeletal: sternum

visceral/soft tissue: urinary

visceral/soft tissue: cardiovascular

other: humerus, tuberositas, lung

Description (incidence and severity):

These findings were considered to be spontaneous in nature.

Overall developmental toxicity

Developmental effects observed:

yes

Lowest effective dose / conc.:

4 mg/kg bw/day

Treatment related:

no

Relation to maternal toxicity:

not specified

Dose response relationship:

not specified

Relevant for humans:

not specified