2-methyl-1-(4-methylthiophenyl)-2-morpholinopropan-1-one

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: OECD guideline 415 (One-Generation Reproduction Toxicity Study)

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

2-methyl-1-(4-methylthiophenyl)-2-morpholinopropan-1-one

EC Number:

400-600-6

EC Name:

2-methyl-1-(4-methylthiophenyl)-2-morpholinopropan-1-one

Cas Number:

71868-10-5

Molecular formula:

C15 H21 N O2 S

IUPAC Name:

2-methyl-1-[4-(methylsulfanyl)phenyl]-2-(morpholin-4-yl)propan-1-one

Details on test material:

- Appearance : White powder
- Storage conditions : Room temperature protected from light

Test animals

Species:

rat

Strain:

other: Sprague Dawley SD

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Italy s.r.l., San Pietro al Natisone (UD), Italy
- Age at study initiation: males: approx. 7 - 8 weeks; females: approximately 10 - 11 weeks
- Weight at study initiation: females: 217.1 - 264.0 g
- Housing: 4 of one sex to a cage
- Diet (e.g. ad libitum): laboratory rodent diet (Altromin MT pelleted diet, Lage, Germany) was offered ad libitum throughout the study
- Water (e.g. ad libitum): Drinking water was supplied ad libitum
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 55 ± 15
- Air changes (per hr): 15 to 20
- Photoperiod (hrs dark / hrs light): 12 hours/12 hours

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: Aqueous solution of 0.5 % carboxymethylcellulose (CMC).

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The required amount of test substance were suspended in the vehicle. The formulation will be prepared daily (concentrations of 4.0, 8.0 and 12.0 mg/ml). Concentrations will be calculated and expressed in terms of test item as received. All test item solutions were protected from light throughout gavage.

DOSE VOLUME: The test item was administered at a dose volume of 10 ml/kg body weight. Control animals received the vehicle alone at the same dose volume. Dose volumes were calculated according to individual body weight on the first day of treatment and adjusted according to individual body weight at weekly intervals thereafter.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of the formulations prepared during the first and the last week of treatment were analysed to check the concentration. The test item was found to be stable in the vehicle for 4 hours at room temperature. Chemical analyses were carried out by the Analytical Chemistry Department at RTC. The results of analyses were within the limits of acceptance of +/- 10% of nominal concentration.

Details on mating procedure:

- Impregnation procedure: cohoused
- M/F ratio per cage: monogamous (1 male to 1 female)
- Length of cohabitation:
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility. One treated male was utilised to mate a maximum of three females.
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy. These data also provided information about the pre-coital interval (i.e. the number of nights paired prior to the detection of mating).

Duration of treatment / exposure:

males: for 10 consecutive weeks prior to mating and thereafter through the day prior to sacrifice.
females: for 2 consecutive weeks prior to mating and thereafter: half of the females per group until post-coitum day 19 and the other half of the females per group which were allowed to give birth until the day before necropsy (Day 21 post-partum).

Frequency of treatment:

once a day, 7 days a week

Duration of test:

Males were killed after 15 weeks of treatment.
Approximately half of the females per group were sacrificed on Day 20 post-coitum.
Approximately half of the females per group were allowed to give birth.

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 40, 80, 120 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

Each group comprised 48 females and 24 males, with the exception of the female control group which comprised 24 females.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels were selected based on information from the preliminary study (RTC Study No.: 9318EXT).

Examinations

Maternal examinations:

MORTALITY: Yes
- Time schedule: twice daily (early in each working day and again in the afternoon). At weekends and Public Holidays a similar procedure was followed except that the final check was carried out at approximately mid-day.

PRE- AND POST-DOSE OBSERVATIONS: Yes
- Time schedule: daily, prior to dosing, immediately after dosing and within 1 hour of dosing
- Observations included: any changes in gait and posture, reactivity to handling, stereotypes or bizarre behaviour and effects on the autonomic nervous system (e.g. lachrymation, piloerection, pupil size, unusual respiratory pattern)

DETAILED CLINICAL OBSERVATIONS: Yes
-Time schedule: once a week
- Observations included: any changes in gait and posture, reactivity to handling, stereotypes or bizarre behaviour and effects on the autonomic nervous system (e.g. lachrymation, piloerection, pupil size, unusual respiratory pattern). In addition, abnormal changes of the skin/fur (abnormal coloration, presence of scabs, hairless, erythema, oedema, necrosis) with relative localisation and presence of palpable masses with relative localisation were recorded.

BODY WEIGHT: Yes
- Time schedule: Males = on the day of allocation to treatment group, on the day that treatment commenced, weekly thereafter and just prior to necropsy. Females = on the day of allocation to treatment group, on the day that treatment commenced, weekly thereafter until pairing. Body weight during the mating period were recorded, but not presented.
The females were weighed on days 0, 3, 6, 9, 12, 15, 18 and 20 post-coitum and on days 1, 7, 14 and 21 post-partum.

FOOD CONSUMPTION:
- Time schedule: weekly from allocation to pairing; females: Individual food consumption was also measured on days 0, 3, 6, 9, 12, 15 and 20 post-coitum and days 1,7, 14 and 21 post-partum.

ORGAN WEIGHTS: From all males completing the scheduled test period, the organs were dissected free of fat and weighed. The ratios of organ weight to body weight were calculated for each animal.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day: on day 20 post-coitum.
- Organs examined: detailed post-mortem examination was conducted (including examination of the external surface and orifices)

Ovaries and uterine content:

The ovaries and uterine content was examined after termination
Examinations included:
- Gravid uterus weight, number of corpora lutea, number and distribution of live young, number and distribution of intra-uterine deaths, individual foetal weight and sex
- External foetal abnormalities: Yes
- Gross evaluation of placentae: Yes

Fetal examinations:

BODY WEIGHT:
- Time schedule: on day 1 post-partum

EXTERNAL EXAMINATIONS: Yes, for dead or abnormal young
- Time schedule: daily
- Observations included: pinna unfolding, hair growth, incisor eruption (upper), startle response to sound, eye opening (complete separation of eyelids), testis descent (testes palpable in the scrotum), air righting reflex, pupil reflex.

STRUCTURAL DEVIATIONS: Observations included: malformations, abnomalities, variations

NECROPSY: all pups found dead were necropsied with the exception of those excessively cannibalized or autolysed.

Statistics:

For continuous variables the significance of the differences amongst group means will be assessed by Dunnett's test or a modified t test, depending on the homogeneity of data.
Statistical analysis of non-continuous variables will be carried out by means of the Kruskal-Wallis test and intergroup differences between the control and treated groups assessed by a non-parametric version of the Williams test.

Historical control data:

yes

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Before pairing, no clinical signs of toxicological significance were noted. During the gestation period, staining on the body surface was observed in some treated females. This observation continued during the post-partum phase in the mid- and high-dose groups. In addition, hair loss, and swollen abdomen were noted in high dose females.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

During the gestation period, statistically significant decreases in body weight were observed in mid- and high dose females from day 15 to 20 and from day 3 to 20 post coitum, respectively. Mean group values for the mid- and high dose terminal body weight were 7.4 and 8.5% lower than the control mean value, respectively (tab. 1). Food intake was unaffected by treatment in both sexes before pairing and in females during post-coitum period.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

A total of 13 females proved not to be pregnant at sacrifice. The number of females with fetuses on day 20 post-coitum was 10 in the control, 21 in the low dose, 21 in the mid dose and 18 in the high dose group (tab 2). The number of corpora lutea was decreased in all treatment groups. Pre-, post and total implantation loss in all treatment groups was comparable to the control. A significant lower uterus weight was observed in the mid-dose group; the absolute uterus weight gain was decreased in mid- and high-dose females (tab. 2).

Maternal developmental toxicity

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

Pre-, post and total implantation loss in all treatment groups was comparable to the control.

Effect levels (maternal animals)

1

Dose descriptor:

LOAEL

Effect level:

40 mg/kg bw/day (nominal)

Basis for effect level:

other: developmental toxicity

2

Dose descriptor:

LOAEL

Effect level:

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

Basis for effect level:

other: maternal toxicity

3

Dose descriptor:

NOAEL

Effect level:

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

Basis for effect level:

other: maternal toxicity

Results (fetuses)

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

Litter weight was statistically significantly decreased in the mid-dose and high dose group. Mean foetal weight was statistically significantly decreased in the high dose group when compared to controls (tab 3).

Changes in sex ratio:

effects observed, treatment-related

Description (incidence and severity):

Statistically significant decreases in the number of viable males and consequently in the percentage of males and litter weight were noted in the mid- and high dose groups (tab 3).

External malformations:

effects observed, treatment-related

Description (incidence and severity):

Micrognathia, cleft palate, anasarca, tail bent, short or swollen, short body, kyphosis, limbs (forelimbs and/or hindlimbs) malrotated, short or flexure and head with domed shape were observed in the high dose foetuses. In addition, cleft palate was also noted in 5 low dose foetuses and in 1 mid-dose foetus out of one litter each. Anasarca was observed in the low dose group as well as head with domed shape and hindlimbs malrotated in the mid-dose group. A total of 13 small foetuses were present; 2 each in the low and mid-dose groups and 9 in the high dose group (tab. 4).

Skeletal malformations:

effects observed, treatment-related

Description (incidence and severity):

Retardation or no ossification of the sternal elements with cases of asymmetrical ossification were observed in all treated groups. In addition, an increased incidence in general incomplete ossification of the head bones was noted in the high dose group compared to controls. Alteration of ossification in the vertebral column (thoracic, cervical lumbar and sacral vertebrae) with cases of scoliosis were observed in the high dose group.
Rudimentary 14th rib was noted in all treated groups. Displaced ribs were also observed in the high dose group. Dose-related metacarpal(s) incomplete ossification or unossified were observed in all treated groups (tab 6). Metatarsal(s) incomplete ossification or unossified and incomplete ossification of the pubis bone were noted in the high dose group.
A variety of abnormalities and/or variations were recorded especially in mid and high-dose groups, e.g. retarded / incomplete ossification at various sites of the skeleton. These abnormalities/ variations were not addressed individually in this summarized data compilation since they are of minor relevance for classification as compared to the severe teratogenic effects.

Visceral malformations:

effects observed, treatment-related

Description (incidence and severity):

A dome-shape observed during external examination was associated with malformations of the brain. In particular, an increased incidence of enlarged lateral, third and fourth vehicles were observed in mid- and high-dose groups. High-dose foetuses also showed cases of anencephaly, anophthalmia and microphthalmia. Cleft palate and abnormal shape of the fore- and hindlimbs as well as short digits were detected in high-dose foetuses.
An increased incidence in pelvic dilatation of the kidneys with ureters enlarged and/or kinked were noted in all treatment groups. In addition, testes and kidneys displaced were also noted. Criptorchism, kyphosis and short body were noted in the high dose, as well as one case of malformation on the septum wall of the heart and on the intestine and an increased incidence of the agenesis of the pituitary gland (tab 5).

Effect levels (fetuses)

Dose descriptor:

NOEL

Effect level:

0 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

skeletal malformations

visceral malformations

Fetal abnormalities

Abnormalities:

effects observed, treatment-related

Localisation:

skeletal: skull

skeletal: forelimb

skeletal: vertebra

Overall developmental toxicity

Developmental effects observed:

not specified

Any other information on results incl. tables

Table1 Body weight females, post-coitum period

	days post-coitum period
group	0	3	6	9	12	15	18	20
Control	242,42	260,88	272,24	284,5	299,46	321,3	368,4	404,05
40 mg/kg	237,25	253,74	266,31	279,01	293,05	311,35	254,78	387,37
80 mg/kg	241,96	258,9	267,06	278,03	291,35	308,84*	343,12**	375,78
120 mg/kg	232,03	248,99*	260,41*	269,16**	282,04**	299,78**	339,10**	371,33**

* = p< 0.05 ** = p< 0.01

Table 2 Pathology data females(females sacrificed on day 20 p.c.)

	control	40 mg/kg bw	80 mg/kg bw	120 mg/kg bw	Historical control data
Corpora lutea	17.6	15.9*	14.8*	15.2*	13.3-17.7
Implantations	16.5	15.1*	14.2*	14.7*	12.7-16.7
Pre-implantation loss %	5.9	5.6	4.0	3.2	3.5-6.8
Post-implantation loss %	3.8	4.6	10.1	6.2	2.6-6.3
Pre-birth loss %	8.03	8.64	17.23	14.63	3.5-6.8
Total implantation loss %	9.6	10.1	13.9	9.2	----
Gravid uterus weight [g]	94	83	77*	82	66.2-88.9
Absolute gain (uterus: BW ratio)	66	64	54*	55*	44.5-72.3
Females pregnant / total	10/12	21/24	21/24	18/24	-----

Table 3 Litter data – sex ratio, litter weight, fetal weight

Dose group	Total viable young / litter	Viable males %	Litter weight (g)	Mean fetal weight (g)
Control	15.9	55.3	62.3	3.9
40 mg/kg	14.3*	50.5	55.0	3.8
80 mg/kg	12.8*	40.8*	48.4*	3.8
120 mg/kg	13.8*	45.2*	48.5*	3.5*
Historical control data	12.0 – 15.6	48.0 – 54.2	42.1 – 59.3	3.5 – 3.8

* = p< 0.05

Table 4 Fetal malformations (1) (external examination, group incidence)

Organ	Malformation	Control	40 mg/kg	80 mg/kg	120mg/kg	Historical control data (%) #
	No. of fetuses (litters) examined	159 (10)	301 (21)	269 (21)	249 (18)
Forelimbs	Malrotated	0	0	0	27 (2)	n.f.
	Short	0	0	0	59 (5)	n.f.
	Flexure	0	0	0	8 (1)	n.f.
Head	Domed	0	0	7 (1)	47 (5)	n.f.
	Micrognathia	0	0	0	14 (1)	n.f.
Hindlimbs	Malrotated	0	0	1	47 (8)	n.f.
	Short	0	0	0	21 (4)	n.f.
Palate	Cleft palate	0	5 (1)	1 (1)	28 (5)	n.f.
Tail	Bent	0	0	0	46 (7)
	Short	0	0	0	29 (3)	n.f.
Whole foetus	Anasarca	0	7(1)	0	36 (4)	n.f.
	Short body	0	0	0	53 (4)	n.f.
	Kyphosis	0	0	0	18 (4)	n.f.

# Historical control data were generated from either the test laboratory or the breeder company n.f. = observation not found in the historical data set

Table 5 Malformations (2) (fixed foetuses)

Organ	Malformation	Control	40 mg/kg	80 mg/kg	120mg/kg	Historical control data (%) #
	No. of fetuses (litters) examined	77 (10)	145 (21)	128 (21)	121 (18)
Brain	Agenesis of the pituitary	0	0	1 (1)	27 (6)	n.f.
	Lateral ventricle enlarged, extreme	0	0	14 (5)	90 (16)	n.f.
	Third ventricle enlarged, extreme	0	0	2 (1)	52 (14)	n.f.
	Fourth ventricle enlarged, extreme	0	0	8 (3)	48 (14)	n.f.
	Anencephaly	0	0	0	10 (3)	n.f.
Eye	Anophthalmia	0	0	0	1 (1)	n.f.
Palate	Cleft palate	0	2 (1)	0	19 (5)	1 (1) = 0.1%
Heart	Interventricular septum wall incomplete	0	0	0	1(1)	n.f.
Abdomen	Diaphragmatic haernia	0	0	0	1	0.04 (0.26)
Limbs	Fore- and hindlimb abnormal shape	0	0	0	26 (6)	n.f.
Kidney	Pelvic dilatation extreme	0	0	0	7 (5)	n.f.
Ureter	Enlarged extreme	0	0	1 (1)	12 (7)	n.f.
	Kinked, extreme	0	0	0	4 (2)	0.37 (2.38)
Testis	Cryptorchism	0	0	0	20 (7)	n.f.
Forelimb	Short digit	0	0	0	8 (2)	n.f.
Intestine	Protrusion intestine from abdominal cavity	0	0	0	1 (1)	n.f.
Whole Foetus	Kyphosis	0	0	0	20 (6)	n.f.
	Short body	0	0	0	16 (3)	n.f.

# Historical control data were generated from either the test Lab or the Breeder company

n.f. = observation not found in the historical data set

Table 6 Malformations (3) (skeletal findings, group incidence)

Organ	Malformation	Control	40 mg/kg	80 mg/kg	120mg/kg	Historical control data (%) #
	No. of fetuses (litters) examined	82 (10)	156 (21)	141 (21)	128 (18)
Cervical vertebrae	Arches fused	0	0	0	1 (1)	0.04 (0.27)
Thoracic vertebrae	Scogliosis	0	0	0	22 (5)	n.f.
Rib	Displaced	0	0	0	21(5)	n.f.

# Historical control data were generated from either the test Lab or the Breeder company

n.f. = observation not found in the historical data set

Applicant's summary and conclusion

Conclusions:

The developmental toxicity NOEL of the test material in rats was determined to be 0 mg/kg bw/day based upon statistically siognificant visceral and skeleton malformations in offspring at all test doses.