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

Toxicity to reproduction

Administrative data

Endpoint:

one-generation reproductive 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 1

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

Reference 2

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 414 (Prenatal Developmental 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

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

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

Test animals

Species:

rat

Strain:

other: Sprague Dawley SD

Sex:

male/female

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 freatment and adjusted according to individual body weight at weekly intervals thereafter.

Details on mating procedure:

- Impregnation procedure: cohoused
- M/F ratio per cage: monogamous (1 male to 1 female)
- 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).

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.

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 daily, 7 days a week

Details on study schedule:

Culled pups were killed on Day 4 post-partum, weaned pups on Day 21 post-partum.

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).

Positive control:

no

Examinations

Parental animals: Observations and 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 testes and epididymides were weighed. The ratios of organ weight to body weight were calculated for each animal.

Oestrous cyclicity (parental animals):

Vaginal smears were taken daily in the morning, starting from 2 weeks before pairing up to the end of the mating period, until a positive identification of mating was made. The vaginal smear data were examined to determine anomalies of the oestrous cycle.

Sperm parameters (parental animals):

Examination confined to testes and epididymides weights

Litter observations:

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: Litter size, sex ratios, number of foetuses affected with structural deviations and the corresponding litter percentage, foetal structural deviations (Malformations, Abnomalities, Variations), stillbirths, total litter loss, litter weight, pre-weaning development
Dead pups were grossly examined for external and internal abnormalities; sex confirmation by gonadal inspection

Postmortem examinations (parental animals):

All females selected to give birth were examined externally and internally, for the number of visible implantation sites and number of corpora lutea (if detectable).
Procedure for females sacrificed on gestation Day 20 - the ovaries and uteri will be examined to determine: a) number of corpora lutea; b) weight of intact gravid uterus; c) number and distribution of live young; d) number and distribution of intra-uterine deaths; e) individual foetal weight and sex; f) external foetal abnormalities and g) gross evaluation of placentae.
A selection of tissues were fixed and preserved. Histopathological examination was not performed.

Postmortem examinations (offspring):

All foetuses will be examined externally as well as for visceral and skeletal abnormalities.

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.
A modified chi-squared test was used for fertility index parameter. The criterion for statistical significance was p<0.05.

Reproductive indices:

Copulatory Index (%), Fertility Index (%), Copulatory Interval, Oestrus cycles, Pre-implantation loss, Post-implantation loss, total implantation loss

Offspring viability indices:

Pup loss, cumulative pup loss, lactation index

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Hairloss and swollen abdomen were noted in the high dose females during the gestation period. Staining on the body surface was observed in mid- and high dose females during gestation and the post partum phase.
One high dose male and one mid-dose female were sacrificed for humane reasons on Day 72 of the study and on Day 0 post-partum, respectively. Difficulty in parturition was noted for the female and reduced activity and pallor were noted in the male. No clinical signs of particular relevance were noted in males, throughout the treatment period, or in females before pairing. Hairloss and swollen abdomen were noted in the high dose females during the gestation period. Staining on the body surface was observed in mid- and high dose females during gestation and the post partum phase. Body weight, body weight gain and food consumption in male and female animals was comparable to control.

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

not examined

Other effects:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

effects observed, treatment-related

Description (incidence and severity):

Pre-coital interval and copulatory index were comparable between groups and sexes. Fertility was decreased in all treated females and significantly decreased in the high dose group. An increase in irregular cycle (not significant, out of historical range) was noted in all treated females. The number of implantation sites decreased in a dose dependent manner, with the effect still being within the historical range and considered of no statistical significance. Gestation periods were similar in the treated groups compared to controls. All dams gave birth between Day 21 and 22 post-coitum.
A total of 18 females which were allowed to give birth proved not to be pregnant at sacrifice (three low dose, three mid dose and twelve high dose group. The number of females with live foetuses on day 20 post-coitum was 12 in the control, 21 in the low dose, 21 in the mid dose and 13 in the high dose group.
There is no indication from this study that male fertility was affected. Testes and epididymides weights were unaffected and repeat-dose studies also did not indicate adverse effects on male reproductive organs.

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Effect levels (P0)

1

Dose descriptor:

LOAEL

Effect level:

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

Sex:

male/female

Basis for effect level:

other: reduced fertility, increased pre-birth loss, reduced number of implantations

2

Dose descriptor:

NOAEL

Effect level:

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

Sex:

male/female

Basis for effect level:

other: slight reduction in the number of implantations and increase in pre-birth loss

Results: F1 generation

General toxicity (F1)

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

Stillbirths or total litter loss was noted at the day of parturition or the day after parturition in all high dose females which gave birth. A significant increase in pup loss on day 1 post-partum, cumulative loss on days 4 and 14 post-partum was observed in the mid-dose group. Litter weight was significantly decreased in the mid-dose group on days 4 and 14 post-partum. In addition, decreases in the number of viable males and consequently in the percentage of males were noted for the mid- group.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Slight retardation in the development of pups (Pinna detachment, hair growth and upper incisor emption) was noted in the mid-dose group (without statistical significance).

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

In decedent pups no milk in the stomach was observed during the necropsy. The incidence of this finding increased with the treatment. In addition, head with domed shape, hydrocephaly, dilatation of the cerebral ventricle and small cerebrum were the findings observed in the decedent pups of the mid-dose group. In the high dose group, these pups showed forelimbs or hindlimbs short and malrotated, head with domed shape, cleft palate, micrognathia, kyphosis, tail short or bent, short body and anasarca. Tail bent was also noted in the mid-dose group. In some cases, autolysis did not allow necropsy to be performed.
Lateral ventricles or third ventricle of the brain dilated and abnormal size of the urinary bladder in weaned pups of the low- and mid-dose groups. In individual cases, innominate artery was not evident.

Histopathological findings:

not examined

Effect levels (F1)

1

Dose descriptor:

LOAEL

Generation:

F1

Effect level:

80 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

other: sign. increase in pup loss, sign. reduced litter size and litter weight

2

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

40 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

other: reduction in total litter size

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

dose group	total litter size at birth	litter size live (day 1 p.p.)	litter size live (day 21 p.p.)	litter weight (day 1 p.p.)	liter weight (day 4 p.p.)	% males at birth	% males (day 4 p.p.
control	15.0	13.1	7.8	83.9	116.4	44.22	45.47
40 mg/kg bw	13.9	12.7	7.7	84.2	120.1	44.14	43.04
80 mg/kg bw	12.3	6.6*	0	48.2	61.5*	52.17	46.31
120 mg/kg bw	11.9	-	-	-	-	54.45	-

* p < 0.05 adjustment of litter size on day 4 p.p. to n = 8 (4 males, 4 females)

dose group	fertility index (M)	fertility index (F)	irregular cycles	implantation	pre-birth loss %	females pregnant/total
control	83.3	91.7	3/24	16.33	8.03	12/12
40 mg/kg bw	100	87.5	12/48	15.4	8.64	21.24
80 mg/kg bw	95.8	87.5	11/48	15.05	17.23	21/24
120 mg/kg bw	82.6	64.6*	14.48	13.91	14.63	13/25
hist. control	83.3 -91.7	83.3 -91.7	2 -5/24	12.7 -16.7	3.5 -6.8

* = significant at p < 0.05

Applicant's summary and conclusion

Conclusions:

The reproduction toxicity NOAEL of the test material in rats was determined to be 40 mg/kg bw/day based upon number of implatations, litter size, and number of still births under the conditions of the test.