Morpholinium sulphamate

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012-05-17 until 2012-07-13

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP and guideline compliant study

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

, adopted 1995

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: OPPTS 870.3550 Reproduction/Developmental Toxicity Screening Test, EPA Health Effects Test Guidelines, July 2000.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source:
Toxi-Coop Zrt. Cserkesz u. 90., H-1103 Budapest, Hungary
- Age at study initiation:
Male animals: 76 - 82 days old
Female animals: 76 - 82 days old
- Weight at study initiation:
Male animals: 288 - 327 g
Female animals: 163 - 194 g
- Housing: Type II polypropylene/polycarbonate
Size: 22 x 32 x 19 cm (width x length x height)
Supplier: Charles River Europe
Before mating: 2 animals of the same sex/cage
Mating: 1 male and 1 female / cage
Pregnant females were housed individually
Males after mating: individually
- Diet:
Animals received ssniff® SM R/M-Z+H "Autoclavable complete feed for rats and mice – breeding and maintenance" produced by ssniff Spezialdiäten GmbH, D-59494 Soest, Germany ad libitum.
- Water:
Tap water from municipal supply, as for human consumption from a 500 mL bottle ad libitum.
- Acclimation period:
14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C):
22 ± 3 °C
- Humidity (%):
30-70 %
- Air changes (per hr):
8-12
- Photoperiod (hrs dark / hrs light):
12 hours daily, from 6.00 a.m. to 6.00 p.m.

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was formulated in the vehicle at concentrations of 50 mg/mL, 150 mg/mL and 500 mg/mL. Formulations were prepared in the formulation laboratory of the Test Facility for not longer than three days.

VEHICLE
- Justification for use and choice of vehicle :
The test item is soluble in water.

- Amount of vehicle (if gavage):
A constant treatment volume of 2 mL dose preparation/kg body weight was administered in all groups. The individual volume of the treatment based on the most recent individual body weight of the animals. In the first week of the pre-mating period, animals received volumes based on the actual body weight on day 0.

Details on mating procedure:

Mating was started 2 weeks after the initiation of treatment. One female and one male of the same dose group (1:1 mating) were placed in a single cage. Females remained with the same male during 14 days. Pairs were changed within a dose group thereafter; females were paired with not mated males then with proven males, if it was necessary,.
Each morning a vaginal smear was prepared and stained with 1 % aqueous methylene blue solution. The smear was examined with a light microscope. The presence of vaginal plug or sperm in the vaginal smear was considered as evidence of copulation (day 0 of pregnancy as defined by OECD 421). Sperm positive females were caged individually.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The suitability of the chosen vehicle for the test item was analytically proven. Recovery was between 101 and 102 % of nominal concentrations at 1 and 500 mg/mL in water, respectively. Morpholinium sulphamate proved to be stable at room temperature for two days (100 and 101 % of the starting value at 1 and 500 mg/mL) and at 5 ± 3°C for 3 days (100 and 99 % of the starting value at 1 and 500 mg/mL).

Duration of treatment / exposure:

The test item was administered in a single dose by oral gavage (stomach tube) on a 7 days/week basis, every day at a similar time. Control animals were treated concurrently with the vehicle only. Animals were not treated on the day of gross pathology.
Dosing of both sexes began after acclimatization and two weeks before mating and was continued up to and including the day before the necropsy. Rats of this strain have already reached full sexual maturity at the age of 12 weeks.
Male animals were dosed for 42 days and then they were subjected to necropsy one day after the last treatment.
Female animals were dosed for 14 days pre-mating, during mating period, through gestation and up to lactation days 3 - 6 (for 42 or 46 days, depending on date of mating. One dam was dosed for 57 due to late conceiving and delivery (Day 31 and Day 53, respectively). The day of delivery (viz. when parturition was complete) was defined as day 0 post-partum. Non-pregnant female animals were treated up to and including the day before necropsy (for 42 days).
Control animals were handled in an identical manner to the test groups receiving 2 mL vehicle/kg bw.

Frequency of treatment:

Animals were treated once per day.

Details on study schedule:

Male animals were dosed for 42 days and then they were subjected to necropsy one day after the last treatment.
Female animals were dosed for 14 days pre-mating, during mating period, through gestation and up to lactation days 3 - 6 (for 42 or 46 days, depending on date of mating. One dam was dosed for 57 due to late conceiving and delivery (Day 31 and Day 53, respectively). The day of delivery (viz. when parturition was complete) was defined as day 0 post-partum. Non-pregnant female animals were treated up to and including the day before necropsy (for 42 days).
Control animals were handled in an identical manner to the test groups receiving 2 mL vehicle/kg bw.

Remarks:

Doses / Concentrations:
0, 100, 300, 1000 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

12 animals

Control animals:

yes, concurrent vehicle

Details on study design:

The dose setting based on findings obtained in a previous oral repeated dose toxicity study: 14-Day Oral Gavage Dose Range Finding Study with Morpholinum Sulphamate in the Rat (IUCLID Section 7.5.1).

Positive control:

Not applicable

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS:
Animals were inspected for signs of morbidity and mortality twice daily (at the beginning and end of each day).
General clinical observations were made once a day, after treatment at approximately the same time.

DETAILED CLINICAL OBSERVATIONS: Yes
More detailed examinations were made at the times of weekly weighing, prior to and during the mating until necropsy. Observations were performed on the skin, fur, eyes and mucous membranes, autonomic activity (lachrymation, piloerection, pupil size, respiratory pattern, occurrence of secretions and excretions), circulatory and central nervous system, somatomotor activity and behaviour pattern, changes in gait, posture and response to handling.

BODY WEIGHT: Yes
All parent animals were weighed with an accuracy of 1 g. Parent male animals were weighed on the first day of dosing (day 0), weekly thereafter and on the day of necropsy.
Parent females were weighed on the first day of dosing (day 0) then weekly, on gestation days 0, 7, 14 and 20 and on postpartal days 0 (within 24 hours after parturition) and 4, as well as on the day of necropsy. Body weight of the female animals was weighed on gestational days 10 and 17 in order to give accurate treatment volumes, but these data were not evaluated statistically.

Oestrous cyclicity (parental animals):

Mating was started 2 weeks after the initiation of treatment. One female and one male of the same dose group (1:1 mating) were placed in a single cage. Females remained with the same male during 14 days. Pairs were changed within a dose group thereafter; females were paired with not mated males then with proven males, if it was necessary.
Each morning a vaginal smear was prepared and stained with 1 % aqueous methylene blue solution. The smear was examined with a light microscope. The presence of vaginal plug or sperm in the vaginal smear was considered as evidence of copulation (day 0 of pregnancy as defined by OECD 421). Sperm positive females were caged individually.

Sperm parameters (parental animals):

Parameters examined in male parental generations:
Detailed histological examination was performed on the testes and epididymides of the animals in the control and high dose groups. For testes and epididymides, examinations were performed with special emphasis on stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure.

Litter observations:

Each litter was examined as soon as possible after delivery to establish the number and sex of pups, stillbirths, live births, runts (pups that are significantly smaller than normal pups), and the presence of gross abnormalities.
Live pups were counted, sexed and weighed within 24 hours of parturition (on the day when parturition was complete) and day 4 post-partum with an accuracy of 0.01 g.
In addition to the observations on parent animals, any abnormal behaviour of the offspring was observed.
All the litters were checked and recorded daily for the number of viable and dead pups. The dead pups found were subjected to necropsy by a macroscopic examination. On day 0 of lactation, a lung flotation test was performed on all pups found dead to separate stillborns from those that died after delivery. The lung flotation test is negative for stillborns (pups that died intrauterine) but positive for pups that died after delivery.

Postmortem examinations (parental animals):

Gross necropsy was performed on each animal one day after the last treatment. Animals were anesthetized by Isoflurane and then were exsanguinated.
After examination of the external appearance the cranial, thoracic and abdominal cavities were opened and the appearance of the tissues and organs were observed, and any abnormality was recorded with details of the location, color, shape and size. Special attention was paid to the organs of the reproductive system. The number of implantation sites and of corpora lutea was recorded.
The uterus with cervix, vagina, testes, epididymides, prostate, and seminal vesicles with coagulating glands, ovaries, pituitary of all adult animals were preserved. Testes and epididymides were preserved in modified Davidson solution, all other organs in 4 % buffered formaldehyde solution.

Postmortem examinations (offspring):

GROSS NECROPSY
Parameters listed below were evaluated.
Litter weight on postnatal days 0 and 4
Mean body weight gain per litter between postnatal days 0-4
Number of live births per litter, and number of viable pups per litter on postnatal days 0 and 4
Survival Index of pups on postnatal day 4
Sex ratio % (on postnatal days 0 and 4)

Statistics:

The statistical evaluation of appropriate data were performed with the statistical program package SPSS PC+4.0.
The homogeneity of variance between groups was checked by Bartlett’s homogeneity of variance test. Where no significant heterogeneity was detected a one-way analysis of variance (ANOVA) was carried out. If the obtained result was significant Duncan Multiple Range test was used to access the significance of inter-group differences. Getting significant result at Bartlett’s test the Kruskal-Wallis analysis of variance was used and the inter-group comparisons were performed using Mann-Whitney U-test. Chi2 test was performed if feasible. The frequency of clinical signs, pathology and histopathology findings were calculated.
Results were evaluated in comparison with values of control group (i.e. control value).

Reproductive indices:

The following reproductive indices were calculated: Male mating index, female mating index, male fertility index, female fertility index, gestation index. The formulas for calculation can be found below in "Any other information on materials and methods incl. tables"

Clinical signs:

effects observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

no effects observed

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Mortality
There was no test item related mortality during the course of study.

Clinical Observations
Daily Observations
Test item related salivation was observed in a slight degree in some animals administered with 1000 mg/kg bw/day. Three male animals (3/12) salivated for 1, 12 and 24 days and a single female animal (1/12) did during the pre-mating and gestation period altogether for 14 days. Yellowish color of the urine was observed in the bedding material in each cage of 1000 mg/kg bw/day dose animals from Day 4 up to the day before necropsy. Yellowish colorization of urine was not detected in the other groups therefore it was considered to be related to the test item.
A slight and moderate alopecia and small scar formations on the abdominal skin were noted for one control male rat between Day 29 and 34.
The same findings were noted in two female animals (2/12) at 300 mg/kg bw/day. In one of them, scar formation was observed from Day 11 up to gestation day 2 and alopecia between gestation days 3 and 16, both under the right ear. In the other one female rat, alopecia was seen on the abdomen from gestation day 20 and scar formation from lactation day 1 up to termination, both.
Alopecia (1/12) and scar formation (1/12) also appeared in 1000 mg/kg bw/day group. Alopecia was observed under the shoulder in one dam in the pre-mating period (on days 11, 12 and 13) and continuing in the gestation period (between gestation days 0 and 16). Scar formation was detected on the nose of another dam (1/12) from gestation day 11 up to lactation day 6.
Alopecia and scar formation are common findings in this strain of experimental rats and are seen also in untreated rats. In this study were present in male control and female test item treated animals with low incidence and were considered to be independent from treatment.

Detailed Weekly Observations
Test item related yellowish colorization of the urine in the bedding material was observed in each cage of animals dosed with 1000 mg/kg bw/day from Day 7 up to the end of observation period (Days 7, 13, 20, 27, 34, 41, where relevant). Observation period included pre-mating, mating and post-mating in male animals and pre-mating, mating, post-mating, gestation and lactation periods in female animals.
The behavior and physical condition of animals was not affected by the test item at any dose level (1000, 300 or 100 mg/kg bw/day) at the weekly detailed clinical observations during the entire treatment period.
Alopecia and scar formation were also detected in animals described above at the detailed weekly observations.

Body Weight
A slight, transient test item influence on the body weight gain was observed in male animals at 1000 mg/kg bw/day during the pre-mating period. The body weight development was undisturbed in the test item treated female animals with respect to controls during the entire treatment period (pre-mating, gestation and lactation).
The mean body weight was similar to that of the control group in the male and female animals during the entire observation period.
The mean body weight gain was slightly less in male animals at 1000 mg/kg bw/day than in the control group during the first two weeks of the treatment and exceeded the control value at 1000, 300 and 100 mg/kg bw/day between days 27 and 34. There was no significant difference in the summarized body weight gain at any dose level.
In female animals, there were no test item related differences in the mean body weight and body weight gain between the control and test item treated groups at any dose level during premating, gestation and lactation periods. The mean body weight gain was slightly higher at 100 mg/kg bw/day during the first week of premating period and at 1000 and 300 mg/kg bw/day during the lactation period with respect to control. All these statistically significant differences with respect to control were not considered to be of toxicologically significant.

Food Consumption
The mean daily food consumption was slightly affected by the treatment with the test item in male animals at 1000 mg/kg bw/day during the pre-mating period.
Slightly less mean food consumption was observed in male animals dosed with 1000 mg/kg bw/day during the first two weeks of treatment in full compliance with the body weight changes.
Statistical significance was noted for less food consumption of female animals at 1000 mg/kg bw/day between Days 0 and 7 and during the second week of gestation however it was not considered to be biologically significant because of the low degree and in the lack of any changes in the body weight gain.
There was no significant difference in the mean daily food consumption in male or female animals with respect to control in the low or mid dose groups (300 and 100 mg/kg bw/day) in the course of entire treatment period.

Delivery Data of Dams
There were no test item related differences between the control and dosed groups in the delivery data of dams.
Statistical significance was found in the slightly less mean number of implantation sites at 1000 mg/kg bw/day comparing to control. However the mean number of implantation sites at high dose was well within the normal (historical control) ranges (mean: 12.1, SD: 2.5; n=67). The higher number of post-implantation loss and stillborns at 300 mg/kg bw/day, as well as less number of live borns at 300 mg/kg bw/day were not considered to be related to the treatment because these changes were not present in the high dose group and there were no significant differences in the mean values of these parameters with respect to control.
The mean of corpora lutea, pre-implantation loss, post-implantation loss, total intrauterine mortality and duration of pregnancy were similar in the control and all test item treated groups. There were no significant differences between the control and test item treated groups in the mean number of total births, live-borns, stillborns or viable pups.

Reproductive Performance
The reproductive performance was not affected in male or female animals. There were no significant differences between the control and test item treated male animals in the examined parameters of reproductive performance (number of mated males, number of fertile and infertile males, copulatory and fertility indices).
There were no significant differences between the control and test item treated groups in female animals in the examined parameters of reproductive performance (number and percentage of sperm positive (mated) animals, the number and percentage of non-pregnant and pregnant animals, number of dams delivered and number of pregnant animals with live born pups, copulatory, fertility and gestation indices, mean pre-coital interval and mean number of conceiving days.

Necropsy
No test item related alterations were found at the macroscopic observations of organs or tissues in male or female animals at any dose level (1000, 300 and 100 mg/kg bw/day) at the necropsy.
Pyelectasia (1/12) and smaller than normal testis and seminal vesicles (1/12, both) were observed in single male animals at 1000 mg/kg bw/day. Alopecia was detected on the abdomen in one control male rat (1/12).
Pyelectasia and smaller than normal testis and seminal vesicles are common findings in experimental rats and were considered to be individual changes in this study. Organ weight measurement and histopathology examination of testis was in full compliance with macroscopic observation of testis.

Organ Weight
The absolute and relative organ weights of testes and epididymides did not demonstrate any test item related alterations.
There were no toxicologically significant differences between the control and test item treated groups in the examined organ weights (testes and epididymides). Testes weight of animal no. 412 was less than normal in compliance with macroscopic and microscopic findings.

Histopathology
Histological examination of male and female genital organs (ovaries, uterus, cervix vagina, testes, epididymides) and pituitary did not reveal any toxic or other test item related alterations at 1000 mg/kg/bw/day dose.
In one male animal dosed with 1000 mg/kg bw/day, decreased intensity of spermatogenesis was observed in one side testis in compliance with macroscopic observation and organ weight measurement. In this organ the lack of mature spermatozoa, spermatids and spermatocytes was detected in a proportion (40%) of testicular tubuli. However the Sertoli cells and the spermatogonia were intact. An active spermatogenesis was seen in the other 60% of testicular tubuli in this testis and in 100 % of testicular tubuli in the other testis of this animal.
In all other male animals dosed with 1000 mg/kg bw/day, the investigated organs of reproductive system (testes, epididymides) were histologically normal and characteristic on the sexually mature organism in all cases of control and treated groups. The various spermatogenic cells (the spermatogonia, the spermatocytes, the spermatids and spermatozoa), representing different phases in the development and differentiation of the spermatozoons and the interstitial cells were the same in quantity and morphologically. Epididymides and pituitary gland were histologically normal in all animals.
Pyelectasia was observed in the kidney of one male animal without significant histological lesion (degeneration, inflammation or fibrosis). This is a common finding occurring sporadically in Wistar rats without toxicological significance. In the female animals including non-pregnant animals, the ovaries had a normal structure characteristic for the species, age and phase of sexual cycle. The cortex contained primary, secondary and tertiary follicles and corpora lutea, indicating the active maturation of oocytes and ovulation. The epithelial capsule and ovarian stroma were normal in all cases as well. The uterus, cervix, and vagina had a normal structure in accordance with the phase of sexual cycle in the investigated animals. In two female animal slight dilatation of uterus (1/1 non pregnant female at 300 mg/kg bw/day, 1/12 dam at 1000 mg/kg bw/day) was observed. This finding - without inflammation or other pathological lesion - is a physiological phenomenon in connection with the normal sexual cycle. The histological picture of pituitary was normal as well in the case of female treated and control animals.

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed.

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Mortality
There was no test item effect on pup’s mortality.
The number and percentage of extra uterine mortality was less in the test item treated groups than in the control group between postnatal days 0 and 4 (no statistical significance). The survival indices were similar in all groups, i.e. it was 98 and 99 % in the 1000 and 300 mg/kg bw/day groups and 97 % in the control group.

Sex Distribution
There were no test item related differences between control and test item treated groups in the ratio or in the litter means of genders on postnatal days 0 or 4. A slightly less litter mean number of female offspring was not considered toxicologically relevant as values remained within the historical control ranges.

Clinical Observations
Test item related clinical signs were not detected in the offspring.
The number and percent of pups with findings (cold, no milk in the stomach, missing) were less in the 1000 mg/kg bw/day group than in the control group.

Body Weight
Test item effect on the body weight development of offspring was not found.
The mean litter weights were similar in the control and in all test item treated groups on postnatal days 0 and 4. The mean pup weight was slightly higher at 1000 and 300 mg/kg bw/day than in the control group without any toxicological relevance at the birth (postnatal day 0).

No test item related macroscopic alterations were found in offspring subjected to gross pathological examination.

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed.

Reproductive effects observed:

not specified

Conclusions:

The reprotoxic properties of the test item were assessed in a study performed according to OECD Guideline 421 in rats. Based on the results obtained from testing the parental NOAEL , the NOAEL for reproductive performance and the NOAEL for the F1 generation were determined to be 1000 mg/kg bw/day.

Executive summary:

The purpose of this reproduction/developmental toxicity screening test was to provide initial information concerning the effect of the test item Morpholinum Sulphamate on male and female reproductive performance such as gonadal function, mating behavior, conception, pregnancy, parturition as well as on development of the F1 offspring from conception to day 4 post-partum associated with oral administration to rats at repeated doses. Four groups of Hsd.Brl.Han:Wist rats (n=12/sex/group) were administered orally (by gavage) once a day at 0 (vehicle only), 100, 300 and 1000 mg/kg bw/day at concentrations of 50, 150 and 500 mg/mL corresponding to 2 mL/kg bw dose volume. The suitability of the chosen vehicle for the test item at the intended concentrations was analytically verified up front. Concentration of the test item in the dosing solutions varied in the range of ± 10 % of the nominal values at both analytical occasions. All animals of the parent (P) generation received test item or vehicle prior to mating (14 days) and throughout mating. Test item or vehicle was administered to male animals post mating up to the day before the necropsy. For females, test item was administered through the gestation period and up to lactation days 3-6, i.e. up to the day before the necropsy. Observations included mortality, clinical signs, body weight, food consumption, mating, pregnancy and delivery process, as well as development of offsprings. The dams were allowed to litter, and rear their young up to day 4 postpartum. Litters were weighed and offsprings were observed for possible abnormalities and were euthanized on postnatal day 4. All parental animals were subjected to gross pathology one day after the last treatment. Histopathology examination was performed on reproductive organs and pituitary in the control and high dose groups. The reproductive organs and pituitary of non-pregnant females dosed with the low and middle doses were also processed and evaluated histologically. The results were interpreted comparing treatment groups with respect to controls, which were treated concurrently with vehicle (aqua purificata) only.

Results:

Mortality

There was no test item related mortality at any dose level (100, 300 and 1000 mg/kg bw/day).

Clinical observation

Test item related salivation and yellowish colored urine were observed at 1000 mg/kg bw/day. Salivation appeared transiently in a slight degree in some animals. Yellowish color of the urine was detected in each cage of 1000 mg/kg bw/day dosed animals from Day 4 up to the termination of the treatment. The behavior and physical condition of animals were normal during the entire observation period (pre-mating, mating, post-mating, gestation and lactation periods) in each group.

Body weight and body weight gain

The body weight gain was slightly reduced in male animals dosed with 1000 mg/kg bw/day with respect to control during treatment weeks 1 and 2. Nevertheless this slight change did not influence significantly the body weight values. The body weight development was undisturbed in the test item treated female animals at each dose level (1000, 300 and 100 mg/kg bw/day) during the entire treatment period (pre-mating, gestation and lactation periods).

Food consumption

The mean daily food consumption was slightly affected by the test item in male animals at 1000 mg/kg bw/day during the pre-mating period. This finding corresponds to the observed changes in body weight gain.

Reproduction

There were no test item related differences between the control and test item treated groups in delivery data of dams and in the reproductive performance of male and female animals.

Necropsy

Specific macroscopic alterations related to the test item were not found during the necropsy.

Organ weight

There were no test item related changes in testes and epididymides weights.

Histopathology

Histopathological examinations of male and female genital organs (ovaries, uterus, cervix vagina, testes and epididymides) and pituitary did not reveal any toxic or other test item related changes at any dose level.

Offspring

Negative effects of the test item on offspring development (mortality, clinical signs, body weight and necropsy findings) were not detected between postnatal days 0 and 4.

Conclusion

Under the conditions of the present study Morpholinum Sulphamate did not cause toxic changes and did not influence male and female reproductive performance (gonad function, mating behavior, conception, pregnancy, parturition) in parental male and female Hsd.Brl.Han: Wistar rats or development of the F1 offspring from conception to day 4 post-partum after repeated dose oral administration at 100, 300 or 1000 mg/kg bw/day. Slight sporadic salivation (male and female) and slight changes in body weight gain and food consumption of male animals administered with 1000 mg/kg bw/day were transient and were not considered to be adverse. Yellowish colorization of the urine at 1000 mg/kg bw/day was not related to any pathological changes in the urinary tract therefore it was not considered to be toxicologically relevant in this study.

Based on these observations the No Observed Adverse Effect Levels (NOAEL) were determined as follows:

NOAEL for male rats: 1000 mg/kg bw/day

NOAEL for female rats: 1000 mg/kg bw/day

NOAEL for reproductive performance of the male rats: 1000 mg/kg bw/day

NOAEL for reproductive performance of the female rats: 1000 mg/kg bw/day

NOAEL for F1 Offspring: 1000 mg/kg bw/day

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

GLP study with the morpholinum sulphamate according to OECD guideline.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

The purpose of this reproduction/developmental toxicity screening test was to provide initial information concerning the effect of the test item Morpholinum Sulphamate on male and female reproductive performance such as gonadal function, mating behavior, conception, pregnancy, parturition as well as on development of the F1 offspring from conception to day 4 post-partum associated with oral administration to rats at repeated doses. Four groups of Hsd.Brl.Han:Wist rats (n=12/sex/group) were administered orally (by gavage) once a day at 0 (vehicle only), 100, 300 and 1000 mg/kg bw/day at concentrations of 50, 150 and 500 mg/mL corresponding to 2 mL/kg bw dose volume. The suitability of the chosen vehicle for the test item at the intended concentrations was analytically verified up front. Concentration of the test item in the dosing solutions varied in the range of ± 10 % of the nominal values at both analytical occasions. All animals of the parent (P) generation received test item or vehicle prior to mating (14 days) and throughout mating. Test item or vehicle was administered to male animals post mating up to the day before the necropsy. For females, test item was administered through the gestation period and up to lactation days 3-6, i.e. up to the day before the necropsy. Observations included mortality, clinical signs, body weight, food consumption, mating, pregnancy and delivery process, as well as development of offsprings. The dams were allowed to litter, and rear their young up to day 4 postpartum. Litters were weighed and offsprings were observed for possible abnormalities and were euthanized on postnatal day 4. All parental animals were subjected to gross pathology one day after the last treatment. Histopathology examination was performed on reproductive organs and pituitary in the control and high dose groups. The reproductive organs and pituitary of non-pregnant females dosed with the low and middle doses were also processed and evaluated histologically. The results were interpreted comparing treatment groups with respect to controls, which were treated concurrently with vehicle (aqua purificata) only.

Results:

Mortality

There was no test item related mortality at any dose level (100, 300 and 1000 mg/kg bw/day).

Clinical observation

Test item related salivation and yellowish colored urine were observed at 1000 mg/kg bw/day. Salivation appeared transiently in a slight degree in some animals. Yellowish color of the urine was detected in each cage of 1000 mg/kg bw/day dosed animals from Day 4 up to the termination of the treatment. The behavior and physical condition of animals were normal during the entire observation period (pre-mating, mating, post-mating, gestation and lactation periods) in each group.

Body weight and body weight gain

The body weight gain was slightly reduced in male animals dosed with 1000 mg/kg bw/day with respect to control during treatment weeks 1 and 2. Nevertheless this slight change did not influence significantly the body weight values. The body weight development was undisturbed in the test item treated female animals at each dose level (1000, 300 and 100 mg/kg bw/day) during the entire treatment period (pre-mating, gestation and lactation periods).

Food consumption

The mean daily food consumption was slightly affected by the test item in male animals at 1000 mg/kg bw/day during the pre-mating period. This finding corresponds to the observed changes in body weight gain.

Reproduction

There were no test item related differences between the control and test item treated groups in delivery data of dams and in the reproductive performance of male and female animals.

Necropsy

Specific macroscopic alterations related to the test item were not found during the necropsy.

Organ weight

There were no test item related changes in testes and epididymides weights.

Histopathology

Histopathological examinations of male and female genital organs (ovaries, uterus, cervix vagina, testes and epididymides) and pituitary did not reveal any toxic or other test item related changes at any dose level.

Offspring

Negative effects of the test item on offspring development (mortality, clinical signs, body weight and necropsy findings) were not detected between postnatal days 0 and 4.

Conclusion

Under the conditions of the present study Morpholinum Sulphamate did not cause toxic changes and did not influence male and female reproductive performance (gonad function, mating behavior, conception, pregnancy, parturition) in parental male and female Hsd.Brl.Han: Wistar rats or development of the F1 offspring from conception to day 4 post-partum after repeated dose oral administration at 100, 300 or 1000 mg/kg bw/day. Slight sporadic salivation (male and female) and slight changes in body weight gain and food consumption of male animals administered with 1000 mg/kg bw/day were transient and were not considered to be adverse. Yellowish colorization of the urine at 1000 mg/kg bw/day was not related to any pathological changes in the urinary tract therefore it was not considered to be toxicologically relevant in this study.

Based on these observations the No Observed Adverse Effect Levels (NOAEL) were determined as follows:

NOAEL for male rats: 1000 mg/kg bw/day

NOAEL for female rats: 1000 mg/kg bw/day

NOAEL for reproductive performance of the male rats: 1000 mg/kg bw/day

NOAEL for reproductive performance of the female rats: 1000 mg/kg bw/day

NOAEL for F1 Offspring: 1000 mg/kg bw/day

Short description of key information:
The reprotoxic properties of the test item were assessed in a study performed according to OECD Guideline 421 in rats. Based on the results obtained from testing the parental NOAEL , the NOAEL for reproductive performance and the NOAEL for the F1 generation were determined to be 1000 mg/kg bw/day.

Justification for selection of Effect on fertility via oral route:
Only one study available.

Effects on developmental toxicity

Description of key information

In a developmental toxicity study with Morpholine hydrochloride (97 % ) at doses of 75, 250 and 750 mg/kg bw/d applied orally to rats, no adverse fetal findings of toxicological relevance were evident. The developmental NOAEL was determined as 750 mg/kg bw/day, whereas the maternal NOAEL was 75 mg/kg body weight/day based on statistically significant haematological changes in the dams at 250 and 750 mg/kg bw/day.

Link to relevant study records

Reference

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

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:


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

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.

Duration of test:

Test substance was administered on gestation days (GD) 6 through 19.

No. of animals per sex per dose:

25 female rats

Control animals:

yes, concurrent vehicle

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.

Details on maternal toxic effects:

Maternal toxic effects:yes

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.

Dose descriptor:

NOAEL

Effect level:

75 mg/kg bw/day

Basis for effect level:

other: maternal toxicity

Dose descriptor:

NOAEL

Effect level:

750 mg/kg bw/day

Basis for effect level:

other: developmental toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

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.

Dose descriptor:

NOAEL

Effect level:

750 mg/kg bw/day (nominal)

Basis for effect level:

other: no effects

Abnormalities:

not specified

Developmental effects observed:

not specified

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.

Conclusions:

In this developmental toxicity study with Morpholine hydrochloride (97 % ) at doses of 75, 250 and 750 mg/kg bw/d applied orally to rats, no adverse fetal findings of toxicological relevance were evident. The developmental NOAEL was determined as 750 mg/kg bw/day, whereas the maternal NOAEL was 75 mg/kg body weight/day based on statistically significant haematological changes in the dams at 250 and 750 mg/kg bw/day.

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.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

750 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

GLP study according to OECD/EU guideline.

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

Listed as read across study, Morpholine hydrochloride (97% a.i.) was administered as an aqueous solution to 25 time-mated female Wistar rats (Crl:WI[Han]) by gavage at doses of 75, 250 or 750 mg/kg bw/day on gestation days 6 through 19 in a developmental toxicity study. The control group, consisting of 25 females, was dosed with the vehicle (drinking water) in parallel. A standard dose volume of 10 mL/kg bw 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 anaemia 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 were noted at high dose. 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 foetuses, foetal sex ratio, and the values calculated for pre- and postimplantation losses were all unaffected. The maternal NOAEL was 75 mg/kg bw/day based on statistically significant haematological changes in the dams at 250 and 750 mg/kg bw/day. Foetal examinations revealed no influence of Morpholine hydrochloride on sex distribution of the foetuses and foetal body weights. Morpholine hydrochloride showed no direct and specific effect on the respective morphological structures. Foetal 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 mirrored common minor effects on foetal 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 were not classified as adverse events. The developmental NOAEL was 750 mg/kg bw/day. No adverse foetal 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.

Justification for selection of Effect on developmental toxicity: via oral route:
Only one read-across study available. For read-across justification please refer to IUCLID section 13.

Justification for classification or non-classification

Based on results obtained in reproduction toxicity testing, the test item was not classified or labeled according to Regulation (EC) No 1272/2008 (CLP) or Directive 67/548/EEC (DSD).