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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2019-01-15 to 2019-09-06
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report Date:
2019

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
Dose Range Fiding Study, reduced number of animals in dose groups
Principles of method if other than guideline:
- Principle of test: The aim of this preliminary study was to detect effects of Methylmorpholine-oxide in the un-mated and pregnant rabbit following oral gavage administration. There were two phases of this study. In the first phase, a preliminary dose range finding phase was performed in un-mated females to assess potential toxicity to the rabbit and establish dose levels for the second phase. The second phase was to provide initial information on possible maternal toxicity and effects on embryo-fetal development when given to pregnant females from Day 6 to 21 of gestation and was used to establish appropriate dose levels for a subsequent study.
- Parameters analysed / observed: The following parameters and end points were evaluated in this study: clinical observations, body weights, food consumption, gross necropsy findings, maternal performance, ovarian and uterine findings and fetal examinations (external abnormalities and body weights).
GLP compliance:
no
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: Lenzing AG, 20.06.2018 10:00, T1
- Expiration date of the lot/batch: 20 Jun 2019
- Purity: 49.2%; dose calculations were corrected for purity.

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Kept in a refrigerator set to maintain 4°C, protected from light
- Stability: The stability of the bulk test item was not determined during the course of this study.
Information to support the stability of each lot of the bulk test item was provided by the
Sponsor.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Test item dosing formulations were prepared based on a method established at the Test Facility at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared by weighing out the required amount for each group and adding the appropriate amount of vehicle to the test item to achieve the requested concentration and then magnetically stirred until a homogenous formulation was obtained. This was done as required for the un-mated phase, and weekly for the mated phase, stored in a refrigerator set to maintain 4C, and dispensed daily. The formulations were stirred continuously during dosing.

Test animals

Species:
rabbit
Strain:
New Zealand White
Remarks:
HsdIf:NZW
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Envigo RMS (UK) Limited, Shaw’s Farm, Blackthorn, Bicester, Oxon, UK.
- Age at study initiation: 4 to 7 months
- Weight at study initiation: 2.5 to 4.0 kg
- Fasting period before study: no
- Housing: Females were housed individually in appropriately sized stainless steel cages with a ‘Noryl’ dual level interior and perforated flooring. Beneath each cage was a suspended tray containing absorbent paper.
- Diet (e.g. ad libitum): Teklad Certified Global Rabbit Diet (Envigo Diet), ad libitum. Each animal was also offered a supplement of hay at least 3 times per week and a small portion of a limited selection of fruits and/or vegetables at least twice per week.
- Water (e.g. ad libitum): public supply, ad libitum
- Acclimation period: The animals were allowed to acclimate to the Test Facility’s rabbit toxicology accommodation for at least 3 days before the commencement of dosing.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 16°C to 21°C
- Humidity (%): 30% to 64%
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light): 12-hour light/12-hour dark

IN-LIFE DATES:
- un-mated animals From: 09 Jan 2019 To: 15 Mar 2019
- time-mated animals From: 22 Feb 2019 To: 15 Mar 2019

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
- Test item dosing formulations were prepared based on a method established at the Test Facility at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared by weighing out the required amount for each group and adding the appropriate amount of vehicle to the test item to achieve the requested concentration and then magnetically stirred until a homogenous formulation was obtained. This was done as required for the un-mated phase, and weekly for the mated phase, stored in a refrigerator set to maintain 4° C, and dispensed daily. The formulations were stirred continuously during dosing. Any residual volumes were discarded.

VEHICLE: Milli Q water
- Concentration in vehicle: 20-75 mg/ml for un-mated phase of experiment
- Amount of vehicle (if gavage): 12.5-50 mg/ml for mated phase of experiment
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Duplicate top, middle and bottom (duplicate middle only for control) sets of samples were transferred at ambient temperature to the analytical laboratory at the Test Facility immediately after sampling. triplicate top, middle and bottom (triplicate middle only for
control) sets of samples were retained at the Test Facility as backup samples. Samples were 0.1 mL (taken by weight), collected into appropriately sized volumetric flasks, and kept in a refrigerator set to maintain 4°C, protected from light.
Concentration results were considered acceptable if sample concentration results were within or equal to ± 10% of theoretical concentration. For homogeneity, the criterion for acceptability was a relative standard deviation (RSD) of concentrations of ≤ 10% for each group. All results were acceptable and within the acceptance criteria.
Details on mating procedure:
Time-mated New Zealand White rabbits were received from Envigo RMS (UK) Limited, Shaw’s Farm, Blackthorn, Bicester, Oxon, UK.
Duration of treatment / exposure:
In unmated phase: 3 days (Dose Levels of 200 and 500 mg/kg/day)/ 7 days (Dose Level 750 mg/kg/day)
In the mated phase: days 6-21 of gestation
Frequency of treatment:
once daily
Duration of test:
unmated phase: up to day 8-15
mated phase: until day 22 of gestation
Doses / concentrationsopen allclose all
Dose / conc.:
200 mg/kg bw/day
Remarks:
unmated phase
Dose / conc.:
500 mg/kg bw/day
Remarks:
unmated phase
Dose / conc.:
750 mg/kg bw/day
Remarks:
unmated phase
Dose / conc.:
0 mg/kg bw/day
Remarks:
mated phase
Dose / conc.:
125 mg/kg bw/day
Remarks:
mated phase
Dose / conc.:
250 mg/kg bw/day
Remarks:
mated phase
Dose / conc.:
500 mg/kg bw/day
Remarks:
mated phase
No. of animals per sex per dose:
unmated phase: 2 female animals per dose
mated phase: 6 female animals per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The first dose for the un-mated phase of the study was set based on observations from a developmental toxicity study in the rat (Test Facility Study No. 498682ii). Within this study the rats were dosed at 30, 100 and 300 mg/kg/day, and the maternal No Observed Adverse Effects Level (NOAEL) was 30 mg/kg/day. At 300 mg/kg/day administration of Methylmorpholine-oxide was associated with body weight loss within the first few days of dosing followed by a reduction in body weight gain and an associated decrease in food consumption. At 100 mg/kg/day a lower body weight gain was also evident during gestation along with lower food consumption over the same period when compared with the controls.
Based on these results a dose level of 200 mg/kg/day was considered to be a suitable first dose level in the un-mated rabbits. The subsequent dose levels (200, 500, 750 mg/kg/d) were determined based on the data collected.
Based on the data from the un-mated phase, 500 mg/kg/day was considered to be a suitable high dose level for the mated phase due to the consistently low food consumption and body weight losses observed at 750 mg/kg/day. Based on these considerations, doses in the second phase were 125, 250, and 500 mg/kg/d, respectively.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were observed twice daily, once at the start and once towards the end of the working
day throughout the study for general health/mortality and moribundity. Animals were observed regularly throughout the day on each day of dosing for signs of reaction to treatment, with particular attention being paid to the animals during and for the first hour after dosing (see Protocol Deviations and Other Events in Appendix 1). The onset, intensity and duration of any signs were recorded, as appropriate.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Animals were subjected to detailed clinical observations daily from Day 1 for un-mated animals and weekly from Day 5 of gestation for mated animals. Animals were observed more frequently, as required, for welfare reasons.

BODY WEIGHT: Yes
- Time schedule for examinations: Un-mated animals were individually weighed once during pretreatment then daily from Day 1.
Mated animals were individually weighed once during pretreatment then daily from Day 6 to Day 22 of gestation

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for un-mated animals was measured daily from Day -3 for Group 3 and Animal 2501F, from Day -6 for Animal 1501F, from Day -7 for Animal 2502F and from Day -10 for Animal 1502F.
Food consumption for mated animals was measured daily from Day 4 of gestation.

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Water consumption was monitored on a regular basis throughout the study by visual inspection of the water bottles.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 22 for mated phase; day 15 for 200 mg/kg bw/day group, day 11 for 500 mg/kg bw/day group and day 8 for 750 mg/kg bw/day group in unmated phase.
- Organs examined: Mated females were subjected to a complete necropsy examination, which included the carcass and musculoskeletal system; all external surfaces and orifices; and thoracic, abdominal, and pelvic cavities with their associated organs and tissues. Necropsy examinations consisted of an external and internal examination and recording of observations for all animals.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included: The reproductive tract was dissected out and the gravid uterine weight recorded. The ovaries
and uterus were examined for number and distribution of the following parameters:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Placentae (size, colour or shape) – only abnormalities were recorded
Fetal examinations:
Each implant was assessed and classified in one of these categories: a live fetus, a dead fetus (dead fetus that showed no sign of maceration), a late embryonic resorption (macerated tissue identifiable as an embryo fetus, with recognisable external features such as tail, limbs, mouth and nares present; attached to distinct identifiable placentae), or an early embryonic death (discrete, formless, discoloured tissue mass attached to the internal uterine wall; may be of varying size).
Fetuses were examined for external abnormalities to the extent possible and weighed individually.
Statistics:
All results presented in the tables of the report are calculated using non-rounded values as per the raw data rounding procedure and may not be exactly reproduced from the individual data presented.
Any data collected during the pretreatment period were tabulated, summarised or statistically analysed. All statistical analyses were performed within the respective study phase, unless otherwise noted. Numerical data collected on scheduled occasions were summarised and statistically analysed as indicated below according to occasion.

Descriptive Statistical Analysis:
Means, standard deviations (or % coefficient of variation or standard error, when deemed appropriate), percentages, numbers and/or incidences have been re orted as appropriate by dataset.

Inferential Statistical Methods:
All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two-sided tests and have been reported at the 1%, and 5% levels unless otherwise noted.
The following pairwise comparisons were made:
Group 6 vs. Group 5
Group 7 vs. Group 5
Group 8 vs. Group 5
Analyses excluded any group with less than 3 observations.

Parametric/Non-Parametric Tests:
Levene’s test was used to assess the homogeneity of group variances.
The groups were compared either using an overall one-way ANOVA F-test if Levene’s test was not significant or using the Kruskal-Wallis test if it was significant. If the overall F-test or Kruskal-Wallis test was found to be significant, then pairwise comparisons were conducted using Dunnett’s or Dunn’s test, respectively.
Indices:
For all litter data, the litter was used as the unit of assessment. Group mean values for each parameter were calculated as a mean of litter percentages.
Pre-implantation loss (%) per litter was calculated as:
((Number of corpora lutea – Number of implantations per female)/Number of corpora lutea per female) x 100

Post-implantation loss (%) per litter was calculated as:
((Number of implantations – Number of live fetuses per female)/Number of implantations per female) x 100

Group mean implantation losses were expressed on a per litter basis according to the
following:
Total individual litter pre or post implantation loss (%)/Number of litters per group
Historical control data:
not specified

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
In the unmated phase at 750 mg/kg/day from Study Day 3 to 8 both animals had clinical observations of decreased faecal output.
In the mated phase at 500 mg/kg/day from Gestation Day 12 to 19 animals had clinical observations of decreased faecal output whereas the control animals had none.
It is considered likely that the decreased faecal output in both phases was a consequence of the lower food consumption.
There were no clinical observations at 200 or 500 mg/kg/day in the unmated phase and no test item-related clinical observations at 125 or 250 mg/kg/day in the mated phase.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
There were no unscheduled deaths.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
In the unmated phase at 500 or 750 mg/kg/day there were mean body weight losses of -102 g and -274 g respectively during in the dosing period.
In the mated phase at 500 mg/kg/day there was a mean bodyweight loss of -84 g from Gestation Day 6 to 11 and mean body weight gain was lower by 25% (195.5 g for the controls vs. 146.4 g for 500 mg/kg/day) from Gestation Day 11 to 22 when compared with the control. This resulted in a lower mean body weight gain of 27% (226.8 g for the controls vs. 62.0 g for 500 mg/kg/day) from Gestation Day 6 to 22 when compared with the control.
There were no effects on body weighs or body weight gains at 200 mg/kg/day in the unmated phase and at 125 or 250 mg/kg/day in the mated phase.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
In the unmated phase at 750 mg/kg/day food consumption was low ranging from 0 to 16 g per day compared with the pre-dose intake of 83 to 119 g per day,.
In the mated phase at 500 mg/kg/day food consumption was lower by 52% (total food consumption: 1848 g for the controls vs. 890 g for 500 mg/kg/day) throughout the dosing period.
There was no effect on food consumption at 200 or 500 mg/kg/day in the unmated phase and at 125 or 250 mg/kg/day in the mated phase.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no findings at 125, 250 and 500 mg/kg/day in the mated phase.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Details on results:
In the unmated phase, there were clinical observations of deceased faecal output at 750 mg/kg/day and body weight losses at 500 and 750 mg/kg/day with lower food consumption seen at 750 mg/kg/day. Based on these results it was considered that 500 mg/kg/day would be a suitable high dose level for the mated phase.

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
effects observed, treatment-related
Description (incidence and severity):
At 125, 250 or 500 mg/kg/day there were lower mean number of implantations of -11%, -21% and -25% respectively.
Post-implantation losses were lower in the treated groups when compared with the control.
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Description (incidence and severity):
There were no dead fetuses observed.
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
At 125, 250 or 500 mg/kg/day there were lower mean numbers of corpora lutea of -12%, -19% and -23% respectively when compared with the controls.
Details on maternal toxic effects:
Administration of Methylmorpholine-oxide by once daily oral gavage in mated rabbits resulted in initial body weight losses and reduced bodyweight gain at 500 mg/kg/day (HD) which were considered to be related to lower food consumption. There were also lower rates of implantation and corpora lutea in the treatment groups however, there were lower mean post implantation losses in the treatment groups when compared with the control

Effect levels (maternal animals)

open allclose all
Key result
Dose descriptor:
LOAEL
Effect level:
750 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
food consumption and compound intake
Key result
Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: no adverse effects observed

Results (fetuses)

Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 250 or 500 mg/kg/day group mean fetal weights were lower by 11% and 21% when compared to the control and were associated with lower mean gravid uterine weights of -14% and -26% respectively when compared with the control.
There were no effects on mean fetal weight at 125 mg/kg/day.
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
not examined
Changes in litter size and weights:
effects observed, treatment-related
Description (incidence and severity):
At 250 or 500 mg/kg/day there were lower gravid uterine weights (-4.15, -14.18 and -25.79 at 125, 250 and 500 mg/kg/day, respectively) correlating with lower fetal weights.
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Description (incidence and severity):
There were no external findings considered to be related to the test item at 125, 250 and 500 mg/kg/day.
Skeletal malformations:
not examined
Visceral malformations:
not examined
Details on embryotoxic / teratogenic effects:
The lower mean gravid uterine weight was considered to be associated with lower fetal weight at 250 and 500 mg/kg/day.

Effect levels (fetuses)

open allclose all
Key result
Dose descriptor:
LOAEL
Effect level:
250 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
fetal/pup body weight changes
changes in litter size and weights
Key result
Dose descriptor:
NOAEL
Effect level:
125 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: no adverse effects on fetuses observed

Applicant's summary and conclusion

Conclusions:
In conclusion, administration of Methylmorpholine-oxide by once daily oral gavage in mated rabbits resulted in initial body weight losses and reduced bodyweight gain at 500 mg/kg/d which is considered to be related to the lower food consumption. There were also lower rates of implantation and corpora lutea and lower mean post implantation losses in the treatment groups when compared with the control. There was also lower mean gravid uterine weight which are considered to be associated with lower fetal weight at 250 and 500 mg/kg/day. There were no fetal findings in the other examinations performed in this study. Based on these results a dose level of 500 mg/kg/day is considered suitable for use on a future developmental toxicity study.
Executive summary:

In a dose range finding study for developmental toxicity in rabbits (similar to OECD 414), the aim was to establish dose levels of Methylmorpholine-oxide in the pregnant rabbit for subsequent developmental toxicity studies in this species.

In the first phase, Methylmorpholine-oxide (49.2 % purity) was administered to 2 un-mated female New Zealand White rabbits/dose at dose levels of 200, 500 and 750 mg/kg bw/day for 3 -7 days by oral gavage to assess potential toxicity to the rabbit for preliminary dose range finding for the second phase.

At 750 mg/kg bw/day, mean body weight reduction and decreased faecal output was observed which can be attributed to lower food consumption. Based on these results, the top dose for the second phase of the experiment was set at 500 mg/kg bw/day.

In the second phase of the experiment, Methylmorpholine-oxide (49.2 % purity) was administered to 6 mated female New Zealand White rabbits/dose at dose levels of 0, 125, 250 and 500 mg/kg bw/day from gestation day 6 -21 by oral gavage in order to provide initial information on possible maternal toxicity and effects on embryo-fetal development.

Within the study an evaluation of clinical observations, body weights, food consumption, gross necropsy findings, maternal performance, ovarian and uterine findings and fetal examinations (external abnormalities and body weights) was performed.

At 500 mg/kg bw/day there were clinical signs of decreased faecal output and an initial body weight loss from Gestation Day 6 to 11 and subsequent lower body weight gain considered to be related to the lower food consumption throughout the dosing period. At 250 or 500 mg/kg bw/day there were lower gravid uterine weights correlating with lower fetal weights.

At 125, 250 or 500 mg/kg bw/day there was a lower mean number of implantations and a lower mean numbers of corpora lutea when compared with the controls. Post-implantation losses were lower in the treatment groups when compared with the control.

There were no effects on gross pathology and fetal findings at 125, 250 or 500 mg/kg bw/day.

Based on these results of the dose range finding study, a top dose level of 500 mg/kg bw/day was considered suitable for use in a subsequent OECD 414 study in rabbit.