Registration Dossier

Toxicological information

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Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Effects on fertility

Description of key information

A NOAEL of 100 mg/kg bw/day for fertility effects could be derived from a reliable extended one generation reproductive study in rats.

Link to relevant study records
Reference
Endpoint:
extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2019-03-08 to 2020-06-25
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Justification for study design:
SPECIFICATION OF STUDY DESIGN FOR EXTENDED ONE-GENERATION REPRODUCTION TOXICITY STUDY WITH JUSTIFICATIONS [please address all points below]:

- Premating exposure duration for parental (P0) animals
prolonged premating exposure up to 10 weeks is recommended to clarify potential effects on reproduction
- Basis for dose level selection
Based on results derived from a previously performed 90 day toxicity study, a combined repeated dose toxicity screening study and a deveopmental toxicity study
- Inclusion of extension of Cohort 1B
clarifiaction on fertility effects observed in P0
- Termination time for F2
n.a.
- Exclusion of developmental neurotoxicity Cohorts 2A and 2B
There were no effects observed in the available studies with Methylmorpholine-oxide that would trigger the inclusion of Cohorts 2A and 2B.
- Exclusion of developmental immunotoxicity Cohort 3
There were no effects observed in the available studies with Methylmorpholine-oxide that would trigger the inclusion of Cohort 3.
- Route of administration
Based on the information provided in the technical dossier and/or in the chemical safety report, ECHA agreed that the oral route - which is the preferred one as indicated in ECHA Guidance on information requirements and chemical safety assessment (version 4.1, October 2015) Chapter R.7a, section R.7.5.4.3 - was the most appropriate route of administration. More specifically, even though the information indicated that human exposure to the registered substance by the inhalation route is likely, the exposure concentrations reported in the chemical safety report for the inhalation route are low. Hence, the test was performed by the oral route.
- Other considerations
The Sprague Dawley rat was chosen as the animal model for this study as it is a rodent species accepted by regulatory agencies for reproductive toxicity testing. The total number of animals used in this study was considered to be the minimum required to properly characterise the effects of the test item. This study was designed such that it did not require an unnecessary number of animals to accomplish its objectives.
Specific details on test material used for the study:
TEST MATERIAL
- lot/batch number of test material:
01.03.2019 12:00, PA
- Expiration date of the lot/batch:
01 Mar 2020
- Purity:
50.5% in aqueous solution (dose calculations were corrected for purity)
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source:
Charles River UK Limited, Margate, Kent, UK
- Age at study initiation: (P) 6 to 9 weeks; (F1) 21 days
- Weight at study initiation: (P) 142 g - 385 g; (F1) 44 g - 74 g
- Housing: Animals were initially housed 2 or 3 per cage by sex (unless reduced by mortality) in appropriately sized suspended polycarbonate cages with stainless steel grid tops and solid bottoms. Bedding material was sterilised white wood shavings.
few days prior to mating, males were transferred to individual cages with solid bottoms. Females were transferred to this cage for mating.
Mated females were transferred to individual solid bottomed cages. White paper tissue was supplied as nesting material from Gestation Day (GD) 20. F0 females with litters were retained in this type of cage until termination. On a suitable day after completion of mating, the males were re-housed with their original cage mates.
F1 animals retained after weaning were housed 2 or 3 per cage by sex in appropriately sized suspended polycarbonate cages with stainless steel grid tops and solid bottoms.
- Diet (e.g. ad libitum):
SDS VRF-1 breeder diet was provided ad libitum
- Water (e.g. ad libitum):
water ad libitum from the public supply
- Acclimation period:
The F0 animals were allowed to acclimate to the test facility rodent toxicology accommodation for a period of approximately 2 weeks before the commencement of dosing.

ENVIRONMENTAL CONDITIONS
- Temperature (°C):
5 to 24°C
- Humidity (%):
32 to 84%
- Air changes (per hr):
>/= 10
- Photoperiod (hrs dark / hrs light):
12/12

IN-LIFE DATES:
From: 18. March To: 24. Oct. 2019
Route of administration:
oral: gavage
Vehicle:
water
Remarks:
Milli-Q
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The dosing formulations were prepared at least weekly, stored in a refrigerator set to maintain 4 °C, or transferred immediately to the animal unit, and dispensed daily.

VEHICLE
- Justification for use and choice of vehicle (if other than water): n.a.
- Concentration in vehicle: 3, 10 and 30 mg/mL
- Amount of vehicle (if gavage): 10 ml/kg
- Lot/batch no. (if required):
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: max. 11 nights
- Proof of pregnancy: Vaginal lavages were taken early each morning from the day of pairing until mating occurred and the stage of oestrus observed in each vaginal lavage was recorded. The day of detection of a copulatory plug in situ and/or of sperm in the lavage was designated GD 0.
- After successful mating each pregnant female was caged: Mated females were transferred to individual solid bottomed cages. White paper tissue was supplied as nesting material from Gestation Day (GD) 20.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dose formulation samples were collected from all groups on day 1, week 14 and 27.
Duplicate sets of top, middle and bottom samples (duplicate middle only for control) were collected for analysis; triplicate top, middle and bottom samples (triplicate middle only for control) were retained at the test facility as backup samples. Sample volumes (0.1 mL; recorded by weight) were 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 mean 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.
Stability analyses performed previously demonstrated that the test item is stable in the vehicle when prepared and stored under the same conditions at concentrations bracketing those used in the present study.
Duration of treatment / exposure:
The F0 males were dosed for at least 10 weeks up to necropsy. The F0 females were dosed for 10 weeks prior to mating, then through mating, gestation and until at least Lactation Day (LD) 21. The F1 animals were dosed from PND 21 up to at least PND 90.
Frequency of treatment:
once daily
Details on study schedule:
- Age at mating of the mated animals in the study (P0): 16-19 weeks (after 10 weeks dosing)
P0 males were treated for 10 weeks prior to mating until necropsy after the termination of the P0 females. F0 females were treated for 10 weeks prior to mating, then through mating, gestation and until at least Lactation Day (LD) 21. F1 animals from Cohorts 1A and 1B were then dosed directly from postnatal day (PND) 21 to at least PND 90.
Dose / conc.:
30 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
25
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
Based on results of a combined repeated dose toxicity screening study, a 90 day toxicity Study and a developmental toxicity study. Based on these studies 500 mg/kg/day was considered not to be suitable due to the effects observed within the reproductive parameters. A high dose level of 300 mg/kg/day was considered to be suitable to induce moderate but not excessive effects of toxicity.
- Fasting period before blood sampling for clinical biochemistry:
yes
Parental animals: Observations and 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 dosing day for signs of reaction to treatment, with particular attention being paid to the animals during and for the first hour after dosing.
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 weekly, beginning Week -1. The animals were removed from the cage for examination. The examinations included, but were not limited to, changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (lacrimation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture, response to handling, as well as the presence of clonic or tonic movements, stereotypy or bizarre behaviour were also examined.

BODY WEIGHT: Yes
- Time schedule for examinations:
Males were weighed weekly beginning Week -1. Females were weighed weekly beginning Week -1 until pairing for mating and then on GD 0, 7, 14 and 20 and on LD 1, 4, 7, 14 and 21. Litters were weighed en masse and by sex on LD 4, 7 and 14 and weighed individually on LD 1 and 21. The final in-life body weight was recorded on the day of scheduled necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
Food consumption was quantitatively measured for both sexes weekly, beginning Week -1 until pairing for mating, and for the mated females on the GD periods 0-7, 7-14 and 14-20 and on LD periods 1-7, 7-14 and 14-21. Food consumption resumed weekly for the males after mating and re-housing.

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

HAEMATOLOGY:
At necropsy day.
Analysed parameters: Red blood cell count, Haemoglobin concentration, Haematocrit, Mean corpuscular volume, Red blood cell distribution width, Mean corpuscular haemoglobin concentration, Mean corpuscular haemoglobin, Reticulocyte count (absolute), Platelet count White blood cell count, Neutrophil count (absolute), Lymphocyte count (absolute), Monocyte count (absolute), Eosinophil count (absolute), Basophil count (absolute), Large unstained cells (absolute), Activated partial thromboplastin time, Fibrinogen, Prothrombin time

CLINICAL CHEMISTRY:
At necropsy day.
Analysed parameters: Alanine aminotransferase, Aspartate aminotransferase, Alkaline phosphatase, Gamma-glutamyltransferase, Creatine kinase, Total bilirubin, Urea, Creatinine, Calcium, Phosphate, Total protein, Albumin, Globulin, Albumin/globulin ratio, Glucose, Cholesterol, Triglycerides, Sodium, Potassium, Chloride

THYROID STIMULATING HORMONE, T3 AND T4:
At necropsy day.

URINALYSIS:
At last week of dosing.
Analysed parameters: Colour, Appearance/Clarity, Specific gravity, Volume, pH, Protein, Glucose, Bilirubin, Ketones, Blood
Oestrous cyclicity (parental animals):
Vaginal lavages were taken early each morning and the stages of oestrus observed were recorded from Study Day 57 (i.e. 2 weeks before pairing for mating) until the day of detection of a copulatory plug in situ and/or of sperm in the lavage.
Sperm parameters (parental animals):
Computer-aided sperm assessment (CASA)
From all males from the P0, the right cauda epididymis was placed in Medium 199 (as per Test Facility SOPs) and the sperm were allowed to “swim out” into the medium. An appropriate dilution of the sperm suspension was prepared and examined using a Hamilton Thorne sperm motility analyser.

Sperm Count and Morphological Analysis
The right cauda epididymis was minced and suspended. Dilutions of this sperm suspension were counted using a haemocytometer to obtain a total sperm count which was expressed per cauda epididymis and per gram of cauda epididymis.
From all samples of the sperm suspension as described in the preceding paragraph, a sperm smear was prepared and stained with eosin Y solution. From the Group 1 and 4 P0 animals, at least two hundred sperm per animal were evaluated for morphological abnormalities using criteria described by Wyrobek and Bruce (1975). As test item-related effects were noted in Group 4 for the P0 animals and Group 3 for the Cohort 1A animals, Groups 2 (and 3) were also examined.

Spermatid Count
One testis was decapsulated and homogenised. The homogenate was sonicated to reduce tissue debris, as if required. The number of homogenisation-resistant spermatids in dilutions of this suspension was counted using a haemocytometer to obtain a total spermatid count which was expressed per testis and per gram of testis.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in [F1] offspring:
The numbers of live and dead pups born in each litter was recorded as soon as possible after completion of parturition on LD 0. The live pups were counted, sexed, weighed individually on PND 1 and examined daily for the presence of milk in the stomach and for any externally visible abnormalities daily up to and including PND 4. On PND 4, litter size was standardised to 8 pups (if possible 4 males and 4 females), by culling of extra pups via random selection. Extra pups were necropsied. If 4 males or 4 females were not available, extra pups of the opposite sex were retained to ensure a total number of 8 pups. When the total number of pups in a litter on PND 4 was ≤ 8 pups, no litter size adjustment occurred. From PND 5, the total live pups were counted daily and were sexed and examined for abnormalities again on PND 7, 14 and 21. These pups were weighed en masse and by sex on PND, 4, 7 and 14. On PND 21 all pups were weighed individually.
Where practicable, any pups that were found dead or were killed during lactation were sexed and appropriately examined as above. Any externally abnormal decedent pups were preserved; externally normal pups were discarded.
Deficiencies in maternal care were recorded: inadequate construction or cleaning of the nest, pups left scattered and cold, physical abuse of pups, or apparently inadequate lactation or feeding. White paper tissue was supplied to each mother for incorporation in the nest. This was replaced when it had become soiled. Females which failed to produce a litter by their expected GD 24 continued to be dosed until termination on their expected GD 25 to 27.
Assessment of ano-genital distance was measured for both sexes on PND 1. Nipple retention was assessed in males on PND 13.

- Selection and weaning of F1 animals for Cohort 1A and 1B
From each group, 40 males and 40 females were selected for post-weaning assessments, nominally by selecting up to 2 males and 2 females from each litter. The selected pups had the median body weight among the pups of that sex in the litter on PND 20 and were individually identified. These pups were removed from their mother on PND 21, and housed in their new cage.
Pups that were not selected for post-weaning assessments remained with their mother until sacrifice on LD 22 (PND 22).

- Assessment of F1 Sexual Maturation (Cohorts 1A and 1B)
Commencing at PND 28, females were examined daily for vaginal opening. The day on which the vagina became open was recorded, along with the body weight on that day. Commencing at PND 35, males were examined daily for balano-preputial separation. The day on which separation occurred was recorded, along with the body weight on that day.

In-life procedures. observations and measurements F1 (Cohorts 1A and 1B)
- Mortality/Moribundity Checks
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.

- Detailed Clinical Observations
Animals were subjected to detailed clinical observations weekly from weaning on PND 21, starting on a suitable day within one week of weaning of the majority of litters. The animals were removed from the cage for examination. The examinations included, but were not limited to, changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (lacrimation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture, response to handling, as well as the presence of clonic or tonic movements, stereotypy or bizarre behaviour were also examined.

- Pre and Postdose Observations
Animals were observed regularly throughout dosing for signs of reaction to treatment, with particular attention being paid to the animals during and for the first hour after dosing. The onset, intensity and duration of any signs were recorded, as appropriate.

- Body weights
Animals were individually weighed weekly, starting on a suitable day within one week of weaning, on PND 21 for the majority of litters. The final body in-life weight was recorded on the day of scheduled necropsy.

- Food consumption
Food consumption was quantitatively measured weekly, starting on a suitable day within one week of weaning, on PND 21 for the majority of litters.

Water consumption
Water consumption was monitored on a regular basis throughout the study by visual inspection of the water bottles.

- Oestrus Cycle Monitoring
Vaginal lavages were taken early each morning and the stages of oestrus observed were recorded from the day after vaginal patency (approximately PND 28) until 1 oestrus cycle was confirmed for Cohort 1A and 1B. Daily vaginal lavages were again taken early each morning and the stages of oestrus observed from PND 76 up to and including the day of necropsy (PND 91 – 93) for the Cohort 1A animals.

- Haematology (Cohort 1A; 10 rats/sex/group):
At day of necropsy
Analysed parameters: Red blood cell count, Haemoglobin concentration, Haematocrit, Mean corpuscular volume, Red blood cell distribution width, Mean corpuscular haemoglobin concentration, Mean corpuscular haemoglobin, Reticulocyte count (absolute), Platelet count, White blood cell count, Neutrophil count (absolute), Lymphocyte count (absolute), Monocyte count (absolute), Eosinophil count (absolute), Basophil count (absolute), Large unstained cells (absolute), Activated partial thromboplastin time, Fibrinogen and prothrombin time

- Clinical Chemistry (Cohort 1A; 10 rats/sex/group):
At day of necropsy
Analysed parameters: Alanine aminotransferase, Aspartate aminotransferase, Alkaline phosphatase, Gamma-glutamyltransferase, Creatine kinase, Total bilirubin, Urea, Creatinine, Calcium, Phosphate, Total protein, Albumin, Globulin, Albumin/globulin ratio, Glucose, Cholesterol, Triglycerides
Sodium, Potassium, Chloride, Sample quality

- Urinalysis (Cohort 1A; 10 rats/sex/group):
Last week of dosing
Analysed parameters: Colour, Appearance/Clarity, Specific gravity, Volume, pH, Protein, Glucose, Bilirubin, Ketones, Blood

- Thyroid stimulating hormone, T3 and T4 (F1 - culled offspring up to 4 rats/sex/group), F1 - unselected pups (1 rat/sex/group), Cohort 1A - 10 rats/sex/group):
culled: PND 4
F1 - unselected pups: PND 22
Cohort 1A: at day of necropsy

Postmortem examinations (parental animals):
SACRIFICE
P0 animals:
- Male animals: study day 127-132
- Maternal animals: LD 22

GROSS NECROPSY
- P0
Animals were subjected to a complete necropsy examination, which included evaluation of the carcass and musculoskeletal system; all external surfaces and orifices; cranial cavity and external surfaces of the brain; and thoracic, abdominal, and pelvic cavities with their associated organs and tissues

ORGAN WEIGHTS
P0:
brain, epididymis, adrenal gland, pituitary, prostate, thyroid, parathyroid, heart, kidney, liver, ovary, spleen, testis, thymus, uterus/cervix. Paired organs were weighed together. Organ to body weight percentages (using the terminal body
weight) were calculated.

MICROSCOPIC EVALUATION
P0:
Aorta, bone marrow (sternum), bone (femur, sternum), brain, cervix, epididymis, esophagus, eye, adrenal gland, Harderian gland, mammary gland, parathyroid, pituitary, prostate, salivary submandibular gland, seminal vesicle/coagulating gland, thyroid, gut-associated lymphoid tissue, heart, femorotibial joint, kidney, cecum, colon, rectum, liver, lung, lymph node (mandibular, mesenteric), skeletal muscle, nerve (optic, sciatic), ovary, pancreas, skin, duodenum, ileum, jejunum, spinal cord, spleen, stomach, testis, thymus, tongue, trachea, urinary bladder, uterus and vagina
Postmortem examinations (offspring):
SACRIFICE
F1 animals:
- Cohort 1A: PND 91-93
- Cohort 1B: a suitable period after PND 98
- non selected pups: PND 4
- non selected weanlings: PND 22

GROSS NECROPSY
- Non selected weanlings PND4
Non-selected pups on PND4 were necropsied. This consisted of external examination followed by macroscopic examination of the tissues and organs of the cranial, thoracic and abdominal cavities in situ. Samples of any grossly abnormal tissues were preserved in 10% formalin or other appropriate fixative. The carcasses were discarded.

- Non selected weanlings PND22
From each litter, 1 male and 1 female pup (where they were available) were necropsied. This consisted of external examination followed by macroscopic examination of the tissues and organs of the cranial, thoracic and abdominal cavities in situ. Samples of any grossly abnormal tissues were preserved in 10% formalin or other appropriate fixative. The carcasses were then discarded.
Where 1 pup of each sex was not available, additional pups of the opposite sex were necropsied/weighed such that 2 animals were necropsied and 2 animals were weighed per litter as far as possible. The remaining pups in each litter were checked for externally visible abnormalities at the
time of euthanasia. Any found to have an abnormality had a gross necropsy performed and any abnormalities were fixed. The remaining carcasses were discarded.

- F1
Where practicable, offspring found dead or killed (unscheduled or scheduled) were sexed internally opening and then checked for the presence of milk in the stomach and for the presence of any externally visible abnormalities. Any abnormal pups were, where practicable, fixed in 10% formalin or methylated ethyl alcohol, as appropriate, for optional further examination. Externally normal pups were discarded.

ORGAN WEIGHTS
F1 (Cohort 1A):
brain, epididymis, adrenal gland, pituitary, prostate, thyroid, parathyroid, heart, kidney, liver, ovary, spleen, testis, thymus, uterus/cervix. Paired organs were weighed together. Organ to body weight percentages (using the terminal body
weight) were calculated.

MICROSCOPIC EVALUATION
F1 (Cohort 1A):
Aorta, bone marrow (sternum), bone (femur, sternum), brain, cervix, epididymis, esophagus, eye, adrenal gland, Harderian gland, mammary gland, parathyroid, pituitary, prostate, salivary submandibular gland, seminal vesicle/coagulating gland, thyroid, gut-associated lymphoid tissue, heart, femorotibial joint, kidney, cecum, colon, rectum, liver, lung, lymph node (mandibular, mesenteric), skeletal muscle, nerve (optic, sciatic), ovary, pancreas, skin, duodenum, ileum, jejunum, spinal cord, spleen, stomach, testis, thymus, tongue, trachea, urinary bladder, uterus and vagina

IMMUNOPHENOTYPING SAMPLE COLLECTION AND ANALYSIS
Spleen samples (approximately half of the spleen) were taken from 10 rats per sex per group of the Cohort 1A F1 animals at necropsy and were collected into tubes containing RPMI (Roswell Park Memorial Institute) medium and stored immediately on wet ice. The remainder of the spleen was preserved in 10% neutral buffered formalin. The samples were analysed by flow cytometry.
Statistics:
Descriptive statistics such as arithmetic means and standard deviations, accuracy and precision were calculated using Microsoft® Excel.

All statistical tests were conducted at the 5% significance level. All pairwise comparisons
were conducted using two-sided tests and were reported at the 1% and 5% levels, unless
otherwise noted.

Parametric/Non-Parametric
Levene’s test was used to assess the homogeneity of group variances.
Datasets with at least 3 groups were compared using an overall one-way ANOVA F-test if Levene’s test was not significant or the Kruskal-Wallis test if it was. If the overall F-test or Kruskal-Wallis test was found to be significant, then the above pairwise comparisons were conducted using Dunnett’s or Dunn’s test, respectively.

Incidence
A Fisher’s exact test was used to conduct pairwise group comparisons of interest.
Clinical signs:
no effects observed
Description (incidence and severity):
There were no clinical observations considered to be related to the test item at 30, 100 or 300 mg/kg/day in the F0 animals.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
There were two unscheduled deaths during the course of this study. One P0 male receiving 30 mg/kg/day was found dead on Study Day 104 and another female P0 animal receiving 0 mg/kg/day was euthanised on Lactation Day 1 due to having difficulty littering
Low dose male:
In this animal there were no clinical observation or effects on body weight or food consumption that were considered to be related to the test-item or cause of death. There were gross pathology findings of mottled discoloured lobes in the lungs and dark focus in the thymus however, these considered to be agonal changes. Histologically, there was minimal cellular debris in the testis, which was also considered not to be related to the death of this animal. The cause of death was therefore not determined. This death was considered not to be related to the test item as there were no deaths in the higher dose levels of 100 or 300 mg/kg/day.

Control female:
This animal had clinical observations of firm inter structure in the abdomen, pallor skin and fur staining, there were no gross pathology findings. Histologically, there was dilatation and minimal inflammatory infiltrate in the uterus, but this was considered normal for an animal giving birth, and no cause of the dystocia was identified.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 300 mg/kg/day body weight gain was 31% lower (299 g for the controls vs. 207 g for the 300 mg/kg/day dose) in the males over the whole study period and 30% lower in the females during in premating period (108 g for the controls vs. 75.2 g for the 300 mg/kg/day dose) when compared with the controls. The bodyweight gain was lowest between Study Day 1 and 29 in both the males and pre-mating females so that by Study Day 29 the mean group bodyweight was lower by 14% (439 g for the controls vs. 379 g for the 300 mg/kg/day dose) in the males and 10% (254 g for the controls vs. 227 g for the 300 mg/kg/day dose) in the females when compared with the controls.

At 100 mg/kg/day bodyweight gain was slightly lower by 9% (108 g for the controls vs. 98 g for the 100 mg/kg/day dose) in the premating period and lower by 10% (139 g for the controls vs. 125 g for the 100 mg/kg/day dose) in the gestation period in the females when compared with the controls. At the start of the lactation period body weights were lower 10% (Lactation Day 1; 340 g for the controls vs. 307 g for the 100 mg/kg/day dose) and remained lower throughout lactation period when compared with the controls.

There were no effects on body weight or body weight gain in the males at 30 or 100 mg/kg/day or females at 30 mg/kg/day.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
At 300 mg/kg/day food consumption was lower by 11% in the males throughout the dosing period and females in the premating period when compared with the control. In both sexes food consumption was particularly lower between Study Day 1 and 29 where it was lower by 13-21% in the males and 16-21% lower in females when compared with the controls.

At 100 mg/kg/day food consumption was lower by between 10-15% during in gestation and 9-13% during lactation in the females when compared with the controls.

There were no effects on food consumption in the males at 30 or 100 mg/kg/day or the females at 30 mg/kg/day.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
At 300 mg/kg/day there were lower levels of white blood cells (×0.68), lymphocytes (×0.58), monocytes(×0.85), eosinophils (×0.79), basophils (×0.75), large unstained cells (0.023), red blood cell count (×0.92) and red blood cell distribution width (×0.92) and increase in the level of reticulocytes (×1.16), mean corpuscular haemoglobin (×1.07) in the F0 males when compared with the control. There were no test item-related effect on haematology parameters at 30, 100 or 300 mg/kg/day in the females or at 30 mg/kg/day in the males.
However, it was concluded that there were no test item-related effects on haematology parameters, since all values were considered to be within normal biological variation.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
At 300 mg/kg/day there was a decrease in triglycerides (x0.52) and an increase alanine aminotransferase (x1.43) in the males and an increase albumin/globulin ratio (x1.20) in the females. At 300 and 100 mg/kg/day there was a decrease in globulin (x0.89 and x0.76 for 100 and 300 mg/kg/day, respectively) and an increase
albumin/globulin ratio (x1.14 and x1.38 for 100 and 300 mg/kg/day, respectively) in the males. There were no test item-related effect on clinical chemistry parameters at 100 or 300 mg/kg/day in the females or at 30 mg/kg/day in the males. However, it was concluded that there were no test item-related effects on clinical chemistry parameters, since all values were considered to be within normal biological variation.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
At 300 mg/kg/day in males and at 100 mg/kg/day in females (both P0), there was a significant increase in specific gravity in males. In the F1 generation, there was a significant increase in specific gravity at 100 mg/kg/day in males and a significant increase in urinary volume at 30 mg/kg/day in females. In summary, there were no test item-related effects on urinalysis parameters. All values were considered to be within normal biological variation.
Behaviour (functional findings):
no effects observed
Immunological findings:
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
There was minimal or mild spermatid retention in the testis in 15/25 animals at 100 mg/kg/day and all 25 animals at 300 mg/kg/day. This finding was evident in tubules at stage IX to XII of spermatogenesis. The incidence and average severity of cellular debris in the testis was also higher than controls at 100 and 300 mg/kg/day. This finding comprised generally oval or sometimes round bodies consisting of densely packed chromatin with adjacent scant cytoplasm, within tubule lumens or amongst maturing spermatids. In some other cases, debris comprised degenerate or multinucleate spermatogenic cells at any spermatogenic stage. Both findings showed a dose level-related increase in incidence and severity
In the epididymis, there was minimal decreased sperm cellularity (comprising decreased numbers of sperm in the tubule lumens, near the head of the epididymis) at 100 and 300 mg/kg/day, and minimal cellular debris at 300 mg/kg/day. The occurrence of cellular debris in 1 animal given 30 mg/kg/day was considered not to be test item-related, because the animal also had mild tubular atrophy in the testis, which is a spontaneous change seen occasionally in rats.
It was considered that the spermatid retention and cellular debris in the testis, and the effects seen on sperm motility, count and morphology were likely to be due to the same effect of the test item and that the epidydimal changes resulted from the testicular changes. These findings were considered to have at least contributed to the decreased fertility. The histological effects of the test item in the testis and epididymis were not seen at 30 mg/kg/day.
In the mesenteric lymph node, minimal or mild macrophage vacuolation occurred at 100 and 300 mg/kg/day in males, with no dose level-response, and (in only 1 animal at the minimal severity) at 100 mg/kg/day in females.
In the spleen, the incidence and average severity of increased brown pigment in the red pulp were higher than controls at 300 mg/kg/day in males and at 100 mg/kg/day in females. The slightly higher incidence of increased pigment at 100 mg/kg/day in males was considered insufficient evidence of an effect at this dose level.
In the Harderian gland, the incidence and average severity of acinar dilatation were higher than controls at 300 mg/kg/day in males. The slightly higher incidences at 30 and 100 mg/kg/day in both sexes were considered insufficient evidence of an effect at these dose levels.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
There were no test item-related effects on thyroid hormone parameter. All values were considered to be within normal biological variation.
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
effects observed, treatment-related
Description (incidence and severity):
At 300 mg/kg/day there was a lower sperm motility (-44%), straight line velocity (-36%), number of sperm per gram of cauda (-76%), total number of sperm per cauda -(84%), number of spermatids per gram of testis (-19%) and total number of spermatids per testis (-15%) in the P0 males when compared with the controls. There was also a higher total number of abnormal sperm (84%; including tail defects, 76%; excluding tail defects) in the P0 males when compared with the controls.
At 100 mg/kg/day there was a lower straight line velocity (-16%), number of sperm per gram of cauda (-18%) and total number of sperm per cauda (-23%) in the P0 males when compared with the controls. There was also a higher total number of abnormal sperm (13%; including tail defects, 13%; excluding tail defects) in the P0 males when compared with the controls.
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
At 300 mg/kg/day there was a lower male and female fertility index of 0% when compare with the control at 96%.
The females dosed at 300 mg/kg/day had regular oestrous cycles prior to mating and there were positive mating signs in all females after 4 nights of pairing.
The uteruses of all the females were stained and there were no signs of implantation indicating that the females had never become pregnant.
There were no effects on mating performance or fertility indices at 30 or 100 mg/kg/day.
There were no effects on duration of gestation, overall litter performance or survival at 30 or 100 mg/kg/day.
There were considered to be no test item-related effects on ovarian counts.
There were no test item-related effects on thyroid hormone parameter. All values were considered to be within normal biological variation.
Key result
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive function (sperm measures)
reproductive performance
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
300 mg/kg bw/day (actual dose received)
System:
male reproductive system
Organ:
other: affected sperm parameters
Treatment related:
yes
Dose response relationship:
yes
Clinical signs:
no effects observed
Description (incidence and severity):
There were no clinical observations considered to be related to the test item at 30 or 100 mg/kg/day in the F1 animals.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Litter weights:
At 100 mg/kg/day litter weights were lower by 11% on Lactation Day 14 (309 g for the controls vs. 274 g for the 100 mg/kg/day dose) and on Lactation Day 21 (257 g for the controls vs. 228 g for the 100 mg/kg/day dose) when compared to the control. This correlated with slightly lower male and female pup weights of up 6% on Lactation Day 21 (males; 63 g, female; 60 g for the controls vs. males; 59 g, females; 56 g for the 100 mg/kg/day dose) when compared with the controls
There were no effects on litter or pup weights at 30 mg/kg/day.
Body weights:
At 100 mg/kg/day body weight gain was lower by 10-12% in F1 males in Cohort 1A (459 g for the controls vs. 414 g for the 100 mg/kg/day dose) and Cohort 1B (563 g for the controls vs. 496 g for the 100 mg/kg/day dose).
At 100 mg/kg/day body weight gain was lower by 7-10% in the F1 females in Cohort 1A (288 g for the controls vs. 206 g for the 100 mg/kg/day dose) and Cohort 1B (266 g for the controls vs. 246 g for the 100 mg/kg/day dose).
There were no body weight or body weight gain effects in the Cohort 1A and 1B animals at 30 mg/kg/day
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
At 100 mg/kg/day food consumption was slightly higher in the Cohort 1A and 1B males and females where on occasion food consumption was lower by up to 13% and 9% in the males and females respectively when compared with the control. There were no effects on food consumption in the Cohort 1A or Cohort 1B animals at 30 mg/kg/day.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
At 100 mg/kg/day, there were lower levels of white blood cells (×0.73), lymphocytes (×0.68) and large unstained cells (×0.38) in the Cohort 1A males.
At 100 and 30 mg/kg/day, there was a lower RDW (×0.96, respectively) in Cohort 1A females. In addition, there was a slightly higher MCHC at 100 mg/kg/day in Cohort 1A females.
However, it was concluded that there were no test item-related effects on haematology parameters. All values were considered to be within normal biological variation.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
At 100 mg/kg/day there was an increase in alanine aminotransferase (x1.19) and inorgainic phosphate (x1.10) in the males. At 100 and 30 mg/kg/day there was an increase in alkaline phosphatase (x1.18 and x1.13 at 100 and 30 mg/kg/day, respectively) in the males and an increase in inorganic phosphate in the females (x1.12 and x1.17 at 100 and 30 mg/kg/day, respectively).
Urinalysis findings:
no effects observed
Description (incidence and severity):
There were no test item-related effects on urinalysis parameters. All values were considered to be within normal biological variation.
Sexual maturation:
no effects observed
Description (incidence and severity):
There was no effect on sexual maturation at 30 or 100 mg/kg/day.
Anogenital distance (AGD):
no effects observed
Description (incidence and severity):
At 100 mg/kg/day anogenital distance was slightly lower by 8% (2.02 g for the controls vs. 1.86 g for the 100 mg/kg/day dose) in the F1 females on PND1 when compared to the control. There was no effect on anogenital distance at 100 or 30 mg/kg/day in the F1 males and no effect on anogenital distance at 30 mg/kg/day in the F1 females.
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
There was no effect on nipple retention at 100 or 30 mg/kg/day in the F1 males.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Cohort 1A
The lower epididymis weight noted at the terminal euthanasia for the F0 males receiving 300 mg/kg/day was also observed at 100 mg/kg/day in the terminal euthanasia for the F1 males – Cohort 1A. Despite the absence of histological effects in the epididymis in the F1 animals, this was considered to be test item-related.

Other intergroup differences in organ weights were considered not be test item-related, because they had no histological correlate. This included the higher prostate weight in males receiving 30 or 100 mg/kg/day.

Cohort 1B
The lower epididymis weight noted at the terminal euthanasia for the F0 males receiving 300 mg/kg/day was also observed at 100 mg/kg/day in the terminal euthanasia for the F1 males – Cohort 1B and was considered to be test item-related.

Intergroup differences in the other organs weighed (pituitary and prostate glands, ovary and testis) were considered not be test item-related. This included the higher prostate weight in males receiving 100 mg/kg/day.

Pups not selected for Cohorts 1A and 1B
There were no intergroup differences in the organs weighed (brain, spleen and thymus) that were considered to be test item-related.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Cohort 1A
There were considered to be no test item-related gross findings. The spleen was not dark in any animals, correlating with the absence of increased pigment in the red pulp histologically.

Cohort 1B
There were considered to be no test item-related gross findings. The only abnormality recorded was a subcutaneous mass in a control female, that was found histologically to be a mammary gland adenocarcinoma.

Pups culled on PND 4
These pups had no abnormalities at necropsy, and all had milk in the stomach.

Pups not selected for Cohorts 1A and 1B
There were no grossly abnormal tissues in these animals.
Histopathological findings:
effects observed, treatment-related
Description (incidence and severity):
Cohort 1A
Test item-related microscopic findings noted in the testis and mesenteric lymph node at 100 and 300 mg/kg/day at the terminal euthanasia for the F0 animals were also observed at 100 mg/kg/day in the terminal euthanasia for the F1 Animals – Cohort 1A.
Decreased sperm cellularity in the epididymis was not seen at 100 mg/kg/day in the F1 Animals – Cohort 1A, although it had been seen in the F0 animals at that dose level.
Additional evaluations of sperm parameters showed that there was a lower straight line velocity (-20%), number of sperm per gram of cauda (-17%), total number of sperm per cauda (-32%), number of spermatids per gram of testis (-13%) and total number of spermatids per testis (-10%) in the Cohort 1A males at 100 mg/kg/day when compared with the controls. There was also a higher total number of abnormal sperm (37%; including tail defects, 36%; excluding tail defects) in the Cohort 1A males when compared with the controls.

The incidence and severity of pigment in the red pulp of the spleen were not increased at 100 mg/kg/day in either sex in the F1 Animals – Cohort 1A, although they had been increased in the F0 females at that dose.
Other effects:
no effects observed
Description (incidence and severity):
Immunophenotyping
The results demonstrate that at necropsy there is no test item or dose dependent effect on the cell populations analysed within the spleen. Both dosing Group 2 (30 mg/kg /day) and Group 3 (100 mg/kg/day), showed comparable percentages of all immune cells analysed with the control Group 1 animals.
There were no test item-related effects on thyroid hormone parameters. All values were considered to be within normal biological variation.
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Key result
Dose descriptor:
NOAEL
Generation:
F1 (cohort 1A)
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: sperm parameters
Key result
Dose descriptor:
NOAEL
Generation:
F1 (cohort 1B)
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: sperm parameters
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
300 mg/kg bw/day (actual dose received)
System:
male reproductive system
Organ:
other: sperm parameters
Treatment related:
yes
Dose response relationship:
yes
Key result
Reproductive effects observed:
yes
Lowest effective dose / conc.:
300 mg/kg bw/day (actual dose received)
Treatment related:
yes
Relation to other toxic effects:
not specified
Dose response relationship:
yes

Please refer to attached tables

Conclusions:
Administration of Methylmorpholine-oxide by oral gavage once daily was well tolerated in rats at levels up to 100 mg/kg/day. A dose level of 300 mg/kg/day showed adverse effects manifesting in a reduction of fertility rate to 0%, which can be attributed to changes in the motility, morphology and number of sperm. Although there were effects on the motility, morphology and number of sperm at 100 mg/kg/day, this observation was considered not to be adverse as it did not affect the fertility of the animals.
Based on these results, the no-observed-adverse-effect level (NOAEL) was considered to be 100 mg/kg/day.
Executive summary:

In an extented one-generation reproductive toxicity study (including Cohorts 1A and 1B) in rats, Methylmorpholine-oxide was administered to groups of 25 animals per sex and dose level by gavage at dose levels of 30, 100 or 300 mg/kg/day (P0) or to groups of 20 animals per sex and dose level by gavage at dose levels of 30 or 100 mg/kg/day (F1, Cohorts 1A and 1B).

P0 males were treated for 10 weeks prior to mating until necropsy after the termination of the P0 females. P0 females were treated for 10 weeks prior to mating, then through mating, gestation and until at least Lactation Day (LD) 21. F1 animals from Cohorts 1A and 1B were then dosed directly from postnatal day (PND) 21 to at least PND 90.

There was a lower body weight gain in the P0 males and in the pre-mating period for the P0 females at 300 mg/kg/day correlating with a lower food consumption. At 100 mg/kg/day, there were lower body weight gains in the premating, gestation and lactation phase in the P0 animals. There was also a lower body weight gain at 100 mg/kg/day in the Cohort 1A and 1B animals. The lower body weight gains correlated with lower food consumption.

At 100 mg/kg/day there was a lower mean litter weight in the P0 litters which correlated to a slightly lower body weight in the pups on PND21 when compared to the control.

There was a slightly lower anogenital distance on PND1 in the F1 females pups when compared with the control.

At 300 mg/kg/day, the fertility rate was decreased to 0%. This was considered to be related to observations of spermatid retention which affectied all males of the 300 mg/kg/day group and a dose-related increase in incidence and severity of cellular debris in the testis. Furthermore, general parameters of sperm quality including motility, velocity, morphology, count and number of abnormal sperm were negatively affected following the treatment with the test material, which was most severe at 300 mg/kg/day. There were no signs of implantation in female uteruses after treatment with 300 mg/kg/day. In addition, epididymis weight was decreased in P0 males at 300 mg/kg/day.

These changes in histology of the testis and epididymis and the change in sperm morphology and motility were also observed in F1 Cohort 1A males dosed at 100 mg/kg/day, but to a lower degree compared to the P0 males. Effects at 100 mg/kg/day did not affect the fertility rate in the P0 animals.

Females had regular oestrus cycles and there were no effects on mating performance, fertility, duration of gestation and overall litter performance or survival at 30 or 100 mg/kg/day.

In P0 males, there was minimal or mild macrophage vacuolation in the mesenteric lymph node at 100 and 300 mg/kg/day as well as increased pigment in the red pulp in the spleen (sometimes seen grossly as darkening) and increased acinar dilatation in the Harderian gland at 300 mg/kg/day. In the P0 females, there was minimal or mild macrophage vacuolation in the mesenteric lymph node and increased pigment in the red pulp in the spleen (sometimes seen grossly as darkening) at 100 mg/kg/day. The incidence and severity of pigment in the red pulp of the spleen were not increased at 100 mg/kg/day in either sex in the F1 Animals.

Based on these results, the no-observed-adverse-effect level (NOAEL) for reproduction was considered to be 100 mg/kg/day due to the adverse effect on fertility observed at 300 mg/kg/day.

The NOAEL for systemic toxicity was considered to be 100 mg/kg/day due to the lower body weight gain observed at the high dose level.

This study is acceptable and satisfies the guideline requirement for an extended one-generation toxicity study (OECD 443) in rats.

Effect on fertility: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
100 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
reliable without restriction
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

In an extented one generation reproductive toxicity study (including Cohorts 1A and 1B) methylmorpholine-oxide was administered to groups of 25 animals per sex and dose level by gavage at dose levels of 30, 100 or 300 mg/kg/day (P0) or to groups of 20 animals per sex and dose level by gavage at dose levels of 30 or100 mg/kg/day (F1, Cohorts 1A and 1B). P0 males were treated for 10 weeks prior to mating until necropsy after the termination of the P0 females. F0 females were treated for 10 weeks prior to mating, then through mating, gestation and until at least Lactation Day (LD) 21. F1 animals from Cohorts 1A and 1B were then dosed directly from postnatal day (PND) 21 to at least PND 90.

There was a lower body weight gain in the P0 males and in the pre-mating period for the P0 females at 300 mg/kg/day correlating with a lower food consumption. At 100 mg/kg/day there were lower body weight gains in the premating, gestation and lactation phase in the P0 animals. There was also a lower body weight gain at 100 mg/kg/day in the Cohort 1A and 1B animals. The lower body weight gains correlated with lower food consumption.

At 300 mg/kg/day there was a 0% male and female fertility rate. It was considered that the spermatid retention and cellular debris in the testis, and the effects seen on sperm motility, count and morphology were likely to be due to the same effect of the test item and that the epidydimal changes resulted from the testicular changes. These changes in histology of the testis and epididymis and change in sperm morphology and motility were also observed but to a lower degree in the P0 males and Cohort 1A males dosed at 100 mg/kg/day. These changes were considered to be toxicological significant but did not affect the fertility rate in the P0 animals dosed at 100 mg/kg/day.

At 100 mg/kg/day there were lower litter and pups weight in the P0 litters as well as a slightly lower anogenital distance in the F1 female pups.

There was an increase in albumin/globulin levels in the P0 animals as well as a decrease in globulin in the P0 males at 300 mg/kg/day.

At 300 mg/kg/day there were lower levels white blood cells, lymphocytes, monocytes, eosinophils, basophils, large unstained cells, red blood cell count and red blood cell distribution width and increase in the level of reticulocytes, mean corpuscular haemoglobin in the P0 males. At 100 mg/kg/day there were lower levels of white blood cells, lymphocytes, basophils, large unstained cells and red blood cell distribution width in the P0 males when compared with the controls. There was lower levels of white blood cells, lymphocytes and large unstained cells in the Cohort 1A males.

In P0 males there was minimal or mild macrophage vacuolation in the mesenteric lymph node at 100 and 300 mg/kg/day as well as increased pigment in the red pulp in the spleen (sometimes seen grossly as darkening) and increased acinar dilatation in the Harderian gland at 300 mg/kg/day In the P0 females was minimal or mild macrophage vacuolation in the mesenteric lymph, increased pigment in the red pulp in the spleen (sometimes seen grossly as darkening) at 100 mg/kg/day. In the P0 animals, there was minimal or mild spermatid retention.

 

The effects on fertility in an EOGRTS after oral application of methylmorpholine 4-oxide are not fully understood and subject to follow up analyses. Because of the Corona associated lock down, subsequent in vitro examinations in rat and human tissue have been delayed. Results will be submitted as soon as they are ready, in approx. 2-3 months. The follow-up tests should allow a decision on classification according to Regulation (EC) No. 1272/2008 (CLP).

Prior to the EOGRTS, a combined repeated dose toxicity study with the reproduction/developmental toxicity screening test was performed in rats, in which male and female rats are exposed to 0 (vehicle), 10, 100, 1000 mg/kg bw/d via gavage (OECD 422) as 50% solution (corresponding to 5, 50 and 500 mg/kg/d methylmorpholine 4 -oxide, respectively).

The NOAELs in Wistar rats are 50 mg/kg/d for both parental fertility and reproduction and for offspring toxicity.

NMMO did not cause mortality or clinical signs of toxicity during the study in parental animals. However, treatment related effects at the high dose level were observed for body weight, body weight gain and food consumption of male and female rats, where a statistically significant decrease was noted during all treatment periods. However, during the recovery period treated satellite males and females showed an increase in all of these endpoints. These increases are indicative of a reversibility of these effects, because 14 days after treatment, treated satellites had higher body weight, body weight gain and food consumption than the control group animals. No test item related changes were observed in the functional observation battery, hematology and clotting parameters, clinical chemistry parameters and organ weights. No treatment-related findings were observed upon necropsy or during the microscopic examination of tissues from treated and control male or female rats.

Dams at the high dose level showed a decreased number of implantations, while corpora lutea, total number of pups and live pups were statistically significantly decreased in comparison to the control group. Also, on post-partum day 4, these dams had a lower total number of pups and live pups. All of these findings are considered to be test item related with toxicological significance. In pups at the high dose level, treatment related effects were noted on post-partum day 4, where the number of pups found dead was statistically significant lower pup body weight was observed. In addition, at necropsy, these pups showed a statistically higher incidence in cyanosis, body trauma and cannibalized pups. Since these findings occurred at the high dose level, they were considered to be treatment related and indicative of slight embryo fetal toxicity. It is assumed that the effects on the offspring are secondary to maternal toxicity.

Effects on developmental toxicity

Description of key information

In New Zealand White rabbits, the maternal as well as fetal No Observed Adverse Effect Level (NOAEL) were established at 500 mg/kg/day. Dosing was based on a preceeding dose range finding study (DRF).

The maternal No Observed Adverse Effect Level (NOAEL) of developmental toxicity study performed in rats was established at 30 mg/kg/day and the foetal NOAEL was established at 100 mg/kg/day. Developmental effects in rat were considered as a secondary non-specific consequence of maternal toxicity effects.

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2019-04-04 to 2020-01-09
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
adopted 25 June 2018
Deviations:
no
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
- Source and lot/batch No.of test material: Source: Lenzing; Batch No.: 20.06.2018 10:00, T1
- Expiration date of the lot/batch: 2019-06-20
- Purity: 49.2% (dose calculations were corrected for purity)
- Purity test date: 2019-01-03 to 2019-01-04
- Storage: kept at 4 °C

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
Test item dosing formulations were prepared based on a method established at the Test Facility (Formulation Process Document: 998849-17-047) at appropriate concentrations to meet dose level requirements using the control item as vehicle. The dose formulations were prepared at least weekly, stored in a refrigerator set to maintain 4 °C, and dispensed daily. The dose formulation was stirred for at least 30 minutes before dosing and continuously
during dosing.
Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Envigo RMS (UK) Ltd Bicester, Oxon, UK
- Age at study initiation: 4 - 5 months old
- Weight at study initiation: 2.5 - 4.0 kg
- Fasting period before study: None
- Housing: Animals were housed individually in appropriately sized stainless steel cages with a ‘Noryl’ dual level interior and perforated flooring. The housing provided an area for hiding. Beneath each cage was a suspended tray containing absorbent paper.
- Diet: Teklad Certified Global Rabbit Diet (Envigo Diet) was available to the animals 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 vegetables at least twice a week. The hay, fruit and vegetables were not analysed.
- Water: The animals had access to water ad libitum from the public supply.
- Acclimation period: The time-mated animals were allowed to acclimate to the Test Facility rodent toxicology accommodation from arrival until dosing on Day 6 of gestation.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 15-22
- Humidity (%): 38-72
- Air changes (per hr): At least 10
- Photoperiod (hrs dark / hrs light): 12 / 12
Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
VEHICLE:
- Vehicle: Water (Milli-Q Grade) was selected as the vehicle for this study.
- Dose volume: 10 mL/kg bw


Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dose Formulation and Analysis:
Dose formulation samples of the test item were collected for analysis on day 1 (gestation day (GD) 6) and week 4 (GD 27) of dosing. The homogeneity of the dose formulation results obtained from the top, middle and bottom for the test item preparations were averaged and utilised as the concentration results. Samples to be analysed were submitted within the established stability period. All samples to be analysed were transferred at ambient temperature to the analytical laboratory at the Test Facility. The analyses were performed by the Test Facility using a validated UPLC-MS procedure.

Concentration and Homogeneity Analysis:
Duplicate top, middle, and bottom samples (duplicate middle only for control) for each sampling time point were taken for analysis. Triplicate top, middle and bottom samples (triplicate middle only for control) were retained at the Test Facility as backup samples. Sample volumes (0.1 mL, taken by weight) were collected into 10 mL (Group 1), 25 mL (Group 2), 50 mL (Group 4) or 100 mL (Group 3) amber volumetric flasks. Concentration results were considered acceptable if sample concentration results were within ± 10% of theoretical concentration. Each individual sample concentration result was considered acceptable if it was within or equal to ± 15%. For homogeneity, the criteria for acceptability was a relative standard deviation (RSD) of concentrations of ≤ 10% for each group. After acceptance of the analytical results, backup samples were discarded.

Stability Analysis:
Stability analyses performed previously in conjunction with Charles River Study No. 438028 demonstrated that the test item is stable in the vehicle when prepared and stored under the same conditions at concentrations bracketing those used in the present study. Stability data have been retained in the study records for Charles River Study No. 438028.
Details on mating procedure:
- Impregnation procedure: purchased timed pregnant (animals were on GD 1, 2 or 3 upon arrival at the test facility)

Duration of treatment / exposure:
Day 6 to 28 of gestation
Frequency of treatment:
Once a day
Duration of test:
29 days (starting with GD0)
Dose / conc.:
125 mg/kg bw/day (nominal)
Remarks:
Adjusted dose of 254.1 mg/kg bw/day (when corrected for purity)
Dose / conc.:
250 mg/kg bw/day (nominal)
Remarks:
Adjusted dose of 508.3 mg/kg bw/day (when corrected for purity)
Dose / conc.:
500 mg/kg bw/day (nominal)
Remarks:
Adjusted dose of 1016.5 mg/kg bw/day (when corrected for purity)
No. of animals per sex per dose:
24 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose levels for this study were set based on the results of the preliminary developmental toxicity study in the rabbit (Charles River Study No. 499398). In this study pregnant rabbits were dosed at 125, 250 and 500 mg/kg bw/day from GD 6 to 21. In the pregnant animals, the treatment was associated with decreases in food consumption at initiation of dosing. This reduction was transient with recovery in the food consumption levels for all dose groups. At 500 mg/kg bw/day, the decrease in food consumption levels at initiation of dosing was more substantial and the mean food consumption levels remained lower than the control group at termination. This was accompanied by lower body weight gain and clinical signs of reduced faeces. These findings were considered indicative of mild to moderate maternal toxicity and, therefore, 500 mg/kg bw/day was selected as the high dose for the current study. Low and intermediate dose levels of 125 and 250 mg/kg/day, respectively, were selected to further characterise this toxicity.
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. In addition, animals were observed regularly throughout the day on each day of dosing for signs of
reaction to treatment, with particular attention paid to the animals during and for the first hour after dosing. The onset, intensity and duration of any signs were recorded.
- Cage side observations checked: mortality, moribundity and general health

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once during pretreatment, daily during the dosing period (from GD 6 to GD 28) and on Day 29 of gestation.

BODY WEIGHT: Yes
- Time schedule for examinations: Animals were individually weighed daily from GD 5 to GD 29. An in-life body weight was recorded on the day of scheduled necropsy.

FOOD CONSUMPTION: Yes
- Food consumption was measured daily from GD 5 to GD 29. In the event that the food consumption for an animal was below 20 g per day, hay consumption and consumption of fruit and vegetables were monitored to assess the welfare of the animals and the animal was given some time (approximately 30 ± 5 minutes) in an exercise area.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 29
- Necropsy: All animals were subjected to a complete necropsy examination, which included evaluation of the carcass and musculoskeletal system; all external surfaces and orifices; cranial cavity and external surfaces of the brain; and thoracic, abdominal, and pelvic cavities with their associated organs and tissues. The reproductive tract was dissected from the abdominal cavity and weighed intact. The maternal carcass was then discarded. Necropsy examinations were conducted by a trained technician and 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:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: Number of live and dead foetuses; placentae (size, colour or shape) - for this observation, only abnormalities were recorded.
Fetal examinations:
- External examinations: Yes: all per litter; live fetuses were examined for external abnormalities including examination of the oral cavity. Dead fetuses were examined for external abnormalities to the extent possible.
- Soft tissue examinations: Yes: half per litter; for half of the fetuses there was macroscopic examination of the eyes and cranial bones, following removal of the skin from these areas, and the cranium was sectioned once through the coronal suture to allow inspection of the brain in that region. For the remaining fetuses, the head was removed from the spine (as close to the head as possible) and placed in Bouin’s fluid for subsequent serial sectioning and evaluation. The internal structure of the heart and kidneys of all fetuses was examined. The thoracic and abdominal viscera were then discarded and the fetuses were fixed in industrial denatured alcohol (IDA) 99%.
- Skeletal examinations: Yes: all per litter; all of the eviscerated carcasses were macerated in potassium hydroxide, the skeletons stained with Alizarin Red S and then the fetuses cleared with aqueous glycerol solutions. All the preparations were then examined for the presence of skeletal abnormalities and for the extent of ossification.
- Head examinations: Yes: half per liter. The heads fixed in Bouin’s fluid were examined for soft tissue abnormalities using a free hand sectioning technique derived from that of Wilson.
- Other: The body weight of each live fetus was recorded following euthanasia. Fetuses were individually identified within each litter. Prior to fixation, the fetuses were sexed and examined by open dissection for abnormalities of the thoracic and abdominal
viscera.

Statistics:
Parametric/ non-parametric:
Levene’s test was used to assess the homogeneity of group variances. Datasets with at least 3 groups were compared using an overall one-way ANOVA F test if Levene’s test was not significant or the Kruskal-Wallis test if it was. If the overall F test or Kruskal-Wallis test was found to be significant, then the above pairwise comparisons (the designated control group and 1 other group) were conducted using Dunnett’s or Dunn’s test, respectively.

Non-Parametric:
The groups will be compared using an overall Kruskal-Wallis test. If the overall Kruskal-Wallis test is found to be significant, then the above pairwise comparisons will be conducted using Dunn’s test.

Incidence:
A Fisher’s exact test was used to conduct pairwise group comparisons of interest. 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.
Indices:
Number of corpora lutea, pregnancy rate, pre-implantation loss, post-implantation loss, sex ratio (% males), litter % of fetuses with abnormalities.
Historical control data:
Yes
Clinical signs:
no effects observed
Description (incidence and severity):
Please refer to Table 1 and 5 in box "Any other information on results incl. tables".
At 250 and 500 mg/kg bw/day, animals had clinical observations of decreased faecal output. At 250 mg/kg bw/day this was observed in 2 animals during dosing period and at 500 mg/kg bw/day in 12 animals. This clinical sign is indicative of lower food intake and in itself is considered not adverse. All other clinical observations at these dose levels and at 125 mg/kg bw/day were considered not related to the administration of Methylmorpholine-oxide due to low incidence, lack of dose-response or the nature of the observations (e.g. broken toe nail).
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Please refer to Table 3 in box "Any other information on results incl. tables".
At 125 mg/kgbw /day, there were 2 decedents: animal 2504F was found dead on GD 22 and animal 2505F on GD 24 (both after dosing). At necropsy, animal 2504F was found with dark red watery liquid accumulated in the thoracic body cavity and dark red frothy liquid accumulated in the trachea. The lungs had dark red foci and appeared spongy. At discovery, animal 2505F had red liquid around nose and mouth but had no gross pathology findings. Prior to these deaths, both animals had relatively normal food consumption levels and no adverse body weight changes or clinical observations. Based on this, it was considered that these deaths were related to a potential technical gavage error and not related to administration of Methylmorpholine-oxide.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Please refer to Table 2 and 4 in box "Any other information on results incl. tables".
At 500 mg/kg bw/day, there was an initial mean body weight loss of -43.5 g between GD 6-10. In the control group the body weight increased by 25.7 g during the same period. From GD 12, the body weight changes were comparable to the control group. The overall mean body weight gain (GD 6-29) was statistically significantly lower when compared with the control group (-42%, 248 g versus 424 g in the control). As a result, the mean body weight at the end of the study (GD 29) was also significantly lower than the control (-6.3%, 3490 g compared with 3726 g in the control). At 250 mg/kg bw/day, the overall body weight gain throughout GD 6-29 was slightly lower (-14%, 366 g versus 424 g in the control), however this difference was considered minor and not toxicologically relevant. The mean daily body weights and the body weight changes were in general comparable to the control group throughout the study. At 125 mg/kg bw/day, there were no effects on body weight or body weight gains.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Please refer to Table 1 in box "Any other information on results incl. tables".
At 500 mg/kg bw/day, the mean total food consumption during the study (GD 6-29) was -27% lower compared with the control group (2068 g versus 2865 g in the control). Daily mean food consumption levels decreased following the start of dosing period until GD 9 when it was -55% lower than the control (58 g compared with 127 g in the control). However, the lower mean food consumption was transient and increased gradually after GD 9 with the food consumption levels being within normal variation for rabbits towards the end of the study. At 250 mg/kg bw/day, the daily mean food consumption levels were slightly lower than the control. However, they were already slightly lower than the control the day before the start of dosing period and were within normal variation for rabbits. Therefore, this was considered not to be an effect of the test item. At 125 mg/kg bw/day, the food consumption levels were comparable to the control throughout the study.
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):
At all dose levels, there were no gross pathology findings related to administration of Methylmorpholine-oxide.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not specified
Number of abortions:
no effects observed
Description (incidence and severity):
Please refer to Table 3 in box "Any other information on results incl. tables".
There were no abortions observed in animals exposed to the test item at dose levels up to and including 500 mg/kg bw/day.
Pre- and post-implantation loss:
effects observed, non-treatment-related
Description (incidence and severity):
Please refer to Table 3 in box "Any other information on results incl. tables".
At 125, 250 and 500 mg/kg bw/day, the group mean post-implantation loss was higher (11%, 10% and 11%, compared with 3.8% in the control). However, all of these parameters were within the facility’s historical control ranges.
Total litter losses by resorption:
effects observed, non-treatment-related
Description (incidence and severity):
Please refer to Table 3 in box "Any other information on results incl. tables".
At 125, 250 and 500 mg/kg bw/day, the number of females with resorptions was higher than the control (12, 9 and 13, respectively compared with 6 in the control). However, these values were within the facility’s historical control ranges.
Early or late resorptions:
effects observed, non-treatment-related
Description (incidence and severity):
Please refer to Table 3 in box "Any other information on results incl. tables".
The mean number of resorptions (early, late and total) was also higher than the control. However, all of these parameters were within the facility’s historical control ranges.
Dead fetuses:
effects observed, non-treatment-related
Description (incidence and severity):
Please refer to Table 3 in box "Any other information on results incl. tables".
At 500 mg/kg bw/day, one animal (4513F) had a total intrauterine death (late resorptions). Due to a single occurrence in one animal this was considered sporadic and not test-item related. This animal has been excluded from the subsequent litter and fetal data summary tables.
Changes in pregnancy duration:
not examined
Changes in number of pregnant:
not examined
Other effects:
not specified
Details on maternal toxic effects:
Administration of Methylmorpholine-oxide at 500 mg/kg bw/day was associated with lower food consumption (particularly at the start of the dosing period), which resulted in transient body weight loss, lower overall maternal body weight gain, lower maternal terminal body weight, reduced faecal output as well as lower fetal and gravid uterus weights. After correcting for the gravid uterus weight, there were no differences in the final maternal body weights.
In conclusion, administration of Methylmorpholine-oxide by once daily oral gavage on GD 6-28 to New Zealand White rabbits was tolerated in the dams at 500 mg/kg bw/day, resulting in transiently lower food consumption, transient body weight loss, reduced faecal output and lower and gravid uterus weights The maternal No Observed Adverse Effect Level (NOAEL) was established at 500 mg/kg bw/day.
Key result
Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: no adverse signs of toxicity observed
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
Please refer to Table 6 in box "Any other information on results incl. tables".
At 500 mg/kg bw/day, the mean fetal body weight was significantly lower for both sexes compared with the control. The group mean male, female and combined sexes body weights were -16% (38.18 g versus 45.51 g in the control), -16% (37.20 g versus 44.18 g in the control) and -17% (37.48 g versus 45.42 g in the control) lower than the control, respectively. At 125 or 250 mg/kg bw/day, there were no differences in the fetal body weights for either sex and they were comparable to the control.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
No adverse effects observed.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
No test item-related effects were noted for sex ratio.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
No test item-related effects were noted for number of foetuses.
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Description (incidence and severity):
No adverse effects observed.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Please refer to Table 7 in box "Any other information on results incl. tables".
At 500 mg/kg bw/day, there was a higher incidence of unossified forepaw and hindpaw phalanges, unossified metacarpal, malpositioned ilium and full thoracolumbar supernumerary ribs compared with the control group. On the other hand, at 250 and 500 mg/kg/day the number of fetuses and litter with unossified sternebra variation was slightly lower than the control. All other findings were considered to be of insufficient incidence to confirm any Methylmorpholine-oxide related effect, occurred in a similar incidence in the control group or were within biological variation.
Visceral malformations:
no effects observed
Description (incidence and severity):
No adverse effects observed.
Other effects:
not specified
Details on embryotoxic / teratogenic effects:
The increased incidence of variations in the ossification parameters (unossified forepaw and hindpaw phalanges; and unossified metacarpal) were considered to be linked with lower fetal body weights indicating a delay in development, and are considered likely to be completed in time postnatally. As the skeleton undergoes developmental changes at the time fetuses are evaluated in this type of study design, transient delays in development can produce apparent findings of abnormal skeletal structure (DeSesso and Scialli, 2018). However, some of the fetuses with delayed ossification findings also had full thoracolumbar supernumerary ribs or malpositioned ilium, which indicates greater pre-sacral development but does not correlate with delayed development. In addition, the incidence of some delayed ossification parameters (unossified sternebra) was higher in the control group than in the animals treated with Methylmorpholine-oxide. As the fetal findings were contradictory there was no overall pattern of change detected that suggests an effect of treatment. In the fetuses, administration of Methylmorpholine-oxide at 500 mg/kg bw/day resulted in lower fetal weights. The fetal No Observed Adverse Effect Level (NOAEL) was established at 500 mg/kg bw/day.
Key result
Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
not specified
Basis for effect level:
other: no adverse effects observed
Abnormalities:
effects observed, non-treatment-related
Localisation:
other: unossified forepaw and hindpaw phalanges and unossified metacarpal
Description (incidence and severity):
There was no overall pattern of change detected that suggests an effect of treatment.
Key result
Developmental effects observed:
no

Dose Formulation Analyses:

Batch 1 Gestation Day 6 Group 3 mean concentration as well as one individual sample in this group were initially outside of the concentration acceptance criteria.  As part of the investigations, the original samples were re-analysed. Following re-analysis, all Group 2-4 formulation prepared for use on Batch 1 Gestation Day 6 and Batch 1 Gestation Day 27 were found to be within the acceptance criteria for group mean concentration (± 10% of theoretical concentration with all individual concentration results within or equal to ±15% of theoretical concentration) and relative standard deviation (≤ 10%), indicating that the formulations were prepared accurately and homogeneously. No Methylmorpholine-oxide was detected in the control samples.

Table 1: Food Consumption in Maternal Animals

Sex: Female   Days of Gestation 6→29
0 mg/kg/day Group 1 Mean 2865.45
SD 402.78
N 22
   
125 mg/kg/day Group 2 Mean 2998.05
SD 321.06
N 20
%Diff 4.63
250 mg/kg/day Group 3 Mean 2587.11
SD 516.87
N 19
%Diff -9.71
500 mg/kg/day Group 4 Mean 2086.10**
SD 322.50
N 21
%Diff -27.20

Anova & Dunnett: ** = p ≤ 0.01

Table 2: Relevant Clinical Observations

From Day 1 to Day 29 of Gestation 0 mg/kg/day Group 1 125 mg/kg/day Group 2 250 mg/kg/day Group 3 500 mg/kg/day Group 4
Feces, Abnormal Output, Decreased  
Number of Animals Affected 0 0 3 12
Number of Times Recorded 0 0 13 60
% of Affected Animals 0 0 13 50
First to Last seen - - 4 - 26 10 - 29

Table 3: Summary of Maternal performance and mortality and Ovarian and Uterine Examinations and Litter Observations

0 mg/kg/day Group 1 125 mg/kg/day Group 2 250 mg/kg/day Group 3 500 mg/kg/day Group 4
Number of dams 24 24 24 24
Number of pregnant dams [f] 23 23 20 22
Number of non-pregnant dams 1 1 4 2
Number of dams found dead during gestation 0 2 0 0
Number of dams with abortions 0 0 0 0
Number of dams with early deliveries 0 0 0 0
Number of dams with stillbirths [f] N+ve 0 0 0 1 [f] Fisher's Exact: * = p ≤ 0.05
% 0 0 0 4.5
Number of dams with resorptions [f] N+ve 6 12 9 13* [f] Fisher's Exact: * = p ≤ 0.05
% 26.1 52.2 45.0 59.1*
Number of dams with dead fetuses [f] N+ve 0 0 0 1 [f] Fisher's Exact: * = p ≤ 0.05
% 0 0 0 4.5
Number of dams with live fetuses [f] N+ve 23 21 20 21 [f] Fisher's Exact: * = p ≤ 0.05
% 100 100 100 100
Number of Corpora lutea [k] Mean 8.7 8.9 8.6 8.8 [k] - Kruskal-Wallis & Dunn: * = p ≤ 0.05
SD 2.1 2.2 2.2 1.6
N 23 21 20 21
% Diff - 1.9 -1.1 1.3
Implantations [k] Mean 7.6 8.0 7.6 7.7 [k] - Kruskal-Wallis & Dunn: * = p ≤ 0.05
SD 2.7 1.9 2.5 2.0
N 23 21 20 21
% Diff - 5.1 -0.2 2.0
Pre-implantation loss (%) [k1] Mean 14.64 9.4 12.46 12.54 [k1] - Kruskal-Wallis & Dunn
SD 18.6 12.43 17.37 15.97
N 23 21 20 21
% Diff - -35.78 -14.85 -14.32
Post-implantation loss (%) [k1] Mean 3.80 11.29 10.00 10.87 [k1] - Kruskal-Wallis & Dunn
SD 6.79 13.69 12.9 11.76
N 23 21 20 21
% Diff - 196.89 163.02 185.87
Terminal Body weight (g) Mean 3736.0 3784.0 3663.9 3498.8* Anova & Dunnett: * = p ≤ 0.05
SD 362.2 268.5 324.7 299.9
N 23 21 20 21
% Diff - 1.3 -1.9 -6.3
Body weight gain GD 6-29 (g) Mean 424.2 423.5 365.5 247.6** Anova & Dunnett: ** = p ≤ 0.01
SD 130.9 109.4 176.6 126.0
N 23 21 20 21
Gravid uterus weight (g) Mean 476.44 453.28 449.84 386.45
SD 143.63 97.56 131.99 76.74
N 23 21 20 21
% Diff - -4.86 -5.58 -18.89
Corrected body weight (g) Mean 3259.6 3330.7 3214.0 3112.4
SD 344.3 229.8 260.9 280.0*
N 23 21 20 21
% Diff - 2.2 -1.4 -4.5
Body weight change from GD 6-29 corrected for gravid uterine weight (g) Mean -52.24 -29.78 -84.34 -138.85
N 23 21 20 21
Live offspring N+ve 23 21 20 21
% 100 100 100 100
Mean foetal body weight males Mean 45.51 44.03 45.43 38.18** Anova & Dunnett: ** = p ≤ 0.01
SD 5.39 5.11 5.1 6.6
N 23 21 20 21
% Diff - -3.26 -0.17 -16.12
Mean foetal body weight females Mean 44.18 43.63 42.58 37.20** Anova & Dunnett: ** = p ≤ 0.01
SD 4.82 5.36 5.89 6.07
N 22 21 20 20
% Diff - -1.26 -3.64 -15.8
Mean foetal body weight combined Mean 45.42 43.79 44.24 37.48** Anova & Dunnett: ** = p ≤ 0.01
SD 5.04 5.08 5.02 6.12
N 23 21 20 21
% Diff - -3.60 -2.59 -17.47

Table 4: Summary of Fetal Abnormalities by Classification

  0 mg/kg/day Group 1 125 mg/kg/day Group 2 250 mg/kg/day Group 3 500 mg/kg/day Group 4
Exam Type: External        
Number of Fetuses Examined 166 148 135 143
Number of Fetuses Evaluated 166 148 135 143
Number of Litters Examined 23 21 20 21
Number of Litters Evaluated 23 21 20 21
Malformation  
Number of Fetuses 3 2 1 3
Litter % of Fetuses  2.19 1.47 0.42 1.67
Number of Litters 3 2 1 2
All Classifications  
Number of Fetuses 3 2 1 3
Litter % of Fetuses  2.19 1.47 0.42 1,67
Number of Litters 3 2 1 2
 
Exam Type: Fixed Head  
Number of Fetuses Examined 78 69 63 66
Number of Fetuses Evaluated 166 148 135 143
Number of Litters Examined 23 21 20 21
Number of Litters Evaluated 23 21 20 21
Incidental        
Number of Fetuses 0 1 0 1.
Litter % of Fetuses  0.00 1.19 0.00 1.19
Number of Litters 0 1 0 1
Variation        
Number of Fetuses 1 0 2 0
Litter % of Fetuses  1.45 0.00 1.83 0.00
Number of Litters 1 0 2 0
Malformation        
Number of Fetuses 0 1 0 0
Litter % of Fetuses  0.00 1.59 0.00 0.00
Number of Litters 0 1 0 0
All classifications        
Number of Fetuses 1 2 2 1
Litter % of Fetuses  1.45 2.78 1.83 1.19
Number of Litters 1 2 2 1
 
Exam Type: FreshVis  
Number of Fetuses Examined 88 79 72 77
Number of Fetuses Evaluated 166 148 135 143
Number of Litters Examined 23 21 20 21
Number of Litters Evaluated 23 21 20 21
Variation        
Number of Fetuses 1 1 2 0
Litter % of Fetuses  1.09 0.95 2.50 0.00
Number of Litters 1 1 2 0
Malformation        
Number of Fetuses 1 1 0 1
Litter % of Fetuses  1.09 0.95 0.00 1.59
Number of Litters 1 1 0 1
All classifications        
Number of Fetuses 1 2 1 1
Litter % of Fetuses  1.09 1.90 2.50 1.59
Number of Litters 1 1 1 1
 
Exam Type: FreshVisBody  
Number of Fetuses Examined 78 69 63 66
Number of Fetuses Evaluated 166 148 135 143
Number of Litters Examined 23 21 20 21
Number of Litters Evaluated 23 21 20 21
Variation        
Number of Fetuses 0 1 0 0
Litter % of Fetuses  0.00 1.19 0.00 0.00
Number of Litters 0 1 0 0
Malformation        
Number of Fetuses 1 0 0 0
Litter % of Fetuses  1.45 0.00 0.00 0.00
Number of Litters 1 0 0 0
All classifications        
Number of Fetuses 1 1 0 0
Litter % of Fetuses  1.45 1.19 0.00 0.00
Number of Litters 1 1 0 0
 
Exam Type: Skeletal  
Number of Fetuses Examined 80 73 66 69
Number of Fetuses Evaluated 166 148 135 143
Number of Litters Examined 21 19 18 19
Number of Litters Evaluated 23 21 20 21
Variation        
Number of Fetuses 71 57 54 58
Litter % of Fetuses  89.92 78.60 81.94 85.70
Number of Litters 21 19 17 19
Malformation        
Number of Fetuses 3 1 1 2
Litter % of Fetuses  2.94 1.05 2.78 3.07
Number of Litters 3 1 1 2
All classifications        
Number of Fetuses 71 57 54 58
Litter % of Fetuses  89.92 78.60 81.94 85.70
Number of Litters 21 19 17 19
 
Exam Type: SkeletalBody  
Number of Fetuses Examined 71 64 58 58
Number of Fetuses Evaluated 166 148 135 143
Number of Litters Examined 21 19 18 19
Number of Litters Evaluated 23 21 20 21
Variation        
Number of Fetuses 60 52 48 48
Litter % of Fetuses  83.57 84.04 85.65 83.80
Number of Litters 20 19 18 18
Malformation        
Number of Fetuses 0 0 1 1
Litter % of Fetuses  0.00 0.00 1.85 1.39
Number of Litters 0 0 1 1
All classifications        
Number of Fetuses 60 52 48 48
Litter % of Fetuses  83.57 84.04 85.65 83.80
Number of Litters 20 19 18 18
 
Mean Fetal Skeletal Ossification Sites         
Ribs, paired        
Mean 12.32 12.30 12.37 12.51
SD 0.31 0.26 0.27 0.24
N 23 21 20 21
%Diff - -0.18 0.41 1.54

Kruskal-Wallis & Dunn

Conclusions:
In this study performed in accordance with OECD Guideline 414, administration of Methylmorpholine-oxide (49.2% purity) by daily oral gavage during GD 6-28 to female New Zealand White rabbits was tolerated in the dams, resulting in transiently lower food consumption, transient body weight loss, reduced faecal output and lower and gravid uterus weights in the high dose of 500 mg/kg bw/day. In the fetuses, administration of Methylmorpholine-oxide at 500 mg/kg bw/day resulted in lower fetal weights. Based on the results, the maternal as well as fetal No Observed Adverse Effect Level (NOAEL) were established to be 500 mg/kg bw/day.
Executive summary:

In a developmental toxicity study conducted according to OECD guideline 414, Methylmorpholine-oxide (49.2% purity) was administered to groups of 24 female New Zealand White rabbits/dose in water at dose levels of 0, 125, 250 or 500 mg/kg bw/day from days 6 through day 28 of gestation.

No maternal mortality occurred. At 500 mg/kg bw/day, the mean food consumption was up to -55% lower than the concurrent control group decreasing until GD 9 but gradually increased thereafter. Consequently, half of the animals in this group had reduced faecal output. There was an initial body weight loss of -43.5 g between GD 6-10, while in the control group the body weight increased by 25.7 g during the same period. From GD 12, the body weight changes were comparable to the control group but the overall body weight gain (GD 6-29) remained lower (248 g versus 424 g in the control).

At 250 mg/kg bw/day, there were 2 animals with reduced faecal output but there were no significant body weight or food consumption effects. At 125 mg/kg bw/day, there were no test-item related clinical observations, food consumption or body weight effects.

At necropsy, there were no test-item related maternal gross pathology findings in any of the groups.

At 500 mg/kg bw/day, the mean gravid uterus and combined fetal weights were -19% and -17% lower than the control, respectively (fetal weight for both sexes separately were also lower). Fetal examinations indicated a higher incidence of unossified forepaw and hindpaw phalanges, unossified metacarpal, malpositioned ilium and full thoracolumbar supernumerary ribs at 500 mg/kg bw/day. At 250 and 500 mg/kg bw/day, the incidence of unossified sternebra was lower than in the control. There were no differences in the maternal performance parameters, ovarian and uterine examinations or the number of paired ribs in any of the groups.

In conclusion, administration of Methylmorpholine-oxide by once daily oral gavage through gestation days 6-28 to female New Zealand White rabbits was tolerated in the dams at 500 mg/kg bw/day, resulting in transiently lower food consumption, transient body weight loss, reduced faecal output and lower and gravid uterus weights. In the fetuses, administration of Methylmorpholine-oxide at 500 mg/kg bw/day resulted in lower fetal weights. Based on the results, the maternal as well as fetal No Observed Adverse Effect Level (NOAEL) were established at 500 mg/kg bw/day.

 

The developmental toxicity study in the rabbit is classified acceptable and satisfies the guideline requirement for a developmental toxicity study (OPPTS 870.3700; OECD 414) in rabbits.

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
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
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.
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
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
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.
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
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.
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
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
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.
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
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.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2017-08-23 to 2018-09-27
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Source: Lenzing; Batch No.: 14.03.2017 14.30, PA1
- Expiration date of the lot/batch: 2017-10-14
- Purity test date: 2017-03-14 to 2017-03-15

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 under test conditions: The dosing formulations were prepared weekly, stored in a refrigerator set to maintain 4 °C, and dispensed daily.
- Solubility and stability of the test substance in the solvent/vehicle: Stability analyses of the test item performed previously by the laboratory via ultra high-performance liquid chromatography demonstrated that the test item is stable in the vehicle.
Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River UK Limited, Margate, Kent, UK
- Age at study initiation: 9-11 weeks old
- Weight at study initiation: 209-342 g
- Fasting period before study: None
- Housing: Females were housed singly in appropriately sized suspended polycarbonate cages with stainless steel grid tops and solid bottoms. Bedding material was sterilised white wood shavings.
- Diet: SDS VRF-1 breeder diet was provided ad libitum throughout the study. However, from the 07 Sep 2017 (i.e. Day 6-9 of gestation), the animals exposed to the highest dose of the test item were given wetted soft pellets as a supplement to encourage the animals to eat due to low food consumption and weight loss.
- Water: The animals had access to water ad libitum from the public supply from water bottles which were changed as necessary throughout the course of the study.
- Acclimation period: The time-mated animals were allowed to acclimate to the Test Facility rodent toxicology accommodation from arrival until dosing on Day 6 of gestation.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-21 °C
- Humidity (%): 40-76%
- Air changes (per hr): At least 10
- Photoperiod (hrs dark / hrs light): 12 hrs dark / 12 hrs light
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.

VEHICLE: Water (Milli-Q Grade) was selected as the vehicle for this study.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dose formulation samples of the test item were collected for analysis on days 1 (gestation day 6) and 14 (gestation day 19) of dosing. The homogeneity of the dose formulation results obtained from the top, middle and bottom for the test item preparations were averaged and utilised as the concentration results. Samples to be analysed were submitted within the established stability period. All samples to be analysed were transferred at ambient temperature to the analytical laboratory at the Test Facility.

The analyses were performed by the Test Facility using a validated UPLC-MS procedure. In particular, duplicate top, middle and bottom (duplicate middle only for control) samples (0.1 mL) for each sampling time point were sent to the analytical laboratory. Triplicate top, middle, and bottom samples (triplicate middle only for control) were retained at the Test Facility as backup samples.

Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 10% of theoretical concentration. Each individual sample concentration result was considered acceptable if it was within or equal to ± 15%. After acceptance of the analytical results, backup samples were discarded.

Homogeneity results were considered acceptable if the relative standard deviation (RSD) of concentration of ≤ 10%. After acceptance of the analytical results, backup samples were discarded.
Details on mating procedure:
- Impregnation procedure: purchased timed pregnant (time-mated)
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of gestation
Duration of treatment / exposure:
Day 6 to 19 of gestation
Frequency of treatment:
Once a day
Duration of test:
Day 0-3 of gestation (arrival of animals) to Day 20 of gestation (scheduled euthanasia)
Dose / conc.:
30 mg/kg bw/day (nominal)
Remarks:
Adjusted dose of 59.6 mg/kg bw/day (when corrected for purity)
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
Adjusted dose of 199 mg/kg bw/day (when corrected for purity)
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
Adjusted dose of 596 mg/kg bw/day (when corrected for purity)
No. of animals per sex per dose:
24 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were selected with the Sponsor after a review of all relevant toxicological data including a combined repeated dose toxicity study with the reproduction/developmental toxicity screening test in rats and a 90-day study in rats. In the combined screening study, 500 mg NMMO/kg/day was associated with effects on female body weights and food consumption along with significant test item-related effects on reproductive parameters in females. These effects included decreases in implantations, corpora lutea, total number of pups born and pups born live, and a decrease in pup survival, reduction in pup body weights and a higher incidence of cyanosis, body trauma and cannibalised pups. The no-observed-adverse-effect-level for this study was considered to be 50 mg/kg/day. In the 90-day study, the high dose level of 300 mg/kg/day was associated with a 33% reduction in body weight gain in females within the first 14 days of dosing.

For this study a high dose level of 300 mg/kg/day was considered suitable to induce moderate but not excessive effects of toxicity in the pregnant rat. Low and intermediate dose levels of 30 and 100 mg/kg/day, respectively, were selected to further characterise this toxicity.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily
- Cage side observations checked: mortality, moribundity and general health

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once during pretreatment (Day 3 of gestation), daily during the dosing period (from Day 6 to Day 19 of gestation) and on Day 20 of gestation.

BODY WEIGHT: Yes
- Time schedule for examinations: All animals were individually weighed once during pretreatment (Day 3 of gestation), daily during the dosing period (from Day 6 to Day 19 of gestation) and on Day 20 of gestation.

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

WATER CONSUMPTION: Yes
- Time schedule for examinations: Water consumption was monitored by visual inspection of the water bottles throughout the study.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: Evaluation of the carcass and musculoskeletal system; all external surfaces and orifices; thoracic, abdominal, and pelvic cavities with their associated organs and tissues. The reproductive tract was dissected out and weighed intact.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: Number of live and dead foetuses; placentae (size, colour or shape) - for this observation, only abnormalities were recorded.
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: all per litter
Statistics:
Levene’s test was used to assess the homogeneity of group variances. Datasets with at least 3 groups were compared using an overall one-way ANOVA F test if Levene’s test was not significant or the Kruskal-Wallis test if it was. If the overall F test or Kruskal-Wallis test was found to be significant, then the above pairwise comparisons were conducted using Dunnett’s or Dunn’s test, respectively. Datasets with 2 groups (the designated control group and 1 other group) were compared using a t-test if Levene’s test was not significant or Wilcoxon Rank-Sum test if it was. All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 0.1%, 1%, and 5% levels.
Historical control data:
Yes
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
See Table 1 in "Any other information on results incl. tables".

Administration at 300 mg/kg/day was associated with several clinical observations including pale faeces (Day 10-20 of gestation), decreased activity (Day 9-13 of gestation), erected fur (Day 9-20 of gestation), ploughing (Day 15-16 of gestation) and fur staining (Day 3-20 of gestation). Any other clinical observations at this dose level during the study were considered not to be related to the administration of Methylmorpholine-oxide.

Any clinical observations at levels up to and including 100 mg/kg/day were considered not to be related to the administration of Methylmorpholine-oxide due to lack of a dose response relationship or to isolated incidences.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
There were no unscheduled deaths during this study.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
See Table 2 in "Any other information on results incl. tables".

Body weight loss was evident in all the animals in the first few days of dosing at 300 mg/kg/day. A slight recovery in body weight was evident from ca Day 15 of gestation in all animals at 300 mg/kg/day; however, mean body weight and body weight gain remained lower from Day 9 until Day 20 and was statistically significant when compared with the control. Overall, the mean body weight gain from Day 6 until Day 20 of gestation was significantly lower than the control (53%; 127.8 g for the controls vs. 59.5 g for the 300 mg/kg/day dose). At Day 20 of gestation, there was a statistically significant decrease in mean body weight observed with the 300 mg/kg/day dose compared with the control (-16%; 402.0 g for the control vs. 337.5 g for the 300 mg/kg/day dose).

At 100 mg/kg/day the mean body weight gain from Day 6 until Day 20 of gestation was significantly lower than the controls (-16%; 127.8 g for the controls vs. 107.5 g for the 100 mg/kg/day dose). At Day 20 of gestation, there was a slight decrease in mean body weight observed with the 100 mg/kg/day dose compared with the control (-4%; 402.0 g for the controls vs. 386.3 g for the 100 mg/kg/day dose) but this decrease was not statistically significant.

At 30 mg/kg/day the mean body weight or body weight gains were comparable with the controls throughout the study.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Administration at 300 mg/kg/day was associated with a statistically significant lower daily food consumption of up to -47% (observed on Day 12; 29.1 g for the controls vs. 15.5 g for the 300 mg/kg/day dose) from Day 7-20 of gestation when compared with the control values.

Lower food consumption was also observed at 100 mg/kg/day, which the daily food consumption was statistically significantly lower between Day 11-14 of gestation (-10%; total food consumption during dosing from Day 11 to 14 of gestation: 114.3 g for the controls vs. 103.3 g for the 100 mg/kg/day dose) and on Day 20 of gestation ( 15%; 26.8 g for the controls vs. 22.9 g for the 100 mg/kg/day dose) when compared to the controls. There was a slightly lower food consumption throughout dosing (-7; total food consumption during dosing from Day 6 to 19 of gestation: 402.9 g for the controls vs. 373.9 g for the 100 mg/kg/day dose).

At 30 mg/kg/day food consumption values were comparable to the control for the duration of the study.
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 gross pathology findings related to administration of Methylmorpholine-oxide at dose levels up to and including 300 mg/kg/day. Any findings were considered to be a natural variation.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not specified
Number of abortions:
no effects observed
Description (incidence and severity):
There were no abortions observed in animals exposed to the test item at dose levels up to and including 300 mg/kg bw/day.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
There were no notable effects on implantation sites after dosing with Methylmorpholine-oxide when compared with the controls.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
There were no notable effects on number of litters after dosing with Methylmorpholine-oxide when compared with the controls.
Early or late resorptions:
no effects observed
Description (incidence and severity):
There were no notable effects on early and late resorptions after dosing with Methylmorpholine-oxide when compared with the controls.
Dead fetuses:
no effects observed
Description (incidence and severity):
There were no notable effects on number of dead foetuses after dosing with Methylmorpholine-oxide when compared with the controls.
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
At dose levels up to and including 300 mg/kg/day, the number of pregnant females on Day 20 of gestation and the type and distribution of pregnancy types were similar to the controls.
Changes in number of pregnant:
not examined
Description (incidence and severity):
Time-mated females were acquired before dosing of test item, so test item did not affect the number of pregnant females.
Other effects:
not specified
Details on maternal toxic effects:
Administration of Methylmorpholine-oxide at 100 or 300 mg/kg/day was associated with a dose-related lower food consumption and a correlating body weight loss/lower weight gain in the dams. There were also several clinical observations such as decreased activity, pale faeces, fur staining and erect fur in the 300 mg/kg/day-exposed dams.

The lower food consumption may have possibly been related to food avoidance caused by chemical irritant after oral dosing causing digestive discomfort and could be related to the reduced body weight gain observed at these dose level; however there were no finding at necropsy to support this.
Key result
Dose descriptor:
NOAEL
Effect level:
30 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
clinical signs
body weight and weight gain
food consumption and compound intake
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
See Table 4 in "Any other information on results incl. tables".

At 300 mg/kg/day both sexes of foetuses had a statistically significant lower mean foetal body weight than the control (male; -15%, 3.85 g for the controls vs. 3.29 g for 300 mg/kg/day dose; females; -17%, 3.70 g for the controls vs. 3.06 g for the 300 mg/kg/day dose). For both the males and females 99% of the foetuses had foetal weights below the mean of the control, and 21% of the male foetuses and 7% of the female foetuses had foetal weights lower than the lowest control foetal weight (3.03 g for males and 2.56 g for females) recorded in this study.

At 100 mg/kg/day the female foetuses had a statistically significant lower mean body weight than the control (-5%, 3.70 g for the controls vs. 3.51 g for the 100 mg/kg/day dose); however, all of the female foetal weights were within the range of the control foetal weights. The mean foetal weight for male foetuses was not statistically different than that of the control.

At 30 mg/kg/day the mean foetal weights for both sexes were comparable to that of the control.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
There were no notable effects on number of live foetuses after dosing with Methylmorpholine-oxide when compared with the controls.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
There were no notable effects on sex ratio after dosing with Methylmorpholine-oxide when compared with the controls.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
There were no notable effects on litter size after dosing with Methylmorpholine-oxide when compared with the controls.
Changes in postnatal survival:
no effects observed
Description (incidence and severity):
There were no notable effects on postnatal survival after dosing with Methylmorpholine-oxide when compared with the controls.
External malformations:
no effects observed
Description (incidence and severity):
There were no observed external malformations of foetuses that are related to Methylmorpholine-oxide.
Skeletal malformations:
effects observed, treatment-related
Description (incidence and severity):
See Table 5 in "Any other information on results incl. tables".

At 300 mg/kg/day there were incidences of bent and short long bones including the radius, ulna, scapula and humerus that were classified as malformations as well as misaligned costal cartilage classified as a non-adverse variation.

At 300 mg/kg/day there was a lower degree of ossification classified as minimal to severe in the skull bones (frontal, interparietal, nasal, parietal, presphenoid, squamosal, supraoccipital and zygomatic arch), the vertebrate (cervical, thoracic, sacral and caudal), pelvic girdle (pubis), metacarpal, ribs and sternebra. Due to the high proportion of lower ossification with some observations being classified as severe these findings were considered to be adverse.

At 100 and 300 mg/kg/day there were increased incidences of short wavy ribs classified as non-adverse variations as they appear likely to be a secondary response to maternal toxicity. Formation of short wavy ribs are classified as non-adverse as they are transient and reversible postnatally.

At 100 mg/kg/day there was a lower degree of ossification classified as minimal to moderate in the skull bones (interparietal, nasal, parietal, squamosal and supraoccipital), the vertebrate (cervical and sacral), pelvic girdle (pubis), metacarpal, ribs and sternebra. All findings were classified as non-adverse variations due to the reduction in the severity of the findings. These findings are likely to be related to the smaller foetal weights and it is considered likely that the development would be completed with time.

At 30 mg/kg/day there was a lower degree complete ossification classified as minimal to moderate in the interparietal and squamosal as well as unossified metacarpal and sternebra. All findings were classified as non-adverse variations due to the reduced severity of the findings and lower number of bones affected.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
See Table 5 in "Any other information on results incl. tables".

At 100 and 300 mg/kg/day there was a higher incidence of misshapen eye lens (oval in shape) when compared with the control. Within historical control data the incidence of misshapen eye lens (oval in shape) ranged from 0(0) to 3(3) (number of foetuses with abnormality (number of litters with abnormality)). As the observation at 100 mg/kg/day was within this range it was considered to be within the natural variation of the species.

At 300 mg/kg/day the incidence misshapen eye lens (oval in shape) was slightly outside the historical control range at 5(4). At 300 mg/kg/day misshapen eye lens (oval in shape) was noted in male foetuses that had a body weights that were lower by 13 to 21% than the control mean weight (body weight for 300 mg/kg/day of 3.05 to 3.33 g vs body weight for controls of 3.03 to 4.66 g) and female foetuses that had a body weight that were lower by 14 to 26% than the control mean weight (body weight for 300 mg/kg/day of 2.74 to 3.17 g vs body weight for controls of 2.56 to 4.59 g). The historical control data shows that the incidence misshapen eye lens (oval in shape) on Day 21 of gestation ranges from 0(0) to 1(1). In the historical control data the lower incidence misshapen eye lens (oval in shape) on Day 21 of gestation at 1(1) compared to Day 20 of gestation at 3(3) is considered most likely due to an increase in the size of the foetuses on Day 21 of gestation. As these abnormalities are generally noted in foetuses with lower body weights it is considered to be most likely related to the size of the foetuses and not related to test item.
Other effects:
not specified
Details on embryotoxic / teratogenic effects:
There were lower body weights in the foetuses at 100 or 300 mg/kg/day compared to the controls. At 300 mg/kg/day these lower foetal weights were associated with a lower gravid uterus weight. At 100 mg/kg/day the female foetuses had a lower mean body weight than the control; however, all of the female foetal weights were within the range of the control foetal weights. The mean foetal weight for male foetuses at 100 mg/kg/day was not statistically different than that of control.

At 300 mg/kg/day there were incidences of bent and short long bones including the radius, ulna, scapula and humerus that classified as malformations as well as misaligned costal cartilage classified as a non-adverse variation.

At 100 and 300 mg/kg/day there were incidences of short wavy rib classified as non-adverse variations. The incidence of wavy ribs and misshapen scapula appear likely to be a secondary response to maternal toxicity. The classification of wavy ribs and changes in scapula has been reported as non-adverse findings as they are transient and reversible postnatally. At 100 mg/kg/day findings were classified as non-adverse variations likely to be related to a delay in the development and are considered likely to be completed in time. At 300 mg/kg/day due to the high proportion of lower ossification with some being classified as severe these findings are considered to be adverse.
The abnormality of misshapen eye lens (oval in shape) at 300 mg/kg/day was generally noted in foetuses with lower body weights and therefore is considered to be most likely related to the size of the foetuses and not related to treatment. At 100 mg/kg/day the abnormality of misshapen eye lens (oval in shape) was within this range of the historical control data and therefore it is considered to be within the natural variation of the species.
Key result
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes
skeletal malformations
Key result
Abnormalities:
effects observed, treatment-related
Localisation:
skeletal: skull
skeletal: rib
skeletal: vertebra
skeletal: pelvic girdle
Description (incidence and severity):
At 300 mg/kg/day there was a lower degree of ossification classified as minimal to severe in the skull bones (frontal, interparietal, nasal, parietal, presphenoid, squamosal, supraoccipital and zygomatic arch), the vertebrate (cervical, thoracic, sacral and caudal), pelvic girdle (pubis), metacarpal, ribs and sternebra. Due to the high proportion of lower ossification with some observations being classified as severe these findings were considered to be adverse.
Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
300 mg/kg bw/day (nominal)
Treatment related:
yes
Relation to maternal toxicity:
developmental effects as a secondary non-specific consequence of maternal toxicity effects
Dose response relationship:
yes
Relevant for humans:
yes

Table 1: Summary of Relevant Maternal Clinical Observations

Group

1

2

3

4

Dose Level mg/kg/day

0

30

100

300

Activity decreased

0

0

0

5/6

Faeces, Pale

0

0

1/1

22/185

Fur, Erected

0

0

0

22/118

Ploughing

0

0

0

2/3

Fur Staining

5/61

4/46

7/53

13/95

Total Number of animals

24

24

24

24

Number of animals showing observation / Number of observations

Table 2: Summary of Maternal Food Consumption (g/animal/day) During Gestation

Gestation Day

Group/Sex

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

1F

Mean

19.7

22.0

21.2

20.5

22.9

24.6

26.5

28.0

29.1

30.0

27.2

27.8

28.6

30.0

29.8

29.9

26.8

SD

4.7

3.5

3.4

2.5

3.5

3.0

3.0

2.4

3.3

3.2

3.9

3.2

3.1

3.9

4.0

4.0

4.0

N

23

23

23

23

23

23

23

23

23

23

23

23

23

23

23

23

23

2F

Mean

18.7

22.7

21.2

19.9

22.1

23.2

24.5

26.6

27.9

27.6

27.7

27.0

28.1

29.0

30.0

27.0

24.9

SD

6.1

2.9

4.4

2.4

3.1

4.3

4.1

2.9

3.5

2.7

2.7

2.7

3.5

2.9

3.3

6.6

4.4

N

24

23

24

24

24

24

24

24

24

24

24

24

24

24

24

24

23

3F

Mean

20.6

22.5

20.4

20.3

20.9

23.2

24.3

25.4d

26.3d

27.0a

24.6d

25.9

27.7

27.9

29.6

27.5

22.9d

SD

5.6

3.8

2.0

2.8

3.6

4.5

2.8

2.6

4.8

2.6

3.2

2.4

2.5

3.5

3.4

3.9

6.3

N

23

23

23

23

23

23

23

23

23

23

23

23

23

23

22

23

23

4F

Mean

19.3

21.8

21.1

12.1f

12.2f

13.6c

15.8f

17.0f

15.5f

18.3c

16.8f

17.6f

21.3f

22.2f

24.1f

20.9f

18.3f

SD

6.2

2.3

3.1

4.4

5.7

6.5

4.9

4.2

3.7

4.2

4.6

3.4

3.8

4.0

3.4

3.4

5.2

N

24

24

24

24

24

24

24

24

24

24

24

24

24

24

24

24

24

Significantly different from control group 1 value: a=p≤0.05, b=p≤0.01, c=p≤0.001 (Dunn); d=p≤0.05, e=p≤0.01, f=p≤0.001 (Dunnett)

SD: standard deviation; N: animals examined; Group 1: Control; Group 2: 30 mg/kg bw/day; Group 3: 100 mg/kg bw/day; Group 4: 300 mg/kg bw/day

Table 3: Summary of Foetal Body Weights

 

Male Foetuses

Female Foetuses

Group

1

2

3

4

1

2

3

4

Dose Level (mg/kg/day)

0

30

100

300

0

30

100

300

Mean Foetal Weight (g)

3.85

3.82

3.71

3.29c

3.70

3.63

3.51a

3.06c

Lowest Weight (g)

3.03

3.08

2.80

2.33

2.56

3.00

2.68

2.06

Highest Weight (g)

4.66

4.62

4.60

3.95

4.59

4.58

4.17

3.90

Number of foetuses below mean control weight (%)

-

98 (51%)

122 (71%)

170 (99%)

-

97 (62%)

121 (78%)

163 (99%)

Number of foetuses below lowest control weight (%)

-

0(0)

5 (3%)

36 (21%)

-

0 (0%)

0 (0%)

11 (7%)

Total number of foetuses

157

193

172

172

167

156

155

165

Significantly different from control group 1 value: a=p≤0.05, b=p≤0.01, c=p≤0.001 (ANOVA)

Table 4: Summary of Maternal performance and mortality and Ovarian and Uterine Examinations and Litter Observations

0 mg/kg/day Group 1 30 mg/kg/day Group 2 100 mg/kg/day Group 3 300 mg/kg/day Group 4
Number of dams 24 24 24 24
Number of pregnant dams  N 23 24 23 24
% 95.8 100 95.8 100
Number of non-pregnant dams N 1 0 1 0
Number of dams found dead during gestation N 0 0 0 0
Number of dams with abortions N 0 0 0 0
Number of dams with early deliveries N 0 0 0 0
Number of dams with stillbirths  N+ve 0 0 0 0
% 0 0 0 0
Number of resorptions  Mean 0.2 0.7 0.9 0.4
SD 0.4 1.4 1.7 1.1
N 23 24 23 24
Number of dams with dead fetuses  N+ve 0 0 0 0
Number of dams with live fetuses  N+ve 23 24 23 24
% 100 100 100 100
Number of Corpora lutea  Mean 14.4 15.3 15.3 14.8
SD 2.5 2.4 1.3 2.4
N 23 24 23 24
Implantations  Mean 14.3 15.2 15.1 14.5
SD 2.7 2.4 1.3 2.3
N 23 24 23 24
Pre-implantation loss (%)  Mean 1.1 0.8 1.1 2.1
SD 3.1 2.1 2.5 4.0
N 23 24 23 24
Post-implantation loss (%)  Mean 1.1 4.3 6.3 2.9
SD 2.4 9.5 13.8 7.4
N 23 24 23 24
Terminal Body weight (g) Mean 402.0 389.0 386.3 337.5*** ***p≤0.001 (Dunnett)
SD 38.1 34.0 20.3 40.2
N 23 24 23 24
Body weight gain GD 6-20 (g) Mean 127.8 120.3 107.5*** 59.5*** ***p≤0.001 (Dunnett)
SD 16.1 17.5 11.0 16.1
N 23 24 23 24
Gravid uterus weight (g) Mean 85.99 84.98 81.76 72.74
SD 13.73 13.06 13.24 14.26
N 23 24 23 24
Corrected body weight (g) Mean 316.01 304.02 304.54 264.76
N 23 24 23 24
% Diff - -3.8 -3.6 -16.2
Body weight change from GD 6-29 corrected for gravid uterine weight (g) Mean 41.81 35.32 25.74 -13.24
N 23 24 23 24
Live offspring Mean 14.1 14.5 14.2 14.0
SD 2.6 2.6 2.5 2.6
N 23 24 23 24
Mean foetal body weight males Mean 3.85 3.816 3.71 3.288*** ***p≤0.001 (Dunnett)
SD 0.248 0.222 0.313 0.247
N 23 24 23 24
Mean foetal body weight females Mean 3.699 3.633 3.508* 3.062*** *p≤0.05 (Dunnett); ***p≤0.001 (Dunnett) 
SD 0.324 0.221 0.264 0.25
N 23 24 23 24
Mean foetal body weight combined Mean 3.771 3.734 3.61 3.176*** ***p≤0.001 (Dunnett)
SD 0.269 0.222 0.281 0.241
N 23 24 23 24

Table 5:Summary of General Foetal Findings

0 mg/kg/day Group 1 30 mg/kg/day Group 2 100 mg/kg/day Group 3 300 mg/kg/day Group 4
Exam Type: External
Number of fetuses examined 324 349 327 337
Numbers of fetuses evaluated 324 349 327 337
Numbers of litters examined 23 24 23 24
Numbers of litters evaluated 23 24 23 24
Incidental Number of fetuses 0 1 1 1
% of fetuses 0 0.3 0.3 0.3
Number of litters 0 1 1 1
Totals Number of fetuses 0 1 1 1
% of fetuses 0 0.3 0.3 0.3
Number of litters 0 1 1 1
Exam Type: Wilsons (Soft tissue)
Number of fetuses examined 156 168 156 164
Numbers of fetuses evaluated 324 349 327 337
Numbers of litters examined 23 24 23 24
Numbers of litters evaluated 23 24 23 24
Variation Number of fetuses 19 22 12 15
% of fetuses 12.2 13.1 7.7 9.1
Number of litters 10 15 10 9
Incidental Number of fetuses 5 7 1 8
% of fetuses 3.2 4.2 0.6 4.9
Number of litters 5 6 1 8
Malformation Number of fetuses 1 3 3 9
% of fetuses 0.6 1.8 1.9 5.5
Number of litters 1 3 3 6
Totals Number of fetuses 23 29 16 32
% of fetuses 14.7 17.3 10.3 19.5
Number of litters 11 18 12 16
Exam Type: Skeletal
Number of fetuses examined 168 181 171 173
Numbers of fetuses evaluated 324 349 327 337
Numbers of litters examined 23 24 23 24
Numbers of litters evaluated 23 24 23 24
Variation Number of fetuses 163 175 156 172
% of fetuses 97 96.7 91.2 99.4
Number of litters 23 24 23 24
Incidental Number of fetuses 3 0 0 8
% of fetuses 1.8 0 0 4.6
Number of litters 3 0 0 8
Malformation Number of fetuses 0 0 2 4
% of fetuses 0 0 1.2 2.3
Number of litters 0 0 1 3
Totals Number of fetuses 163 175 156 172
% of fetuses 97 96.7 91.2 99.4
Number of litters 23 24 23 24

Table 6: Summary of Relevant Foetal Abnormalities

Group

1

2

3

4

Dose Level mg/kg/day

0

30

100

300

Visceral:

 

 

 

 

Eye lens, misshapen

0/0

0/0

2/2

5/4a

Skeletal:

 

 

 

 

Skull

 

 

 

 

Frontal, incomplete ossification, minimal

0/0

0/0

3/1

4/4a

Interparietal, incomplete ossification, minimal

24/13

32/17a

41/15a

45/18a

Interparietal, incomplete ossification, moderate

8/5

11/7

8/7

15/8a

Interparietal, incomplete ossification, severe

0/0

0/0

0/0

1/1a

Nasal, incomplete ossification, minimal

0/0

0/0

4/1a

3/2a

Parietal, incomplete ossification

6/5

14/10

31/10a

21/11a

Presphenoid, incomplete ossification

1/1

3/3

5/4

7/6a

Presphenoid, unossified

0/0

1/1

0/0

9/5a

Squamosal, incomplete ossification, moderate

3/2

4/3a

7/3a

11/6a

Supraoccipital, incomplete ossification, minimal

13/8

18/10

31/14a

45/19a

Supraoccipital, incomplete ossification, moderate

2/2

3/3

3/3

17/10a

Supraoccipital, incomplete ossification, severe

0/0

0/0

0/0

1/1a

Zygomatic arch, incomplete ossification

2/2

6/5

6/5

14/7a

Vertebrae

 

 

 

 

Atlas, ventral arch, increased ossification

30/15

22/10

33/20

7/5

Cervical arch, incomplete ossification

2/2

3/2

5/3a

9/7a

Cervical arch, increased ossification

22/11

25/12

19/11

7/6

Cervical centrum, increased ossification

16/5

14/5

4/2

1/1

Thoracic arch, incomplete ossification

0/0

0/0

0/0

1/1a

Thoracic centrum, incomplete ossification. moderate

7/5

15/8

2/2

11/10

Sacral arch, incomplete ossification

12/8

13/8

32/12a

40/16a

Sacral arch, increased ossification

19/11

17/8

2/2

1/1

Caudal arch, increased ossification

20/8

16/9

2/2

1/1

Mean number of ossified caudal vertebrae

3.80

3.56

3.42

2.98a

Ribs and Sternabrae

 

 

 

 

Costal cartilage, misaligned

7/7

7/7

9/7

14/11a

Rib, short, minimal

7/5

6/5

4/3

12/8a

Rib, short, moderate

0/0

1/1

1/1

2/2a

Rib, wavy, moderate

0/0

0/0

9/3a

6/3a

Rib, wavy, severe

0/0

0/0

1/1a

1/1a

Rib, incomplete ossification

0/0

0/0

1/1a

3/3a

Sternebra, unossified

38/14

48/19a

43/15a

91/24a

Limbs

 

 

 

 

Scapula, bent

0/0

0/0

2/1

4/3a

Scapula, misshapen, minimal

1/1

1/1

3/1

7/4

Humerus, bent

0/0

0/0

0/0

1/1a

Humerus, short

0/0

0/0

0/0

2/2a

Radius, bent

0/0

0/0

0/0

1/1a

Radius, short

0/0

0/0

0/0

1/1a

Ulna, bent

0/0

0/0

0/0

1/1a

Ulna, short

0/0

0/0

0/0

1/1a

Forepaw phalanges, increased ossification

8/2

2/2

0/0

0/0

Metacarpal, unossified

76/21

81/20a

97/19a

138/24a

Metacarpal, incompletely ossified

2/2

3/3

9/6a

5/4a

Pelvic girdle

 

 

 

 

Ischium, incomplete ossification

1/1

1/1

7/4

3/3

Pubis, incomplete ossification

6/4

4/3

15/7a

25/10a

 Thyroid gland, minimal malformation 0/0  0/0 0/0 1/1

Number of foetuses with abnormality / Number of litters with abnormality

a – Outside of the control and/or historical control data

Conclusions:
In this study performed in accordance with OECD Guideline 414, oral administration of Methylmorpholine-oxide (50.3% purity) given once a day to rat dams from Day 6 to 19 of gestation was not well tolerated at 100 or 300 mg/kg/day for the dams due to a reduction in body weight gain and food consumption and at 300 mg/kg/day for the foetuses due to the lower foetal weights as well as a significant increase in foetal abnormalities and incomplete ossification. Therefore, the maternal No-Observed-Adverse-Effect-Level (NOAEL) was established at 30 mg/kg/day and the foetal NOAEL was established at 100 mg/kg/day. Developmental effects were considered as a secondary non-specific consequence of maternal toxicity effects.
Executive summary:

In a prenatal developmental toxicity conducted according to OECD guideline 414 Methylmorpholine-oxide (50.3% purity) was administered to 24 female Sprague-Dawley rats/dose in water at dose levels of 0, 30, 100 or 300 mg/kg bw/day from day 6 to day 19 of gestation. The following parameters and endpoints were evaluated in this study: clinical observations, body weights, body weight gains, food consumption, gross necropsy findings, foetal examinations, ovarian and uterine findings and gravid uterine weights.

In the maternal animals, administration of Methylmorpholine-oxide at 300 mg/kg/day was associated with clinical observations including decreased activity, pale faeces, erect fur, ploughing and fur staining. Body weight loss was also observed in the first few days of dosing followed by a reduction in body weight gain and an associated lower food consumption. At 100 mg/kg/day a lower body weight gain was evident during gestation along with lower food consumption over the same periods when compared with the controls. There were no gross necropsy findings related to the administration of Methylmorpholine-oxide at any dose level. There were no notable effects on mean numbers of corpora lutea, implantation, and intrauterine deaths after dosing with Methylmorpholine-oxide compared to the controls. 

 

Administration of Methylmorpholine-oxide was associated with lower gravid uterus weights at 300 mg/kg/day only. Lower foetal weights and a higher incidence of foetal abnormalities and variations at 100 and 300 mg/kg/day along with a lower degree of skeletal ossification at 30, 100 or 300 mg/kg/day. These findings were only considered to be adverse at 300 mg/kg/day due to higher proportion of lower ossification along with an increase in the severity of the findings.

 

In conclusion, oral administration of Methylmorpholine-oxide given once a day from Day 6 to 19 of gestation was not well tolerated at 100 or 300 mg/kg/day for the dams due to a reduction in body weight gain and food consumption and at 300 mg/kg/day for the foetuses due to the lower foetal weights as well as an increase in foetal abnormalities and incomplete ossification. Therefore, the maternal No Observed Adverse Effect Level (NOAEL) was established at 30 mg/kg/day and the foetal NOAEL was established at 100 mg/kg/day.

The developmental toxicity study in the rat is classified acceptable and satisfies the guideline requirement for a developmental toxicity study (OECD 414) in rats.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
30 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
reliable without restriction
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

In a developmental toxicity study Methylmorpholine-oxide was administered to groups of 24 pregnant female New Zealand White rabbits by gavage at dose levels of 0, 125, 250 or 500 mg/kg/day each from days 6 through 28 of gestation. 

At 500 mg/kg/day, the mean food consumption was up to -55% lower than the concurrent control group decreasing until GD 9 but gradually increased thereafter. Consequently, half of the animals in this group had reduced faecal output. There was an initial body weight loss of -43.5 g between GD 6-10, while in the control group the body weight increased by 25.7 g during the same period. From GD 12, the body weight changes were comparable to the control group but the overall body weight gain (GD 6-29) still remained lower (248 g versus 424 g in the control). At 250 mg/kg/day, there were 2 animals with reduced faecal output but there were no significant body weight or food consumption effects.

At 125 mg/kg/day, there were no test-item related clinical observations, food consumption or body weight effects. At necropsy, there were no test-item related maternal gross pathology findings in any of the groups.

At 500 mg/kg/day, the mean gravid uterus and combined fetal weights were -19% and -17% lower than the control, respectively (fetal weight for both sexes separately were also lower). Fetal examinations indicated a higher incidence of unossified forepaw and hindpaw phalanges, unossified metacarpal, malpositioned ilium and full thoracolumbar supernumerary ribs at 500 mg/kg/day.

The increased incidence of variations in the ossification parameters (unossified forepaw and hindpaw phalanges; and unossified metacarpal) were considered to be linked with lower fetal body weights indicating a delay in development and are considered likely to be completed in time postnatally. As the skeleton undergoes developmental changes at the time fetuses are evaluated in this type of study design, transient delays in development can produce apparent findings of abnormal skeletal structure (DeSesso and Scialli, 2018iv).

The maternal as well as fetal No Observed Adverse Effect Level (NOAEL) were established at 500 mg/kg/day.

In another prenatal developmental toxicity conducted according to OECD guideline 414 Methylmorpholine-oxide (50.3% purity) was administered to 24 female Sprague-Dawley rats/dose in water at dose levels of 0, 30, 100 or 300 mg/kg bw/day from day 6 to day 19 of gestation. The following parameters and endpoints were evaluated in this study: clinical observations, body weights, body weight gains, food consumption, gross necropsy findings, foetal examinations, ovarian and uterine findings and gravid uterine weights.

In the maternal animals, administration of Methylmorpholine-oxide at 300 mg/kg/day was associated with clinical observations including decreased activity, pale faeces, erect fur, ploughing and fur staining. Body weight loss was also observed in the first few days of dosing followed by a reduction in body weight gain and an associated lower food consumption. At 100 mg/kg/day a lower body weight gain was evident during gestation along with lower food consumption over the same periods when compared with the controls. There were no gross necropsy findings related to the administration of Methylmorpholine-oxide at any dose level. There were no notable effects on mean numbers of corpora lutea, implantation, and intrauterine deaths after dosing with Methylmorpholine-oxide compared to the controls.

Administration of Methylmorpholine-oxide was associated with lower gravid uterus weights at 300 mg/kg/day only. Lower foetal weights and a higher incidence of foetal abnormalities and variations at 100 and 300 mg/kg/day along with a lower degree of skeletal ossification at 30, 100 or 300 mg/kg/day. These findings were only considered to be adverse at 300 mg/kg/day due to higher proportion of lower ossification along with an increase in the severity of the findings.

In conclusion, oral administration of Methylmorpholine-oxide given once a day from Day 6 to 19 of gestation was not well tolerated at 100 or 300 mg/kg/day for the dams due to a reduction in body weight gain and food consumption and at 300 mg/kg/day for the foetuses due to the lower foetal weights as well as an increase in foetal abnormalities and incomplete ossification. Therefore, the maternal No Observed Adverse Effect Level (NOAEL) was established at 30 mg/kg/day and the foetal NOAEL was established at 100 mg/kg/day. Developmental effects were considered as a secondary non-specific consequence of maternal toxicity effects.

The developmental toxicity study in the rat is classified acceptable and satisfies the guideline requirement for a developmental toxicity study (OECD 414) in rats.

Mode of Action Analysis / Human Relevance Framework

High dose effects on fertility in an EOGRTS and its relevance for humans are not fully understood and subject to follow up analyses. Because of the Corona associated lock down, subsequent in vitro/ex vivo examinations in rat and human tissue have been delayed. Results will be submitted as soon as they are ready, in approx. 2-3 months.

Justification for classification or non-classification

Methylmorpholine 4-oxide does not elicit adverse effects on prenatal development in the absence of maternal toxicity effects in two species (rat and rabbit). Therefore, classification is not warranted according to Regulation (EC) No. 1272/2008 (CLP).

High dose effects on fertility in an EOGRTS after oral application of methylmorpholine 4-oxide are not fully understood and subject to follow up analyses. Because of the Corona associated lock down, subsequent in vitro examinations have been delayed. Results will be submitted as soon as they are ready, in approx. 2-3 months. Preliminary results are presented in IUCLID section 13. The follow-up tests should allow a decision on classified as reproductive toxicant Cat. 1B (H360F) according to Regulation (EC) No. 1272/2008 (CLP).