Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020

Materials and methods

Test guideline
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.

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- KocWin is based on the chemical structure of 4-methylmorpholine 4-oxide to make the predictions
- input Smiles: O=N(CCOC1)(C1)C
- a Log Kow of -1.2 was used as input value for the second model
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)

Test animals

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

Administration / exposure

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.
Doses / concentrationsopen allclose all
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

Examinations

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.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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 / performance (P0)

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.

Effect levels (P0)

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

Target system / organ toxicity (P0)

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

Results: F1 generation

General toxicity (F1)

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.

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Effect levels (F1)

open allclose all
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

Target system / organ toxicity (F1)

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

Overall reproductive toxicity

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

Any other information on results incl. tables

Please refer to attached tables

Applicant's summary and conclusion

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.