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Description of key information

NOAEL systemic parental toxicity: 250 mg/kg bw/d
NOAEL reproduction toxicity: 250 mg/kg bw/d
NOAEL developmental toxicity: 250 mg/kg bw/d

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant study according to guideline
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA OPPTS 870.3650: Combined Repeated Dose Toxicity Study With the Reproduction/Developmental Toxicity Screening Test (July 2000)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Age at study initiation: about 10 - 11 wks
- Housing: 1 animal / cage; except pairs during mating and dams with their litter
- Diet: ground Kliba maintenance diet mouse-rat “GLP”, meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerlandad libitum
- Water: ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature: 20 - 24°C
- Humidity: 30 - 70%
- Air changes: 15 per hour
- Photoperiod: 12 h light / 12 h darkness (6:00 to 18:00 h)

Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Acetylmorpholin was applied as a solution. To prepare this solution the appropriate amount of test substance was weighed out depending on the desired concentration. Then, drinking water was filled up to the desired volume, subsequently released with a high speed homogenizer. During administration of the test substance, preparations were kept homogeneous by stirring with a magnetic stirrer. The test substance preparations were produced at least once a week.


Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The analyses of the test substance preparations were carried out at the Analytical Chemistry Laboratory of Experimental Toxicology and Ecology of BASF SE, Ludwigshafen, Germany.

The stability of the test substance in drinking water for a period of 7 days at room temperature was proven before the start of the study.

Homogeneity analyses of the test substance preparations were performed in samples of the highest and lowest concentrations at the start of the administration period. These samples also served for concentration control analyses. In samples of the middle concentration only concentration control analyses were performed.

Analysis of the dose preparations
Stability analyses: The stability of the test substance in drinking water was demonstrated over a period of 7 days at room temperature. As the mixtures were stored no longer than this time period, the stability was guaranteed.

Homogeneity control analyses: Considering the low relative standard deviation in the homogeneity analysis, it can be concluded that Acetylmorpholin was distributed homogeneously in drinking water.

Concentration control analyses: The concentration control analyses of all concentrations revealed that the values were in the expected range of the target concentrations, i.e. were always in a range of about 98.5% - 102.9% of the nominal concentrations.These results demonstrate the correctness of the concentrations of Acetylmorpholin.
Duration of treatment / exposure:
Males: for 14 days (during pre-mating and mating period), followed by treatment approx 3 weeks post-mating
Females: for 14 days (during pre-mating and mating period), throughout entire gestation and 4 days after littering, followed by an additional treatment until one day before sacrifice
Frequency of treatment:
daily
Remarks:
Doses / Concentrations:
50, 250 or 750 mg/kg bw/d
Basis:
actual ingested
No. of animals per sex per dose:
10 males, 10 females
Control animals:
yes, concurrent vehicle
Positive control:
No
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily for any signs of morbidity, pertinent behavioral changes and signs of overt toxicity.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once before administration period (day 0), weekly thereafter.
The littering and lactation behavior of the dams was generally evaluated in the mornings in combination with the daily clinical inspection of the dams. Only particular findings (e.g. inability to deliver) were documented on an individual dam basis.
On weekdays (except public holidays) the parturition behavior of the dams was inspected in the afternoons in addition to the evaluations in the mornings.


BODY WEIGHT: Yes
- Time schedule for examinations: in general weekly (preferrably in the morning)
females: Gestational day (GD) 0, 7, 14 and 20; on the day of parturition (PND 0) and four days later (PND 4), weekly thereafter where applicable

FOOD CONSUMPTION: Yes
- Time schedule: Generally, food consumption was determined once a week for male and female parental animals, with the following exceptions:
- Food consumption was not determined during the mating period (male and female F0 animals).
- Food consumption of the F0 females with evidence of sperm was determined on GD 0, 7, 14 and 20.
- Food consumption of F0 females, which gave birth to a litter was determined for PND 4.
Food consumption was not determined in females without positive evidence of sperm (during the mating period of dams used in parallel) and females without litter (during the lactation period of dams used in parallel).

MORTALITY: Yes
-Time schedule: twice daily on working days and once daily on Saturdays, Sundays and public holidays.

OTHER:
- FOB (Functional Observational Battery): performed in all animals at the end of the administration period.
- MA (Motor activity): performed in all animals at the end of the administration period.
- additional blood and serum samples from each individual animal, stored in an N2 atmosphere at -80°C for reseach purposes.

- Blood and urin sampling: In the morning blood was taken from the retrobulbar venous plexus from fasted animals. The animals were anaesthetized using isoflurane (Isoba, Essex GmbH Munich, Germany). The blood sampling procedure and subsequent analysis of blood and serum samples were carried out in a randomized sequence. For urinalysis the individual animals were transferred to metabolism cages (withdrawal of food and water) and urine was collected overnight. Urine samples were evaluated in a randomized sequence.

From five randomly selected parental males and females the following parameters were investigated:
- Haematology: leukocytes, erythrocytes, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelets, differential blood count, reticulocytes, prothrombin time
- Clinical chemistry: alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, serum gamma-glutamyl transferase, sodium, potassium, chloride, calcium, urea, creatinine, glucose, total protein, total bilirubin, albumin, globulin, triglycerides, cholesterol, magnesium
- Urinalaysis: pH, protein, glucose, ketones, urobilinogen, bilirubin, blood, specific gravity, sediment, color, turbidity, volume

- Sperm parameters: Immediately after necropsy and organ weight determination the right testis and cauda epididymis were taken from all male animals.
Sperm motility examinations were carried out in a randomized sequence.
Following paraemters were investigated: sperm motility, sperm morphology, sperm head count (cauda epididymis), sperm head count (testis)



Sacrifice and pathology:
SACRIFICE
- Male animals: All surviving animals: approximately 3 week post-mating.
- Maternal animals: All surviving animals: at PND 4.

GROSS NECROPSY
- Gross necropsy consisted of: external surfaces, all orifices, the cranial cavity, the external surfaces of the brain, the thoracic, abdominal and pelvic cavities with their associated organs and tissues and the neck with its associated organs and tissues, special attention was given to the reproductive organs.

ORGAN WEIGHTS
- Organ weights: epididymides, testes (all animals); adrenals, brain, heart, kidneys, liver, spleen, thymus (5 animals/sex and test group (females with litters, same animals as used for clinical pathology examinations)).

PRESERVATION OF TISSUES
Tissues preserved: adrenals, aorta, bone marrow (femur), brain, cecum, cervix, coagulating gland, colon, duodenum, epididymides, esophagus, eyes with optic nerve, extraorbital lacrimal glands, female and male mammary gland, femoral bone with articulation, heart, ileum, jejunum (with Peyers patches), kidneys, larynx, liver, lungs, lymph nodes (axillary and mesenteric), nose (nasal cavity), ovaries, oviducts, pancreas, parathyroid gland, pharynx, pituitary gland, prostate, rectum, salivary glands (sublingual and mandibular), sciatic nerves, seminal vesicles, skeletal muscle, spinal cord (cervical, thoracic and lumbar), spleen, sternum with bone marrow, stomach with forestomach and glandular stomach, target organs, testes, thymus, thyroids, trachea, urinary bladder, uterus, vagina, all gross lesions

HISTOPATHOLOGY: Yes, Mainly 5 animals/dose group from the control and high dose group, except all gross lessions, epididymis, kidneys, liver and testes (all dose groups).
- Organs examined: adrenals, bone marrow (femur), brain, cecum, cervix, coagulating gland, colon, duodenum, epididymides, heart, ileum, jejunum (with Peyers patches), kidneys, liver, lungs, lymph nodes (axillary and mesenteric), ovaries, oviducts, prostate, rectum, sciatic nerves, seminal vesicles, spinal cord (cervical, thoracic and lumbar), spleen, stomach with forestomach and glandular stomach, testes, thymus, thyroids, trachea, urinary bladder, uterus, vagina, all gross lesions
Statistics:
Clinical examinations:
Food consumption (parental animals), body weight and body weight change (parental animals and pups; for the pup weights, the litter means were used), number of mating days, duration of gestation, number of implantation sites, postimplantation loss and % postimplantation loss, number of pups delivered per litter: DUNNETT-test (two-sided).
Male and female mating indices, male and female fertility indices, gestation index, females with liveborn pups, females with stillborn pups, females with all stillborn pups, live birth index, pups stillborn, pups died, pups cannibalized, pups sacrificed moribund, viability index, number of litters with affected pups at necropsy: FISHER'S EXACT test.
Proportions of affected pups per litter with necropsy observations: WILCOXON-test (one-sided).
Feces, rearing, grip strength of forelimbs and hindlimbs, landing foot-splay test, motor activity: KRUSKAL-WALLIS test (two-sided), WILCOXON-test (two-sided).

Clinical pathology:
Blood parameters: bidirectional changes: KRUSKAL-WALLIS test, WILCOXON-test (two-sided); unidirectional changes: WILCOXON-test (one-sided).
Urinalysis parameters (apart from urine color and turbidity): WILCOXON-test (one-sided).
Spermanalysis parameters: WILCOXON-test (one-sided).

Pathology: weight parameters: KRUSKAL-WALLIS test (two-sided), WILCOXON test

Details on results:
CLINICAL EXAMINATIONS
750 mg/kg bw/d:
- Significantly decreased food consumption in males and females during premating from study weeks 0 to 1.
- Significantly decreased food consumption during lactation from study days 0 to 4.
- Significantly decreased body weights in males during premating on study weeks 1 to 4.
- Significantly decreased body weight change in males during premating from study week 0 to 1.
- Significantly decreased body weight during premating in females on study week 1 and 2.
- Significantly decreased body weight change during premating from study week 0 to 1 and 0 to 2.
- Significantly decreased body weights during gestation from study days 0 to20.
- Significantly decreased body weight change during gestation from study day 7 to 14, 14 to 20 and from 0 to 20.
- Decreased body weights during lactation on study day 4.

250 mg/kg bw/d:
- No test substance-related, adverse findings were noted.

50 mg/kg bw/d:
- No test substance-related, adverse findings were noted.

REPRODUCTIVE PERFORMANCE
750 mg/kg bw/d:
- The male/female fertility index was reduced to 22% .

250 mg/kg bw/d:
- No test substance-related, adverse findings were noted.

50 mg/kg bw/d:
- No test substance-related, adverse findings were noted.

CLINICAL PATHOLOGY
750 mg/kg bw/d:
- Increased hemoglobin and hematokrit values in males.
- Increased total bilirubin and triglyceride values in both sexes.
- Decreased glucose and chloride values in males.
- Increased calcium levels in males.
- Reduced sperm motility, total sperm counts per gram cauda epidymidis and spermatid counts per gram testis in males.
- Increased counts of abnormal sperms from the cauda epidymidis in males.

250 mg/kg bw/d:
- No treatment-related effects were measured regarding clinical pathology parameters

50 mg/kg bw/d:
- No treatment-related effects were measured regarding clinical pathology parameters

PATHOLOGY
750 mg/kg bw/d:
- Decrease in terminal body -7% weight in males.
- Decrease in absolute testes weights (-16%).
- Decrease in absolute and relative epididymal weights (up to -24% absolute; up to – 19% relative).
- Minimal to slight tubular degeneration, Sertoli cell vacuolation and multinucleated giant cells in up to 8/10 males.
- Minimal to slight oligospermia and cellular detritus in up to 3/10 males.
- Minimal to slight centrilobular hypertrophy in the liver of males and females with minimal to slight increase in apoptosis in two males.

250 mg/kg bw/d:
- No adverse effects regarding weight parameters, gross lesions or histopathologic findings.

50 mg/kg bw/d:
- No adverse effects regarding weight parameters, gross lesions or histopathologic findings.
Dose descriptor:
NOAEL
Effect level:
250 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Based on adverse effects on body weight, food consumption and organ weights as well as adverse effects observed in clinical pathology and histopathology at 750 mg/kg bw/d.
Critical effects observed:
not specified
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
250 mg/kg bw/day
Study duration:
subacute
Species:
rat

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Oral

In a combined repeated dose toxicity study with the reproduction/developmental toxicity screening test Acetylmorpholine was administered orally by gavage to groups of 10 male and 10 female Wistar rats (F0 animals) at dose levels of 0 mg/kg bw/day (test group 0), 50 mg/kg bw/d (test group 1), 250 mg/kg bw/d (test group 2) and 750 mg/kg bw/d (test group 3). The animals of the control group were treated in the same way with the vehicle only (drinking water). Fourteen days after the beginning of treatment, males and females from the same test group were mated. The duration of treatment covered a 2-week pre-mating and mating period in both sexes, approximately 3 week post-mating in males, and the entire gestation period as well as 4 days of lactation in females followed by an additional treatment until one day before sacrifice.

Clinical and detailed clinical signs, food and water consumption as well as body weight development and gain were determined during the study period. Towards study end, clinicochemical and haematological examinations, urinalysis and a functional observation battery incl. motor activity measurements were performed.

After sacrifice, parent animals were examined by gross pathology. Selected organs were weighed and a histopathological examination was performed. Pups were sexed and macroscopically examined on PND0. Viability was recorded. They were weighed on PND1 and PND4. Necropsy was performed on PND4. All pups were examined macroscopically for external and visceral findings.

Obtained results:

750 mg/kg bw /d:

CLINICAL EXAMINATIONS

- Significantly decreased food consumption in males and females during premating from study weeks 0 to 1.

- Significantly decreased food consumption during lactation from study days 0 to 4.

- Significantly decreased body weights in males during premating on study weeks 1 to 4.

- Significantly decreased body weight change in males during premating from study week 0 to 1.

- Significantly decreased body weight during premating in females on study week 1 and 2.

- Significantly decreased body weight change during premating from study week 0 to 1 and 0 to 2.

- Significantly decreased body weights during gestation from study days 0 to20.

- Significantly decreased body weight change during gestation from study day 7 to 14, 14 to 20 and from 0 to 20.

- Decreased body weights during lactation on study day 4.

REPRODUCTIVE PERFORMANCE

- The male/female fertility index was reduced to 22%.

CLINICAL PATHOLOGY

- Increased hemoglobin and hematokrit values in males.

- Increased total bilirubin and triglyceride values in both sexes.

- Decreased glucose and chloride values in males.

- Increased calcium levels in males.

- Reduced sperm motility, total sperm counts per gram cauda epidymidis and spermatid counts per gram testis in males.

- Increased counts of abnormal sperms from the cauda epidymidis in males.

PATHOLOGY

- Decrease in terminal body -7% weight in males.

- Decrease in absolute testes weights (-16%).

- Decrease in absolute and relative epididymal weights (up to -24% absolute; up to – 19% relative).

- Minimal to slight tubular degeneration, Sertoli cell vacuolation and multinucleated giant cells in up to 8/10 males.

- Minimal to slight oligospermia and cellular detritus in up to 3/10 males.

- Minimal to slight centrilobular hypertrophy in the liver of males and females with minimal to slight increase in apoptosis in two males.

250 and 50 mg/kg bw/d - No test substance related adverse effects regarding clincial examinations, reproductive performance, clinical pathology and pathology.

Under the conditions of this reproduction/developmental toxicity screening test the NOAEL (no observed adverse effect level) for reproductive performance and fertility was 250mg/kg bw/d for the F0 parental rats.

The NOAEL for developmental toxicity in the F1 progeny was found to be 250 mg/kg bw/d.

The NOAEL for general, systemic toxicity was 250 mg/kg bw/d based on the clinical findings, the findings in clinical pathology and histopathology.


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
The key study was selected (GLP compliant study according to guideline).

Justification for classification or non-classification

Based on the results of the combined dose toxicity study with the reproduction/developmental toxicity screening test, Acetylmorpholine is not subject to classification and labelling for specific target organ toxicity (STOT RE) according to Directive 67/548/EEC and Regulation 1272/2008/EC.

The adverse effects observed on male reproductive organs are relevant for reproductive toxicity classification as discussed in the respective section.