Registration Dossier

Administrative data

Endpoint:
one-generation reproductive toxicity
Remarks:
based on generations indicated in Effect levels (migrated information)
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.
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
(March 22, 1996)
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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid, colorless, clear
Details on test material:
- Name of test material: Acetylmorpholin

Test animals

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)

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on 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.
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: 14 days
- Proof of pregnancy: vaginal smear. The day on which sperm was detected was denoted gestation day (GD) 0 and the following day "GD 1".

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
Details on study schedule:
- Age at mating of the mated animals in the study: about 13 to 14 weeks
Doses / concentrations
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

Examinations

Parental animals: Observations and examinations:
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
Oestrous cyclicity (parental animals):
Not investigated.
Sperm parameters (parental animals):
- 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).
Litter observations:
PUP NUMBER AND STATUS AT DELIVERY
All pups delivered from the F0 parents were examined as soon as possible on the day of birth to determine the total number of pups and the number of liveborn and stillborn pups in each litter. Pups, which died before the first determination of their status on the day of birth, were defined as stillborn pups.

SEX RATIO
On the day of birth (PND 0) the sex of the pups was determined by observing the distance between the anus and the base of the genital tubercle; normally, the anogenital distance is considerably greater in male than in female pups. The sex of the pups was finally confirmed at necropsy.

The sex ratio was calculated at day 0 and day 4 after birth according to the following formula:
Sex ratio = (number of live male or female pups on day 0/4 /number of live male and female pups on day 0/4) x 100

PUP CLINICAL OBSERVATIONS
The live pups were examined daily for clinical symptoms (including gross-morphological findings) during the clinical inspection of the dams. If pups showed particular findings, these were documented with the dam concerned.

PUP BODY WEIGHT DATA
The pups were weighed on the day after birth (PND 1) and on PND 4.
Pups body weight change was calculated from these results.
The individual weights were always determined at about the same time of the day (in the morning).
“Runts” were defined on the basis of the body weights on PND 1. "Runts" are pups that weigh less than 75% of the mean weight of the respective control pups.
Postmortem examinations (parental animals):
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.

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
Postmortem examinations (offspring):
GROSS NECROPSY
- All surviving pups (sacrificed on PND 4 under isoflurane anesthesia with CO2), all stillborn pups and those pups, which died ahead of schedule, were examined externally, eviscerated and their organs were assessed macroscopically.

All pups were discarded after their evaluation

HISTOPATHOLOGY / ORGAN WEIGTHS
- Not performed.
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.
Reproductive indices:
Male mating index
Male mating index (%) = (number of males with confirmed mating*/number of males placed with females) x 100
* defined by a female with vaginal sperm or with implants in utero

Male fertility index
Male fertility index (%) = (number of males proving their fertility*/number of males placed with females) x 100
* defined by a female with implants in utero

Female mating index
Female mating index (%) = (number of females mated*/number of females placed with males) x 100
* defined as the number of females with vaginal sperm or with implants in utero

Female fertility index
Female fertility index (%) = (number of females pregnant*/number of females mated**) x 100
* defined as the number of females with implants in utero
** defined as the number of females with vaginal sperm or with implants in utero

Gestation index
Gestation index (%) = (number of females with live pups on the day of birth/number of females pregnant*) x 100
* defined as the number of females with implants in utero

Live birth index
Live birth index (%) = (number of liveborn pups at birth/total number of pups born) x 100

Postimplantation loss
Postimplantation loss (%) = (number of implantations - number of pups delivered/number of implantations) x 100
Offspring viability indices:
Pup viability/mortality
In general, a check was made for any dead or moribund pups twice daily on workdays (once in the morning and once in the afternoon) or as a rule, only in the morning on Saturdays, Sundays or public holidays.

The number and percentage of dead pups on the day of birth (PND 0) and of pups dying between PND 1 - 4 (lactation period) were determined. Pups which died accidentally or were sacrificed due to maternal death were not included in these calculations. The number of live pups/litter was calculated on the day after birth, and on lactation day 4. The viability index was calculated according to the following formula:
Viability index (%) = (number of live pups on day 4 after birth/number of live pups on the day of birth) x 100

Results and discussion

Results: P0 (first parental generation)

Details on results (P0)

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.

Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
250 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Based on reduced fertility at 750 mg/kg bw/d.

Results: F1 generation

Details on results (F1)

CLINICAL EXAMINATIONS/GROSS FINDINGS
750 mg/kg bw/d:
- One animal delivered 3 stillborn pups and one animal delivered one liveborn pup which was cannibalized on study day 1
- Zero pups surviving study days 0 to 4 (viability index: 0).

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

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
250 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Mainly based on reduced viability index at 750 mg/kg bw/d.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion