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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
toxicity to reproduction
Remarks:
other: subchronic study incl. reprotox parameters
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2011-11-29 untill 2012-04-04
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP and guideline compliant study.

Data source

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

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
other: OECD 408
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
2-hydroxy-N,N-dimethylpropanamide
EC Number:
609-066-0
Cas Number:
35123-06-9
Molecular formula:
C5H11NO2
IUPAC Name:
2-hydroxy-N,N-dimethylpropanamide
Test material form:
other: liquid

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sandhofer Weg 7, 97633 Sulzfeld
- Age at study initiation: 42 ± 1 days
- Weight at study initiation: 122.2 - 132.4 g (females); 145.8 -146.5 g (males)
- Housing: The animals were housed together (5 animals per cage) in H-Temp polysulfonate cages
supplied by TECNIPLAST, Hohenpeißenberg, Germany (floor area about 2065 cm2).
- Diet: Dround Kliba maintenance diet mouse/rat “GLP”, meal - ad libitum- supplied by
Provimi Kliba SA, Kaiseraugst, Switzerland
- Water: drinking water - ad libitum
- Acclimation period: 6-8 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 30-70%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 h light/ 12 h dark

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
The test item was applied as a solution in drinking water. 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 magnetic stirrer. The test substance preparations were produced at least once a week (depending on the stability of 7 days) and stored in the refrigerator. The administration volume was 10 mL/kg body weight. The concentration in the vehicle was: 100, 50, 20, 10 mg/mL
Details on mating procedure:
Not applicable
Details on analytical verification of doses or concentrations:
The stability of the test substance in drinking water over a period of up to 7 days at room temperature was proven during the study.
Concentration control analyses of the test substance preparations were carried out in samples of all concentrations at the start and towards the end of the administration period.
Duration of treatment / exposure:
90 days
Frequency of treatment:
Once daily
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 100, 200, 500, 1000 mg/kg/bw/d
Basis:
actual ingested
No. of animals per sex per dose:
10 animals
Control animals:
yes, concurrent vehicle
Details on study design:
On the day of arrival the animals were subjected to an acclimatization period during which they received ground diet and drinking water ad libitum. Prior to the first detailed clinical observation, the animals were distributed according to weight among the individual test groups, separated by sex. The weight variation of the animals used did not exceed 20 percent of the mean weight of each sex.
The list of randomization instructions was compiled with a computer.
At the start of the administration period (day 0) the rats were 42±1 days old. The test substance was administered daily by gavage for about 3 months (all rats per sex and group). Control animals received the vehicle only. At the end of the administration period, the animals were sacrificed after a fasting period (withdrawal of food) for at least 16-20 hours.
Positive control:
Not required

Examinations

Parental animals: Observations and examinations:
Mortality
A check for moribund and dead animals was made twice daily on working days and once daily on Saturdays, Sundays and public holidays. If animals were in a moribund state, they were sacrificed and necropsied.

Clinical observations
All animals were checked daily for any abnormal clinically signs before the administration as well as within 2 hours and 5 hours after the administration. Abnormalities and changes were documented for each animal.

Detailed clinical observations
Detailed clinical observations (DCO) were performed on all animals prior to the administration period and thereafter at weekly intervals. The findings were ranked according to the degree of severity, if applicable. The animals were transferred to a standard arena (50 × 37.5 cm with side heights of 25 cm). The following parameters were examined:
1. abnormal behavior when handled
2. fur
3. skin
4. posture
5. salivation
6. respiration
7. activity/arousal level
8. tremors
9. convulsions
10. abnormal movements
11. impairment of gait
12. lacrimation
13. palpebral closure
14. exophthalmus
15. feces (appearance/consistency)
16. urine
17. pupil size

Food consumption
Group food consumption was determined weekly (as representative value over 1 day) for each cage. The average food consumption per cage was used to estimate the mean food consumption in grams per animal per day.

Drinking water consumption
Drinking water consumption was monitored by daily visual inspection of the water bottles for any changes in volume.

Body weight data
Body weight was determined before the start of the administration period in order to randomize the animals. During the administration period body weight was determined on study day 0 (start of the administration period) and thereafter at weekly intervals. The difference between the body weight on the respective day of weighing and the body weight on study day 0 was calculated as body weight change.

Hematology
The following parameters were determined in blood with EDTA-K3 as anticoagulant using a particle counter (Advia 120 model; Bayer, Fernwald, Germany):
Leukocyte count (WBC)
Erythrocyte count (RBC)
Hemoglobin (HGB)
Hematocrit (HCT)
Mean corpuscular volume (MCV)
Mean corpuscular hemoglobin (MCH)
Mean corpuscular hemoglobin concentration (MCHC)
Platelet count (PLT)
Differential blood count
Reticulocytes (RET)
Prothrombin time (Hepato Quick’s test) (HQT)

Clinical chemistry
Alanine aminotransferase (ALT)
Aspartate aminotransferase (AST)
Alkaline phosphatase (ALP)
Oestrous cyclicity (parental animals):
Estrous cycle determination
Vaginal smears for cycle determination were prepared in the morning and evaluated according to the timetable for at least 3 weeks.
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 mobility
Sperm morphology
Sperm head count
Litter observations:
Not applicable
Postmortem examinations (parental animals):
Necropsy
The animals were sacrificed by decapitation under isoflurane anesthesia. The exsanguinated
animals were necropsied and assessed by gross pathology.

Organ weights
The following weights were determined in all animals sacrificed on schedule:
1. Anesthetized animals
2. Adrenal glands
3. Cauda epididymis
4. Brain
5. Epididymides
6. Heart
7. Kidneys
8. Liver
9. Ovaries
10. Pituitary gland
11. Prostate
12. Seminal vesicles with coagulating glands
13. Spleen
14. Testes
15. Thymus
16. Thyroid glands
17. Uterus with cervix

Organ/tissue fixation
The following organs or tissues were fixed in 4% formaldehyde solution or in modified
Davidson’s solution:
1. All gross lesions
2. Adrenal glands
3. Aorta
4. Bone marrow (femur)
5. Brain
6. Cecum
7. Cervix
8. Coagulating glands
9. Colon
10. Duodenum
11. Epididymides, left (modified Davidson’s solution)
12. Esophagus
13. Extraorbital lacrimal glands
14. Eyes with optic nerve (modified Davidson’s solution)
15. Femur with knee joint
16. Harderian glands
17. Heart
18. Ileum
19. Jejunum (with Peyer’s patches)
20. Kidneys
21. Larynx
22. Liver
23. Lung
24. Lymph nodes (mesenteric and axillary lymph nodes)
25. Mammary gland (male and female)
26. Nose (nasal cavity)
27. Ovaries
28. Oviducts
29. Pancreas
30. Parathyroid glands
31. Pharynx
32. Pituitary gland
33. Prostate
34. Rectum
35. Salivary glands (mandibular and sublingual glands)
36. Sciatic nerve
37. Seminal vesicles
38. Skeletal muscle
39. Skin
40. Spinal cord (cervical, thoracic and lumbar cord)
41. Spleen
42. Sternum with marrow
43. Stomach (forestomach and glandular stomach)
44. Testis, left (modified Davidson’s solution)
45. Thymus
46. Thyroid glands
47. Trachea
48. Urinary bladder
49. Uterus
50. Vagina

Histopathology
Fixation was followed by histotechnical processing, examination by light microscopy and assessment of findings on following organs:
1. All gross lesions
2. Adrenal glands
3. Aorta
4. Bone marrow (femur)
5. Brain
6. Cecum
7. Cervix
8. Coagulating glands
9. Colon
10. Duodenum
11. Epididymis, left
12. Esophagus
13. Eyes with optic nerve
14. Female mammary gland
15. Heart
16. Ileum
17. Jejunum
18. Kidneys
19. Liver
20. Lung
21. Lymph nodes (mesenteric and axillary lymph nodes)
22. Ovaries
23. Pancreas
24. Parathyroid glands
25. Peyer’s patches
26. Pituitary gland
27. Prostate
28. Rectum
29. Salivary glands (mandibular and sublingual glands)
30. Sciatic nerve
31. Seminal vesicles
32. Skin
33. Spinal cord (cervical, thoracic and lumbar cord)
34. Spleen
35. Stomach (forestomach and glandular stomach)
36. Testis, left
37. Thymus
38. Thyroid glands
39. Trachea
40. Urinary bladder
41. Uterus
42. Vagina
Postmortem examinations (offspring):
Not applicable
Statistics:
Means and standard deviations of each test group (main groups) were calculated.

A comparison of each group with the control group was performed using DUNNETT's test (two-sided) for the hypothesis of equal means

Non-parametric oneway analysis using KRUSKAL-WALLIS test (two-sided).If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using Wilcoxon-test (twosided) for the equal medians
Reproductive indices:
Not applicable
Offspring viability indices:
Not applicable

Results and discussion

Results: P0 (first parental generation)

Details on results (P0)

Mortality
In the present study no animal died ahead of schedule.


Clinical examinations
All male and female animals of test group 4 (1000 mg/kd bw/d) showed a yellow discolored fur towards the end of the study. This finding was related to the test substance administration and was considered to be not adverse.

Food consumption
No test substance-related findings were observed.

Drinking water consumption
No test substance-related findings were observed.

Body weight data
Body weight values of male and female animals showed no significant deviations in comparison to the control.
The body weight in females of test group 2 (200 mg/kg bw/d) on day 70 and the corresponding body weight changes value were significantly higher to the control group. Due to the lack of a dose response relationship this was assessed as being incidental. The body weight change values in males of test group 4 (1000 mg/kg bw/d) in comparison to day 0 were significantly reduced from study days 7 to 49 (up to -15.8%) and on study day 91 (-14.0%). These findings were related to the test substance administration.

Functional observational battery
Deviations from "zero values" were obtained in several animals. However, as most findings were equally distributed between treated groups and controls, were without a dose-response relationship or occurred in single animals only, these observations were considered to be incidental.

Motor activity measurement
In male animals of test groups 1 (100 mg/kg bw/d) and 3 (500 mg/kg bw/d) the single interval 3 values as well as the overall motor activity were significantly decreased. Due to the lack of a dose response relationship this was assessed as being incidental.

Ophthalmological examinations
All findings were incidental in nature, due to the occurrence in single animals, only, and/or the lack of a dose-response relationship. There were no treatment-related findings.

Estrous cycle determination
No substance-related findings were observed.

Hematology
At the end of the study in females of test group 4 (1000 mg/kg bw/d), platelet counts and relative eosinophil counts were decreased. In males of test groups 2 up to 4 (200, 500 and 1000 mg/kg bw/d) prothrombin time (hepato quick’s time, HQT) was reduced. However, in males of the mentioned test groups HQTs were within the historical control range. In addition, higher relative reticulocyte counts in males of test groups 3 and 4 (500 and 1000 mg/kg bw/d) as well as higher hematokrit and hemoglobin values in females of the same test groups were also within historical control ranges (males relative reticulocyte counts: 0.9-2.5 %; females hemoglobin: 8.7-9.5 g/L; hematocrit: 0.376- 0.424 L/L). Therefore, the alterations of HQT in males of test group 2 up to 4 as well as the other changes in test groups 3 and 4 of both sexes apart from the decreased platelet counts and relative eosinophil counts in females of test group 4 (1000 mg/kg bw/d) were regarded as incidental and not treatment-related.

Clinical chemistry
At the end of the study, in rats of both sexes of test groups 3 and 4 (500 and 1000 mg/kg bw/d) creatinine values were decreased. Additionally, in males of the mentioned test groups triglyceride levels were increased and in females of test group 4 (1000 mg/kg bw/d) cholesterol values were higher compared to controls. All dosed males showed also increased cholesterol values, but compared to the historical controls, only the cholesterol values in test groups 3 and 4 were above the range (cholesterol 1.51-2.23 mmol/L). The statistically higher cholesterol values in males of test group 1 and 2 (100 and 200 mg/kg bw/d) were due to low cholesterol levels in the study controls compared to the historical control range. Therefore, the changes in the latter test groups were regarded as not adverse.
In females of test groups 2 up to 4 (200, 500 and 1000 mg/kg bw/d aspartateaminotransferase (AST) activities were lower compared to controls, and in males of test groups 1, 3 and 4 (100, 500 and 1000 mg/kg bw/d) albumin values were higher compared to controls, but both parameters were not dose-dependently changed and therefore these alterations were regarded as incidental and not treatment-related. In rats of both sexes of test groups 3 and 4 (500 and 1000 mg/kg bw/d) and additionally in females of test groups 1 and 2 (100 and 200 mg/kg bw/d) chloride concentrations were
decreased. In females of test groups 2 and 4 (200 and 1000 mg/kg bw/d) glucose levels were higher, and in females of test groups 3 and 4 (500 and 1000 mg/kg bw/d) albumin levels were lower compared to controls. In males of test groups 4 (1000 mg/kg bw/d) total bilirubin levels were increased. All mentioned values were within historical control ranges (females: chloride 100.2-107.8 mmol/L, glucose 4.93-6.11mmol/L, albumin 39.13-42.59 g/L; males: chloride 99.9-107.4 mmol/L, total bilirubin 1.60-2.76 μmol/L) and therefore the mentioned alterations were regarded as incidental and not treatment-related.

Urinalyses
In males of test groups 3 and 4 (500 and 1000 mg/kg bw/d) and in females of test groups 1 and 4 (100 and 1000 mg/kg bw/d) urine specific gravity was higher compared to controls. But in the mentioned males the values were within the historical control range (specific gravity 1036-1077 g/L) and in the females the parameter was not dosedependently changed. Therefore, this alteration was regarded as incidental and not treatment-related.

In all dosed males increased incidences of granular and epithelial cell casts and in males of test groups 2 up to 4 (200, 500 and 1000 mg/kg bw/d) higher counts of transitional epithelial cells were found in the urine sediment. These findings correspond to the histological finding of α2u-globulins (eosinophilic droplets) in the proximal convoluted kidney tubuli and were discussed as a rat specific finding not relevant for humans. Therefore, these findings were regarded as treatment-related, but not adverse.

Sperm parameters
Concerning the motility of the sperms and the incidence of abnormal sperms in the cauda epididymidis as well as the sperm head counts in the testis and in the cauda epididymidis no treatment-related effects were observed.

Weight Parameters

Absolute organ weights
A significant liver weight increase in males, as well as the significant kidneys weight increase in females of test group 4 (1000 mg/kg bw/d) was regarded as treatment-related. The significant kidneys weight increase in females of test groups 2 and 3 (200 and 500 mg/kg bw/d) occurred without any dose-dependency and is thus regarded as incidental. The significant weight decrease in the pituitary gland of males had no histopathological correlate and is considered to be incidental. All other mean absolute weightparameters did not show significant differences when compared to the control group 0.

Relative organ weights
A significant liver and kidneys weight increase in males and females is regarded as treatment-related. No histopathological correlate was found for the significant relative weight increase in the seminal vesicles of males in test group 4 (1000 mg/kg bw/d) which is considered to be related to a minimal but not significant terminal body weight decrease of -9% in those males. All other mean relative weight parameters did not show significant differences when compared to the control group 0.

Gross lesions
All males and females of test group 4 (1000 mg/kg bw/d) showed a yellow discoloration of the fur in the ano-genital region. This finding was related to the excretion of the test substance, physically characterized as yellowish, and was considered to be treatment-related but not adverse. Two males and one female of test group 4 (1000 mg/kg bw/d) showed in the liver single or few yellow foci of 2 – 8 mm in diameter. Although a histopathological correlate was observed, this type of necrosis is considered to belong to the spectrum of spontaneous lesions and is not regarded as treatment-related. All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Histopathology
Dose dependent centrilobular hypertrophy was detected in the liver of all male and female treatement groups. Furthermore, a dose dependent occurrence of eosinophilic droplets were detected in the kidneys of all male treatement groups. This findings were regarded as treatement related. All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They are considered to be incidental or spontaneous in origin and without any relation to treatment.

Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
ca. 200 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: see 'Remark'

Results: F1 generation

Details on results (F1)

Not applicable

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
other: Parent
Effect level:
ca. 200 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: Toxicity observed at 1000 mg/kg

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

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