Urea, reaction products with diethylene glycol, formaldehyde and glyoxal

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP Guideline Study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Fixapret PC, vor Magnesiumchlorid-Zugabe

IUPAC Name:

Fixapret PC, vor Magnesiumchlorid-Zugabe

Details on test material:

- Name of test material (as cited in study report): Fixapret PC, vor Magnesiumchlorid-Zugabe
- Physical state: liquid
- Analytical purity: Reaction product of urea, formaldehyde, glyoxal and diethylene glycol, content: 62.1 % in water
- Impurities (identity and concentrations): 37.9 % water (solvent)
- Lot/batch No.: 153
- Stability under test conditions: The stability of the test item under storage conditions over the study period was guaranteed by the sponsor,
and the sponsor holds this responsibility.
- Storage condition of test material: Room temperature

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
- Age at study initiation: 11-12 weeks
- Weight at study initiation: the animals were of comparable size and weight
- Housing: individually with the following exceptions:
• During overnight matings, male and female mating partners were housed together in Makrolon type M III cages.
• Pregnant animals and their litters were housed together until PND 4 (end of lactation).
• For motor activity (MA) measurements the animals were housed individually in polycarbonate cages supplied by TECNIPLAST, Hohenpeißenberg, Germany with wire covers from Ehret, Emmendingen, Germany (floor area of about 800 cm2) and small amounts of bedding material
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least 5 days

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

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
“Fixapret PC, vor Magnesiumchlorid-Zugabe” 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 weight, subsequently released with a magnetic stirrer. The test substance preparations were produced at least once a week.


VEHICLE
- Justification for use and choice of vehicle (if other than water): water
- Concentration in vehicle: 0, 1, 3, 10 g/100 ml

Details on mating procedure:

In general, each of the male and female animals was mated overnight in a 1:1 ratio for a maximum of 2 weeks. Throughout the mating period, each female animal was paired with a predetermined male animal from the same test group.

The animals were paired by placing the female in the cage of the male mating partner from about 16.00 h until 07.00 - 09.00 h of the following morning. Deviations from the specified times were possible on weekends and public holidays and were reported in the raw data. A vaginal smear was prepared after each mating and examined for the presence of sperm. If sperm was detected, pairing of the animals was discontinued. 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 analytical investigations of the test substance preparations were carried out as a separate study at the test facility Competence Center Analytics of BASF SE, 67056 Ludwigshafen, Germany, under the responsibility of a Study Director of this test facility. This study was carried out in compliance with the Principles of Good Laboratory Practice.

The stability of the test substance in drinking water for a period of a maximum of 7 days at room temperature was confirmed.

Concentration control Analysis of the test substance preparations were performed in samples of all concentrations at the start and towards the end of the administration period.

Duration of treatment / exposure:

The duration of treatment covered a 2-week pre-mating and mating period in both sexes, approximately 1 week post-mating in males, and the entire gestation period as well as 4 days of lactation and two weeks thereafter in females.

Frequency of treatment:

once daily

Details on study schedule:

N/A

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 100, 300, 1000 mg/kg
Basis:
actual ingested

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: on request of the sponsor
- Rationale for animal assignment (if not random): random, the list of randomization instructions was compiled with a computer.

Positive control:

N/A

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS / DETAILED CLINICAL OBSERVATIONS: Yes
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. The examinations of these animals were carried out according to the methods established at the pathology laboratory.

A cageside examination was conducted at least once daily for any signs of morbidity, pertinent behavioral changes and signs of overt toxicity. Abnormalities and changes were documented daily for each affected animal.

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.

The day of littering was considered the 24-hour period from about 15.00 h of one day until about 15.00 h of the following day.

Detailed clinical observations (DCO) were performed in 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 sides of 25 cm high). The following parameters were examined:

1. abnormal behavior during “handling”
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

BODY WEIGHT: Yes
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 once a week at the same time of the day (in the morning).

The body weight change of the animals was calculated from these results.

The following exceptions are notable for the female animals:

• During the mating period the parental fe¬males were weighed on the day of positive evidence of sperm (GD 0) and on GD 7, 14 and 20.
• Females with litter were weighed on the day of parturition (PND 0) and on PND 4.
• Females without a litter and without positive evidence of sperm in the vaginal smear were weighed weekly. These body weight data were solely used for the calculations of the dose volume.

FOOD CONSUMPTION:
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, 7 - 14 and 14 - 20.
• Food consumption of F0 females, which gave birth to a litter was determined for PND 1 - 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) and in males after the premating period.

Oestrous cyclicity (parental animals):

N/A

Sperm parameters (parental animals):

N/A

Litter observations:

LITTER DATA
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. At the same time, the pups were also being examined for macroscopically evident changes. Pups, which died before the first determination of their status on the day of birth, were defined as stillborn pups.

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.

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.

Pup clinical observations
The live pups were examined daily for clinical symptoms (including gross-morphological findings) during the clinical inspection of the dams.

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).
Pup body weights and pup body weight change are listed for males, females and males + females.
“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):

GROSS PATHOLOGY / HISTOPATHOLOGY: Yes
Necropsy
All animals were sacrificed by decapitation under isoflurane anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology.

Weight parameters
Weight assessment was carried out on all animals. The following weights were determined:

1. Anesthetized animals
2. Epididymides
3. Testes

The following weights were determined in 5 animals/sex and test group (females with litters, same animals as used for clinical pathology examinations):

1. Adrenal glands
2. Brain
3. Heart
4. Kidneys
5. Liver
6. Spleen
7. Thymus

Organ / Tissue preservation list
The following organs / tissues were preserved in neutral-buffered 4% formaldehyde or in modified Davidson’s solution:

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

Paraffin embedding, sectioning and staining
After the organs were fixed, processing, examination by light microscopy and evaluation of findings were performed, staining with Hematoxylin-eosin (H&E):
all gross lesions (all affected animals per group),
control and high dose animals only:
Adrenal glands (5 animals per sex per group, females with litters only, same animals as used for clinical pathology examination)
Bone marrow (femur) (5 animals per sex per group, females with litters only, same animals as used for clinical pathology examination)
Brain (5 animals per sex per group, females with litters only, same animals as used for clinical pathology examination)
Cecum (5 animals per sex per group, females with litters only, same animals as used for clinical pathology examination)
Cervix (all animals per group)
Coagulation glands (all animals per group)
Colon (5 animals per sex per group, females with litters only, same animals as used for clinical pathology examination)
Duodenum (5 animals per sex per group, females with litters only, same animals as used for clinical pathology examination)
Epididymides (all animals per group)
Heart (5 animals per sex per group, females with litters only, same animals as used for clinical pathology examination)
Ileum (5 animals per sex per group, females with litters only, same animals as used for clinical pathology examination)
Jejunum (5 animals per sex per group, females with litters only, same animals as used for clinical pathology examination)
Kidneys (5 animals per sex per group, females with litters only, same animals as used for clinical pathology examination)
Liver (5 animals per sex per group, females with litters only, same animals as used for clinical pathology examination)
Lung (5 animals per sex per group, females with litters only, same animals as used for clinical pathology examination)
Lymph nodes (mesenteric and axillary lymph nodes) (5 animals per sex per group, females with litters only, same animals as used for clinical pathology examination)
Ovaries (all animals per group)
Oviducts (all animals per group)
Peyer’s patches (5 animals per sex per group, females with litters only, same animals as used for clinical pathology examination)
Prostate (all animals per group)
Rectum (5 animals per sex per group, females with litters only, same animals as used for clinical pathology examination)
Sciatic nerve (5 animals per sex per group, females with litters only, same animals as used for clinical pathology examination)
Seminal vesicles (all animals per group)
Spinal cord (cervical, thoracic and lumbar cords) (5 animals per sex per group, females with litters only, same animals as used for clinical pathology examination)
Spleen (5 animals per sex per group, females with litters only, same animals as used for clinical pathology examination)
Stomach (forestomach and glandular stomach) (5 animals per sex per group, females with litters only, same animals as used for clinical pathology examination)
Testes (all animals per group)
Thymus (5 animals per sex per group, females with litters only, same animals as used for clinical pathology examination)
Thyroid glands (5 animals per sex per group, females with litters only, same animals as used for clinical pathology examination)
Trachea (5 animals per sex per group, females with litters only, same animals as used for clinical pathology examination)
Urinary bladder (5 animals per sex per group, females with litters only, same animals as used for clinical pathology examination)
Uterus (all animals per group)
Vagina (all animals per group)

Postmortem examinations (offspring):

Pup necropsy observations
All pups with scheduled sacrifice on PND 4 were sacrificed under isoflurane anesthesia with CO2. All pups were examined externally and eviscerated; their organs were assessed macroscopically.
All stillborn pups and all pups that died before PND 4 were examined externally, eviscerated and their organs were assessed macroscopically.
All pups were discarded after their evaluation.

Statistics:

Detailed statistical analyses were conducted

Reproductive indices:

For the males, mating and fertility indices were calculated. For the females, mating, fertility and gestation indices were calculated. The live birth index was calculated for F1 litters and the postimplantation loss (in %) was calculated.

Offspring viability indices:

The viability index and the sex ratio were calculated

Results and discussion

Results: P0 (first parental generation)

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
No animal died prematurely. One female animal (No. 103) of control group showed red discolored urine on study day 35. Due to the lack of a treatment this was assessed as being incidental.
No other abnormal clinical signs were observed.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
No deviations in the body weights of all test groups were observed.

Body weight change was significantly decreased in females of test group 2 (300 mg/kg bw/d) on lactation days 0 - 4 (-59%) and in females of test group 1 (100 mg/kg bw/d) on lactation days 0 - 4 (-58.4%).
Because no deviation in body weight and no reduced food consumption was observed in both test groups, this finding was assessed as being incidental.

During lactation period the food consumption was significantly decreased in females (-17.9%) of test group 3 (1000 mg/kg bw/d) and in females (-18.1%) of test group 2 (300 mg/kg bw/d).
In test group 3 one animal (No. 140) showed a particularly significant reduction (15.4g/day) while the other animals were in a higher range (26.3-36.6g/day).
The food consumption in test group 2 was lower than in test group 3 therefore no dose response relationship was observed and this finding was assessed as being incidental.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
Male mating index
For F0 parental males, which were placed with females to generate F1 pups, mating was confirmed. Thus, the male mating index was 100% in all test groups.

Male fertility index
Fertility was proven for all of the F0 parental males within the scheduled mating interval to produce F1 litter.

One male of test group 0 (No. 2 mated with female No. 102), one male of test group 1 (No. 12 mated with No. 112) and two males of test group 2 (No. 28 and 29 mated with No.128 and 129) did not generate F1 pups.

The male fertility index was 90% in test group 0, 90% in test group 1, 80% in test group 2 and 100% in test group 3.

This finding reflected the normal range of biological variation inherent in the strain of rats used for this study as all respective values were within the range of the historical control data.

Female mating index
The female mating index calculated after the mating period for F1 litter was 100% for all test groups The mean duration until sperm was detected (GD 0) was 2.8, 1.9, 1.7 and 1.8 days in test groups 0-3.

Female fertility index
All sperm positive rats delivered pups with the exception of female Nos. 102 (test group 0), 112 (test group 1) and 128, 129 (test group 2), which were mated with male Nos. 2, 12, 28 and 29 did not become pregnant. The female fertility index was 90% in test group 0, 90% in test group 1, 80% in test group 2 and 100% in test group 3.
Female animals Nos. 102, 112, 128 and 129 which delivered no pups, showed no implantation sites.
These data reflect the normal range of biological variation inherent in the strain of rats used for this study as all respective values were within the range of the historical control data .

Gestation index
The gestation index was 100% in all test groups.

Live birth indices
The live birth index was 100% in test group 0, 77.8% in test group 1, 100% in test group 2 and 90% in test group 3.
Two damns with stillborn pups were seen in test group 1 (100 mg/kg bw/d) and one damn with stillborn pups was seen in test group 3 (1000 mg/kg bw/d).
This was assessed as being incidental. In addition, the live birth index was in the historical control range of the rat strain used.

Postimplantation loss
The postimplantation loss was 1.64% in test group 0, 4.39% in test group 1, 4.71% in test group 2 and 4.10% in test group 3.
All values were in the range of the historical control data.

ORGAN WEIGHTS (PARENTAL ANIMALS)
All mean weight parameters (absolute and relative) did not show significant differences when compared to the control groups.

GROSS PATHOLOGY (PARENTAL ANIMALS)
All gross lesions noted were single observations and they were regarded to have developed spontaneously and unrelated to compound and treatment.
One female that was not pregnant (No. 129 of test group 2) revealed up to 3 mm, white foci on the ovaries of solid consistency.

All other females that were not pregnant and all male mating partners did not show gross lesions in reproduction relevant organs.

HISTOPATHOLOGY (PARENTAL ANIMALS)
The female with the macroscopic finding on the ovaries (No. 129) revealed histopathologically a multifocal sex cord stromal hyperplasia of the sertoli cell type. In addition, no corpora lutea were observed in the ovaries. This might have had a disturbing influence on the normal ovarian cycle and prevented cohabitation in this animal. As it was a single finding in one animal of the test group 2 (300 mg/kg bw/day) it is regarded to have developed spontaneously and unrelated to treatment.
All other organs of the reproduction tract did not show any relevant finding.

All other investigated not pregnant females as well as the male mating partners did not show relevant histopathological findings that could explain infertility.

All other findings noted were either single observations or they were biologically equally distributed between control and treatment group. All of them were considered to be incidental or spontaneous in origin and without any relation to treatment.

Effect levels (P0)

Dose descriptor:

NOAEL

Remarks:

parental, reproductive performance, fertility and development

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Results: F1 generation

Details on results (F1)

LITTER DATA
Pup number and status at delivery
The mean number of delivered F1 pups was 13.3 in test group 0, 12.1 in test group 1, 10.1 in test group 2 and 11.7 in test group 3.
The two stillborn pups in different litters in test group 1 were incidental and in the normal range of biological variation inherent in the strain of rats used for this study.
The ten stillborn pups in test group 3 were observed all in one litter (Animal No. 140). Because a single litter was affected, this finding was assessed as being incidental.
In addition all respective values were within the range of the historical control data (PART III, Supplement).

Pup viability/mortality
The viability index indicating pup mortality during lactation (PND 0 - 4) was 100% in test group 0, 100% in test group 1, 100% in test group 2 and 88.2% in test group 3.
However, litter No. 140 contributed disproportionally high to this decrease, since it contained only one surviving pup which was cannibalized one day after parturition. Thus, the overall decrease in pup viability in this group is considered of no biological relevance.

Sex ratio
The sex distribution and sex ratios of live F1 pups on the day of birth and PND 4 did not show substantial differences between the control and the test substance-treated groups; slight differences were regarded to be spontaneous in nature

CLINICAL SIGNS (OFFSPRING)
The surviving F1 pups of any test group did not show adverse clinical signs up to scheduled sacrifice on PND 4.

BODY WEIGHT (OFFSPRING)
Mean pup weight was significantly increased in females of test group 2 (300 mg/kg bw/d) on lactation days 1 and 4.
Mean pup body weights/pup body weight changes of all pups in all other test groups were comparable to the control group.
Due to the lack of dose response relationship this was assessed as being incidental.

In test group 0 (100 mg/kg bw/d) only one male runt was seen (Animal No. 103).

GROSS PATHOLOGY (OFFSPRING)
Ten stillborn pups from animal 140 of test group 3 (1000 mg/kg bw/d) showed post mortem autolysis.

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

“Fixapret PC, vor Magnesiumchlorid-Zugabe” was administered orally via gavage to groups of 10 male and 10 female Wistar rats (F0 animals) at dose levels of 0 mg/kg bw/d (test group 0), 100 mg/kg bw/d (test group 1), 300 mg/kg bw/d (test group 2) and 1000 mg/kg bw/d (test group 3).

Regardingclinical examinations, no signs of general systemic toxicity were observed in male or female parental animals of test groups 1-3 (100, 300 and 1000 mg/kg bw/d) during the entire study period.

Regardingfertility and reproductive performance, no signs of toxicity were observed in male or female parental animals of all test groups (100, 300 and 1000 mg/kg bw/d) during the entire study.

Regardingdevelopmental toxicity, no biologically relevant signs of toxicity were observed in male or female pups of all test groups (100, 300 and 1000 mg/kg bw/d).

Regardingclinical pathology, no treatment-related, adverse effects were observed up to a dose of the compound of 1000 mg/kg bw/d.

Regardingpathology, the treatment of male and female Wistar rats with up to 1000 mg/kg bw of the test substance did not lead to adverse findings regarding organ weights, macroscopy or histopathology. Especially no findings on the reproduction tract were observed.