Urea, reaction products with diethylene glycol, formaldehyde and glyoxal

Repeated dose toxicity: oral

Administrative data

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 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 oral 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

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

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

Observations and examinations performed and frequency:

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.

HAEMATOLOGY: Yes
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.
Clotting tests were carried out using a ball coagulometer (AMAX destiny plus model; Trinity biotech, Lemgo, Germany).
Prothrombin time (Hepato Quick’s test) (HQT)
Furthermore, blood smears were prepared and stained according to WRIGHT without being evaluated, because of non-ambiguous results of the differential blood cell counts measured by the automated instrument. (reference: Hematology: Principles and Procedures, 6th Edition, Brown AB, Lea & Febiger, Philadelphia, 1993, page 101).

CLINICAL CHEMISTRY: Yes
An automatic analyzer (Hitachi 917; Roche, Mannheim, Germany) was used to examine the clinicochemical parameters :
Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Alkaline phosphatase (ALP), gamma-Glutamyltransferase (GGT), Sodium (NA), Potassium (K), Chloride (CL), Inorganic phosphate (INP), Calcium (CA), Urea (UREA), Creatinine (CREA), Glucose (GLUC), Total bilirubin (TBIL), Total protein (TPROT), Albumin (ALB), Globulins (GLOB), Triglycerides (TRIG), Cholesterol (CHOL), Bile acids (TBA)

URINALYSIS: Yes
The dry chemical reactions on test strips (Combur 10 test M, Roche, Mannheim, Germany) used to determine urine constituents semiquantitatively were evaluated with a reflection photometer (Miditron M; Roche, Mannheim, Germany).
Urine-Parameters examined: pH, Protein, Glucose, Ketones, Urobilinogen, Bilirubin, Blood, Specific gravity, Sediment, Color, turbidity, Volume

NEUROBEHAVIOURAL EXAMINATION: Yes
Functional observational battery
A functional observational battery was performed in the first five male animals per test group, the first 5 female animals with litter of test groups 0 (control group), 1 (100 mg/kg bw/d), 2 (300 mg/kg bw/d) and 3 (1000 mg/kg bw/d) at the end of the administration period starting at about 10.00 h. The FOB started with passive observations without disturbing the animals, followed by removal from the home cage, open field observations in a standard arena and sensorimotor tests as well as reflex tests. The findings were ranked according to the degree of severity, if applicable. The observations were performed at random. A detailed description of the methods, the ranking and documentation system can be found in PART III (Supplement).

Home cage observations:

The animals were observed in their closed home cages; any disturbing activities (touching the cage or rack, noise) were avoided during these examinations in order not to influence the behavior of the animals. Attention was paid to:

1. posture
2. tremor
3. convulsions
4. abnormal movements
5. impairment of gait
6. other findings


Open field observations:

The animals were transferred to a standard arena (50 × 50 cm with side heights of 25 cm) and observed for at least 2 minutes. The following parameters were examined:

1. behavior when removed from cage
2. fur
3. skin
4. salivation
5. nose discharge
6. lacrimation
7. eyes/pupil size
8. posture
9. palpebral closure
10. respiration
11. tremors
12. convulsions
13. abnormal movements
14. impairment of gait
15. activity/arousal level
16. feces (number of fecal pellets/appearance/consistency) within two minutes
17. urine (appearance/quantity) within two minutes
18. number of rearings within two minutes

Sensorimotor tests/reflexes:

The animals were removed from the open field and subjected to following sensorimotor or reflex tests:

1. approach response
2. touch response
3. vision ("visual placing response")
4. pupillary reflex
5. pinna reflex
6. audition ("startle response")
7. coordination of movements ("righting response")
8. behavior during "handling"
9. vocalization
10. pain perception ("tail pinch")
11. grip strength of forelimbs
12. grip strength of hindlimbs
13. landing foot-splay test
14. other findings


Motor activity assessment
The motor activity assessment (MA) was carried out in the first five parental males and females (with litter) per group at the end of the administration period. Motor activity was measured on the same day as FOB was performed. The examinations were performed using the TSE Labmaster System supplied by TSE Systems GmbH, Bad Homburg, Germany. For this purpose, the animals were placed in new clean polycarbonate cages for the time of measurement. Eighteen beams were allocated per cage. The numbers of beam interrupts were counted over 12 intervals of 5 minutes. The sequence at which the animals were placed in the polycarbonate cages was selected at random. Motor activity measurements were carried out starting at 14:00 h. On account of the measuring variant "staggered", the starting time varied according to the time needed to place the animals in the cages. For each animal, measurement started individually when the 1st beam was interrupted and was finished exactly 1 hour later. The animals did not receive any food or water during the measurements. After the transfer of the last animal the room of measurement was darkened. The program required a file name for the measured data to be stored. This name consisted of the reference number and a serial number.

Sacrifice and pathology:

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)

Statistics:

Detailed statistical analyses were conducted

Results and discussion

Results of examinations

Details on results:

CLINICAL SIGNS AND MORTALITY
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 WEIGHT GAIN
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.

FOOD CONSUMPTION
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.

HAEMATOLOGY
No treatment-related changes among hematological parameters were observed.

CLINICAL CHEMISTRY
No treatment-related changes among clinical chemistry parameters were observed.

URINALYSIS
No treatment-related changes among urinalysis parameters were observed.

NEUROBEHAVIOUR
Functional observational battery
Deviations from "zero values" were obtained in several rats. However, as most findings were equally distributed between test-substance treated groups and controls, were without a dose-response relationship or occurred in single rats only, these observations were considered as incidental.
The following examinations were performed during FOB and are assessed individually:

Home cage observations:
No test substance-related effects were observed.

Open field observations:
No test substance-related effects were observed.

Sensorimotor tests/reflexes:
No test substance-related effects were observed.

Quantitative Parameters:
The values of rearing in females of test group 2 (300 mg/kg bw/d) and test group 3 (1000 mg/kg bw/d) were significantly decreased. Due to the lack of any clinical observations this was assessed as being incidental.

Motor activity measurement:
Male animals of test group 1 (100 mg/kg bw/d) showed a significantly lower value at interval 8 when compared to the males of the control group.

Female animals of test group 3 (1000 mg/kg bw/d) showed significantly lower value at interval 1 when compared to the females of the control group.

There were no significant deviations concerning the overall motor activity (summation of all intervals) in male and female animals. All described findings were assessed as being incidental and not related to treatment.

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

GROSS PATHOLOGY
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:
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

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Target system / organ toxicity

Critical 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).

Regarding clinical 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.

Regarding fertility 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.

Regarding developmental 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).

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

Regarding pathology, 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.