Registration Dossier

Toxicological information

Toxicity to reproduction

Currently viewing:

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:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

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)
Qualifier:
according to
Guideline:
other: EPA OPPTS 870.3650 (Combined Repeated Dose Toxicity Study With the Reproduction/Developmental Toxicity Screening Test)
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test substance: 1-Methylimidazol
- Test substance No.: 11/0492-001
- Batch identification: 84009124U0
- Purity/composition: 99.9 area-%
- Homogeneity: Given
- Storage stability: The stability of the test substance under storage conditions over the test period was guaranteed by the sponsor, and the sponsor holds this responsibility.
- Physical state, appearance: Liquid/colorless to yellowish, clear
- Storage conditions: Room temperature

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: 11-13 weeks
- Weight at study initiation: 329.9-361.9 g (males); 186.2-215.6 g (females)
- Fasting period before study: No
- Housing: During the study period, the rats were housed individually in Makrolon type M III cages supplied by Becker & Co., Castrop-Rauxel, Germany (floor area of about 800 cm²), 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.
Pregnant females were provided with nesting material (cellulose wadding) toward the end of gestation.
For enrichment wooden gnawing blocks (Typ NGM E-022; supplied by Abedd® Lab. and Vet. Service GmbH, Vienna, Austria) were added.
- Diet: Ground Kliba maintenance diet mouse/rat “GLP” meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland, which was available to the animals ad libitum throughout the study
- Water: Drinking water was supplied from water bottles (ad libitum)
- Acclimation period: About 6 days

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

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test substance solutions in drinking water were prepared at the beginning of the administration period and thereafter in intervals, which took into account the analytical results of the stability verification.
From visual inspection, it seemed that the test substance was not completely miscible with drinking water. Therefore, for the preparation of the administration solution the test substance was weighed in a calibrated beaker depending on the dose group, topped up with drinking water and subsequently intensely mixed with a homogenizer. During administration, the preparations were kept homogeneous with a magnetic stirrer.

VEHICLE
- Concentration in vehicle: 0, 100, 300 and 900 mg/100mL
- Amount of vehicle (if gavage): 10 mL/kg bw/day
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: Maximum of 2 weeks (the female was placed in the cage of the male mating partner from about 16.00 h until 07.00 - 09.00 h of the following morning)
- Proof of pregnancy: Sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged: Individually
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The analyses were carried out at the Analytical Chemistry Laboratory of Experimental Toxicology and Ecology of BASF SE, Ludwigshafen, Germany.
Analytical verifications of the stability of the test substance in drinking water for a period of 7 days at room temperature were carried out during the study. From visual inspection, it seemed that the test substance was not completely miscible with drinking water. Therefore, in order to prove that the test substance preparations were true solutions, a homogeneity analysis was carried out. Samples of the test substance solutions were sent to the analytical laboratory once at the beginning of the study for verification of the concentrations.
Concentration control analysis: The mean values of 1-Metylimidazol in drinking water in the low-dose group (samples 3R-5R and 12-14 ) were found in the range of 122.1% to 129.0% of the nominal concentrations. The mean found concentrations at 100 mg/100 mL are above the specification limit of 110%. This slight departure from the nominal concentration range presumably brought about an insignificantly higher dose for the low-dose group than intended. However, since this dose did not produce any effect in the respective animals, the validity of the study is not regarded to be compromised. All other values of 1-Metylimidazol in drinking water were found to be in the range of 90% - 110% of the nominal concentration.
Duration of treatment / exposure:
The duration of treatment covered a 2-week premating and a mating period in both sexes, approximately 2 days post-mating in males, and the entire gestation period as well as approximately 2 weeks of the lactation period.
Frequency of treatment:
Once daily at approximately the same time in the morning. Females in labor were not treated.
Doses / concentrationsopen allclose all
Dose / conc.:
10 mg/kg bw/day (nominal)
Dose / conc.:
30 mg/kg bw/day (nominal)
Dose / conc.:
90 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The doses for the OECD422 study were selected based on two sequential 14-d range finding studies [01R0492/11R088 and 01R0492/11R136].
In the first 14-d range finding study [01R0492/11R088] 1-methylimidazole was administered daily to groups of 3 male and 3 female Wistar rats by gavage at doses of 60, 125 and 250 mg/kg bw/d. Severe effects on body weight and food consumption were observed in the mid and high dose groups at study day 3 (= day 4 of administration). Compared to the control the food consumption was statistically significantly decreased in males and females [125 mg/kg bw/d: -52.9 % (m), -35.3% (f); 250 mg/kg bw/d: -73.0% (m), -39.4% (f)]. The body weight at SD3 was reduced compared to the control group at SD 3 [125 mg/kg bw/d: -10.1 % (m), -6.2% (f); 250 mg/kg bw/d: -12.9% (m), -7.9% (f)]. Body weight loss at SD 3 compared to SD0 occurred in both sexes, but was more prominent in males than in females [125 mg/kg bw/d: -20.0 g (m), -8.2g (f); 250 mg/kg bw/d: -36.3 g (m), -4.0 (f)]. Thus, the doses were reduced from 125 to 15 mg/kg bw/d and from 250 to 30 mg/kg bw/d, respectively, as from study day 4. A continuous administration of 125 mg/kg bw/d and above would have caused severe suffering and most likely end in death of the animals and had to be stopped because of animal welfare reasons. Compared to the controls body weights of male and female rats remained marginally reduced until termination of the study, but no further severe findings were observed. In conclusion, a dose of 125 mg/kg bw/d or more exceeded the maximal tolerated dose (MTD), but 60 mg/kg bw/d was too low.
Therefore, a second 14-d range finding study [01R0492/11R136] was performed with daily gavage of 90 mg/kg bw/d to 4 male and 4 female Wistar rats. Females showed piloerection of the fur on several study days and again food consumption was statistically significantly decreased at study day 3, but recovered afterwards [-41.5% (m), -26.8% (f)]. Additionally performed clinical pathology showed increased cholesterol levels [control vs. 90 mg/kg bw/d: 1.45 +/- 0.2 vs. 2.25 +/- 0.25 mmol/L (m); 1.17 +/- 0.42 vs. 2.05 +/- 0.45 mmol/L (f)] indicating systemic toxicity. Again, male rats were more susceptible, as additionally their body weight gain was statistically significantly reduced at study day 3 compared to SD0 [-11.6 g] and their urea levels were increased at study day 14 [control vs. 90 mg/kg bw/d: 6.51 +/- 0.51 vs. 10.43 +/- 0.51 mmol/L]. Based on these observed adverse effects and taking into account the results of the first range-finding study, 90 mg/kg bw/d was selected as the top dose for the OECD422 study.

Examinations

Parental animals: Observations and examinations:
MORTALITY:
A check for moribund or dead animals was made twice daily on working days or once daily (Saturday, Sunday or on public holidays). If animals were in a moribund state, they were sacrificed and necropsied.

CAGE SIDE OBSERVATIONS:
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 animal. The parturition 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 Saturday, Sunday and public holidays) the parturition behavior of the dams was inspected in the afternoons in addition to the evaluations in the mornings. The day of parturition 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):
Detailed clinical observations were performed in all animals once prior to the first administration and at weekly intervals during the administration period. The examinations started in the morning. The findings were ranked according to the degree of severity, if applicable. For observation, the animals were removed from their cages by the investigator and placed in a standard arena (50 x 37.5 cm wide, with side borders which are 25 cm high). The following parameters listed were assessed: 1. Abnormal behavior in “handling”, 2. Fur, 3. Skin, 4. Posture, 5. Salivation, 6. Respiration, 7. Activity/arousal level, 8. Tremors, 9. Convulsions, 10. Abnormal movements, 11. Gait abnormalities, 12. Lacrimation, 13. Palpebral closure, 14. Exophthalmos, 15. Assessment of the feces discharged during the examination (appearance/consistency), 16. Assessment of the urine discharged during the examination, 17. Pupil size

BODY WEIGHT:
In general, the body weight of the male and female parental animals was determined once a week at the same time of the day (in the morning) until sacrifice. 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 females 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 positive evidence of sperm, without litter or waiting for necropsy, 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 after the 2nd premating week (male parental animals) and during the mating period (male and female F0 animals).
• Food consumption of the F0 females with evidence of sperm was determined on gestation days (GD) 0, 7, 14 and 20.
• Food consumption of F0 females which gave birth to a litter was determined on PND 1 and 4.
Food consumption was not determined in females without positive evidence of sperm during the gestation period and in females without litter during the lactation period.

HAEMATOLOGY:
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 and Reticulocytes (RET).
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). Clotting tests were carried out using a ball coagulometer (AMAX destiny plus model; Trinity biotech, Lemgo, Germany). Parameter and method: Prothrombin time (Hepato Quick’s test) (HQT).

CLINICAL CHEMISTRY:
An automatic analyzer (Hitachi 917; Roche, Mannheim, Germany) was used to examine the clinicochemical parameters. Parameters:
- Enzymes: Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Alkaline phosphatase (ALP), γ-Glutamyltransferase (GGT).
- Blood chemistry parameters: 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:
The dry chemical reactions on test strips (Combur-10-test M, Roche, Mannheim, Germany) used to semi quantitatively determine urine constituents were evaluated with a reflection photometer (Miditron M; Roche, Mannheim, Germany). Parameters: pH, Protein, Glucose, Ketones, Urobilinogen, Bilirubin, Blood, Specific gravity, Sediment, Color, turbidity, Volume.

NEUROBEHAVIOURAL EXAMINATION:
A functional observational battery was performed in 5 parental male and 5 parental female animals (with litter) per group at the end of the administration period starting at about 10.00h. 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.
- 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. Tremors, 3. Convulsions, 4. Abnormal movements, 5. Impairment of gait, 6. Other findings
- Open field observations: The animals were transferred to a standard arena (50 x 50 cm wide, with side borders which are 25 cm high) 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/stereotypy, 14. Impairment of gait, 15. Activity/arousal level, 16. Feces excreted within 2 minutes (number of scybala discharged/appearance/consistency), 17. Urine excreted within 2 minutes (amount/color), 18. Number of rearings within 2 minutes
- Sensory motor tests/Reflexes: The animals were removed from the open field and subjected to following sensory motor 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. Other findings, 12. Grip strength of forelimbs and hindlimbs, 13. Landing foot-splay test
- Motor activity measurement (MA): The MA was measured on the same day as FOB was performed in 5 parental males and females (with litter) per group. The examinations were performed using the Multi-Varimex system supplied by Columbus Instruments Int. Corp., Ohio, U.S.A. For this purpose, the animals were placed in clean polycarbonate cages for the time of measurement. The number of beam interrupts was counted over 12 intervals for 5 minutes in each case. The sequence at which the animals were placed in the polycarbonate cages was selected at random. The measurement was started at about 14.00 h. On account of the measuring variant "staggered", the starting time was varied by the time needed to place the animals in the cages. For each animal, measurement was started individually when the 1st beam was interrupted and ended exactly 1 hour later. The animals received no food or water during the measurements. After the transfer of the last animal in each case, the room where the measurements were carried out was darkened.
Sperm parameters (parental animals):
Parameters examined in male parental generation: testis weight, epididymis weight
Litter observations:
- Pup number and status at delivery: All pups delivered from the F0 parents (F1 litter) were examined as soon as possible on the day of birth to determine the total number of pups, the sex 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 this initial examination, 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.
- 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.
- Pup clinical observations: The live pups were examined daily for clinical symptoms (including gross-morphological findings) during the clinical inspection of the dams and documented for each pup.
- 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):
GROSS PATHOLOGY: Yes
The following weights were determined in all animals sacrificed on schedule: Anesthetized animals, epididymides, testes
The following weights were determined in 5 animals per sex/test group sacrificed on schedule (females with litters only, same animals as used for clinical pathological examinations): Adrenal glands, brain, heart, kidneys, liver, spleen, thymus
The following organs or tissues of all parental animals were fixed in 4% buffered formaldehyde solution or modified Davidson’s solution: All gross lesions, adrenal glands, aorta, bone marrow (femur), brain, cecum, cervix, coagulating glands, colon, duodenum, eyes with optic nerve, esophagus, extraorbital lacrimal glands, epididymides (modified Davidson’s solution), femur with knee joint, heart, ileum, jejunum (with Peyer’s patches), kidneys, larynx, liver, lungs, lymph nodes (axillary and mesenteric), mammary gland (male and female), nose (nasal cavity), ovaries (modified Davidson’s solution), oviducts, pancreas, parathyroid glands, pharynx, pituitary gland, prostate gland, rectum, salivary glands (mandibular and sublingual), sciatic nerve, seminal vesicles, skeletal muscle, spinal cord (cervical, thoracic and lumbar cord), spleen, sternum with marrow, stomach (fore stomach and glandular stomach), target organs, testes (modified Davidson’s solution), thymus, thyroid glands, trachea, urinary bladder, uterus, vagina
At necropsy additional liver samples (Processus papillaris and caudatus) were taken from all F0 animals per sex and group. The weight of these liver samples was recorded (per animal) for the females only. All samples were then frozen in liquid nitrogen and stored at -80°C.
The uteri of all cohabited female F0 parental animals were examined for the presence and number of implantation sites. The uteri of apparently non-pregnant animals or empty uterus horns were placed in 1% ammonium sulfide solutions for about 5 minutes in order to be able to identify early resorptions or implantations (SALEWSKI's method (1)). Then the uteri were rinsed carefully with 0.9% NaCl-solution. When the examinations were completed, the uteri were transferred to the Pathology Laboratory for further processing.
(1)SALEWSKI, E.: Färbemethode zum makroskopischen Nachweis von Implantationsstellen am Uterus der Ratte; Naunyn-Schmiedeberg’s Arch. Exp. Pathol. Pharmakol. 247, 367 (1964)

HISTOPATHOLOGY: Yes
For the following tissues fixation was followed by histotechnical processing, examination by light microscopy and assessment of findings: Adrenal glands, all gross lesions, bone marrow (femur), brain, cecum, cervix, coagulating glands, colon, duodenum, epididymides, heart, ileum, jejunum, kidneys, liver, lungs, lymph nodes (axillary and mesenteric), ovaries, oviducts, prostate gland, peyers patches, rectum, sciatic nerve, seminal vesicles, spinal cord (cervical, thoracic, lumbar), spleen, stomach (forestomach and glandular stomach), testes, thymus, thyroid glands, trachea, urinary bladder, uterus, vagina.
Special attention was given on stages of spermatogenesis in the male gonads.
Postmortem examinations (offspring):
- 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 without notable findings or abnormalities were discarded after their macroscopic evaluation. Animals with notable findings or abnormalities were evaluated on a case-by-case basis, depending on the type of finding.
Statistics:
- Blood parameters: For parameters with bidirectional changes: Non-parametric one-way analysis using KRUSKAL-WALLIS test. 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 (two-sided) for the hypothesis of equal medians For parameters with unidirectional changes: Pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) for the hypothesis of equal medians.
- Urinalysis parameters (apart from pH, urine volume, specific gravity, color and turbidity): Pairwise comparison of each dose group with the control
group using the WILCOXON-test (one-sided) for the hypothesis of equal medians.
- Urine pH, volume, specific gravity, color and turbidity: Non-parametric one-way analysis using KRUSKAL-WALLIS test. 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 (two-sided) for the hypothesis of equal medians. Urine color and turbidity are not evaluated statistically.
Statistics of pathology:
- Weight parameters: Non-parametric one-way 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 (two-sided) for the equal medians.
Statistics of the clinical examinations:
- Food consumption, body weight and body weight change (parental animals); Simultaneous comparison of all dose groups with the control group using the DUNNETT test (two-sided) for the hypothesis of equal means
Reproductive indices:
The pairing partners, the number of mating days until vaginal sperm was detected in the female animals, and the gestational status of the females were recorded for F0 breeding pairs.
For the males, mating and fertility indices were calculated for F1 litters according to the following formulas:
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 (%) = (number of males proving their fertility* / number of males placed with females) x 100
* defined by a female with implants in utero

For the females, mating, fertility and gestation indices were calculated for F1 litters according to the following formulas:
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 (%) = (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 (%) = (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
The total number of pups delivered and the number of liveborn and stillborn pups were noted, and the live birth index was calculated for F1 litters according to the following formula:
Live birth index (%) = (number of liveborn pups at birth / total number of pups born) x 100
The implantations were counted and the postimplantation loss (in %) was calculated according the following formula:
Postimplantation loss (%) = ((number of implantations – number of pups delivered) / number of implantations) x 100
Offspring viability indices:
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

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

Results and discussion

Results: P0 (first parental animals)

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
There were no test substance-related mortalities in any of the male and female parental animals in any of the groups.
No clinical signs or changes of general behavior, which may be attributed to the test substance, were detected in any male or female F0 generation parental animals during the whole study including gestation and lactation periods.
Several male and female animals of the high-dose group (90 mg/kg bw/day) showed salivation after treatment during mating (males), post-mating, gestation and lactation. This transient salivation for a few minutes immediately after treatment was likely to be induced by the unpleasant taste of the test substance or by local irritation of the upper digestive tract. It is not considered to be a sign of systemic toxicity.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Mean body weights of the male and female F0 generation parental animals in all test substance-treated groups were comparable to the concurrent control group during the entire study period.
The high-dose parental males had a statistically significantly lower body weight change during premating days 0 - 7 (about 37% below control).
The body weight change of the mid and low-dose parental males was comparable to the concurrent control group during the whole study period.
The high-dose parental females had statistically significantly lower body weight change during PND 0 - 4 (about 65% below control).
The mean body weight change of the high-dose parental females was comparable to the concurrent control group during premating and gestation. The body weight change of the mid and low-dose parental females was comparable to the concurrent control group during the whole study period.
Food consumption of the high-dose F0 males (90 mg/kg bw/day) was statistically significantly below control during premating days 0 - 7 (about 11%).
The mid and low-dose F0 males (30 and 10 mg/kg bw/day) did not show any test substancerelated changes in food consumption during the whole treatment period.
Food consumption of the female F0 generation parental animals in all test substance-treated groups (10, 30 and 90 mg/kg bw/day) was comparable to the concurrent control group during the entire study period.

DETAILED CLINICAL OBSERVATIONS (DCO) (PARENTAL ANIMALS)
Male and female animals of all dose groups (90, 30 and 10 mg/kg bw/d) did not show any abnormalities.

FUNCTIONAL OBSERVATIONAL BATTERY (FOB) (PARENTAL ANIMALS)
- Home cage observations: No test substance-related or spontaneous findings were observed in male and female animals of all test groups during the home cage observation.
- Open field observations: The open field observations did not reveal any test substance-related findings in male and female animals of all test groups.
- Sensorimotor tests/reflexes: There were no test substance-related findings in male and female animals of all test groups.Any deviations from "zero values" were equally distributed between test substance-treated groups and controls or occurred in single animals only. Therefore, these observations were considered as being incidental.
- Quantitative Parameters: No test substance-related impaired parameters were observed in male and female animals of all test groups.
- Motor activity measurement (MA): No statistically significant changes on motor activity data (summation of all intervals) was observed in the male and female animals of all dose groups in comparison to the concurrent control group.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
For all F0 parental males, which were placed with females to generate F1 pups, copulation was confirmed. Thus, the male mating index was 100% in all groups including the controls. Fertility was proven for all F0 parental males within the scheduled mating interval for F1 litter. Thus, the male fertility index was 100% in all groups including the controls.
The female mating index calculated after the mating period for F1 litter was 100% in all test groups. The mean duration until sperm was detected (GD 0) varied between 3.0 and 4.3 days without any relation to dosing. All sperm positive rats delivered pups. The fertility index was 100% in all test groups.
The mean duration of gestation was similar in all test groups (i.e. between 22.4 and 22.5 days). The gestation index was 100% in all test groups.
Implantation was not affected by the treatment since the mean number of implantation sites was comparable between all test substance-treated groups and the controls, taking normal biological variation into account (14.0 / 12.0 / 11.9 and 13.5 implants/dam in all test groups (0, 10, 30 and 90 mg/kg bw/day)). There were no statistically significant differences in post-implantation loss between the groups (4.1% / 7.7% / 8.8% / 5.1%). The mean number of F1 pups delivered per dam remained unaffected and was comparable between the test substance-treated groups and the control, taking normal biological variation into account (13.4 / 11.1 / 10.9* [* = p-level of significance ≤0.05] and 12.8 pups/dam at 0, 10, 30 and 90 mg/kg bw/day). The slight, statistically significant reduction in the mean number of F1 pups delivered per dam in the mid-dose females was a result of a small litter size in only two of the females of this group. If these litters are excluded, the remaining average litter size, 12.0, is not different than that of the other dose groups. Since the other litters were similar in size to those found in the control group, there did not appear to be a dose-response relationship. Furthermore, 10.9 pups/litter is within the range of the historical control data at this test facility. All together, these results suggest that the litter size reduction in dose group 2 is spontaneous in nature.
The rate of liveborn pups was also not affected by the test substance, as indicated by live birth indices of 100% control and low-dose group), 99.1% (mid-dose group) and 97.7% (high-dose group). Moreover, the number of stillborn pups was comparable between the groups.

ORGAN WEIGHTS (PARENTAL ANIMALS)
Absolute weights
When compared to the control group, the mean absolute weight of the liver was significantly increased in the high-dose male and female animals. All other mean absolute weight parameters of treated male and female animals did not show relevant differences when compared to the control group and are therefore considered to be within the normal range.
Relative weights
When compared to the control group, the mean relative weight of the liver was significantly increased in the high-dose male and female animals. All other mean relative organ weights of treated male and female parental animals did not show relevant differences when compared to the control group and are therefore considered to be within the normal range.
The significant absolute and relative weight increase in the liver of males and females in the high-dose group (90 mg/kg bw/day) was consistent with histopathological findings and was considered to be treatment-related.

CLINCAL PATHOLOGY (PARENTAL ANIMALS)
- Hematology: No treatment-related, adverse changes among hematological parameters were observed. In males of test group 3 (90 mg/kg bw/d), hematocrit and prothrombin (Hepatoquick’s test = HQT) values were decreased, whereas relative reticulocyte counts were increased. All mentioned parameter means were within historical control ranges (hematocrit 0.396-0.448 L/L; prothrombin time 30.2-37.9 sec; relative reticulocyte counts 0.9-2.5 %). Therefore, the changes were regarded as incidental and not treatment-related. In males of test group 1 (10 mg/kg bw/d) total white blood cell counts, as well as absolute lymphocyte counts, were decreased, but the alterations were not dose-dependent and therefore, the changes were regarded as incidental and not treatment-related. In males of test group 3 (90 mg/kg bw/d) absolute large unstained cell (LUC) counts were increased and
the mean was slightly above the historical control range (LUC 0.01-0.04 Giga/L). However, this was the only changed differential blood cell parameter in these rats. No change occurred in the females of the corresponding dose group. Therefore, the change was regarded as maybe treatment-related, but not adverse.
- Clinical chemistry: In males of test group 3 (90 mg/kg bw/d), urea, cholesterol and inorganic phosphate levels were increased, but chloride concentrations were decreased. Additionally, in females of the same test group urea levels were higher compared to controls.
- Urinalyses: In males of test group 3 (90 mg/kg bw/d), more phosphate crystals and transitional epithelial cells were observed in the urine sediment.
Additionally, in females of the same test group urine volume was higher. This alteration is per se not regarded as an adverse effect.

GROSS PATHOLOGY (PARENTAL ANIMALS)
All gross findings noted at necropsy in parental animals are regarded as incidental and spontaneous and are not related to treatment.

HISTOPATHOLOGY (PARENTAL ANIMALS)
Treatment-related findings were observed in the liver of parental male and female animals; in 5 out of 5 high-dose male and 5 out of 5 high-dose female animals centrilobular hypertrophy was observed. This finding correlated with a slight absolute and relative liver weight increase. However, this finding is regarded as adaptive and not adverse.
Two out of ten parental female animals of the high-dose group (90 mg/kg bw/day) showed a minimal follicular hypertrophy/hyperplasia in the thyroid gland when compared with the ten females of the control group. Due to the minimal magnitude and the low incidence of this change it was not regarded as treatment related and was considered to be an incidental finding.
All other findings noted in parental animals were either single observations, or were biologically equally distributed between controls and treated rats. All of them are considered to be incidental and/or spontaneous in origin.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Remarks:
parental
Effect level:
30 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Dose descriptor:
NOAEL
Remarks:
reproduction
Effect level:
90 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed at the highest dose tested.

Results: F1 generation

Details on results (F1)

VIABILITY (OFFSPRING)
The viability index indicating pup mortality during lactation (PND 0 - 4) varied between 94.4% (high-dose group), 90.0 (mid-dose group), 100.0 (low-dose group) and 99.3% (control), well within historical control values.
The reduction in pup numbers in the mid-dose group was amplified by PND 4. Eleven pups died/were cannibalized before PND 4, resulting in a small, but statistically significantly decreased, average live litter size of 9.7 (compared to 13.3 in the concurrent control group). A further 5 pups in the high dose group also died/were cannibalized intercurrently. All 16 pups originated from only two litters, nos. 123 and 133 (1 each in the 30 and 90 mg/kg bw/day dose groups, respectively), which showed signs of poor maternal care, such as findings of pups not fed/no milk in stomach and reduced nutritional condition in the pups. As the incidence of pup deaths is restricted to a small number of litters, lies within the normal range of biological variation inherent in the rat strain used and is not dose-dependent, the death of these pups was probably incidental.

CLINICAL SIGNS (OFFSPRING)
There were no test substance-related adverse clinical signs observed in any of the F1 generation pups of the different test groups.

BODY WEIGHT (OFFSPRING)
Mean body weights and mean body weight change of the male and female pups in all test substance-treated groups were comparable to the concurrent control group during the entire study period.
One female runt was seen in the high-dose group.
Two male runts were seen in the mid-dose group.
One female runt was seen in the low-dose group.
Five male and two female runts were seen in the control.

GROSS PATHOLOGY (OFFSPRING)
A few pups showed spontaneous findings at gross necropsy, such as post mortem autolysis, empty stomach and discolored liver lobe. These findings occurred without any relation to dosing and/or can be found in the historical control data at comparable or even higher incidences. None of these findings were considered to be associated with the test substance.

HISTOPATHOLOGY (OFFSPRING)
In one pup of the control group and one pup of mid-dose group (30 mg/kg bw/day), dissecting aneurysms of the ductus arteriosus and aorta correlated with macroscopic changes observed at necropsy. Since these findings occurred without a dose-dependency they were considered to be incidental and not related to treatment.

OTHER FINDINGS (OFFSPRING)
The mean number of delivered F1 pups per dam and the rates of liveborn and stillborn F1 pups were comparable between the dose groups. The respective values reflect the normal range of biological variation inherent in the strain used in this study. The slight, statistically significant reduction in the mean number of F1 pups delivered per dam in the mid-dose females was considered to be spontaneous in nature.

Effect levels (F1)

Dose descriptor:
NOAEL
Remarks:
developmental
Generation:
F1
Effect level:
90 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed at the highest dose tested.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion