(3-acetyloxy-2,2-dimethylpropyl) acetate

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 Jun 2018 - 14 Feb 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

developmental toxicity

Remarks:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 Jun 2018 - 14 Feb 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

29 Jul 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: ZH 472 redestilliert.
- Storage stability: until Jan 2020.

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature.
- Stability under test conditions: The stability of the test substance in solution in corn oil over a period of 7 days at room temperature was proven. the mixtures were stored no longer than this time period, the stability was guaranteed.
- Solubility and stability of the test substance in the solvent/vehicle: The test substance is completely miscible with corn oil Ph.Eur 8.0 and thus a solution. Therefore, the test-substance preparation was considered to be homogenous. Consequently, no further homogeneity analysis was performed.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test substance was applied as a solution. To prepare this solution, the appropriate amount of test substance was weighed out depending on the desired concentration. Then, Corn oil Ph.Eur. 8.0 was filled up to the desired volume and intensely mixed with a magnetic stirrer. The test substance preparations were produced weekly, at least.

FORM AS APPLIED IN THE TEST (if different from that of starting material): solution in corn oil.

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI(Han)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH.
- Females (if applicable) nulliparous and non-pregnant: yes.
- Age at study initiation: age at supply about 12 - 13 weeks (male animals), about 11 weeks (female animals).
- Weight at study initiation: mean weight 393 g (males) and 212 g (females).
- Fasting period before study: no.
- Housing: During pre-treatment: Polysulfonate cages Typ 2000P (H-Temp), floor area about 2065 cm2 (610 x 435 x 215 mm); supplied by TECHNIPLAST, Hohenpeißenberg, Germany During premating, mating, gestation, lactation, males after mating and females after weaning: Polycarbonate cages type III. Pregnant animals and their litters were housed together until PND 13 (end of lactation). For motor activity (MA) measurements the animals were housed individually in polycarbonate cages type III supplied by TECNIPLAST, Hohenpeißenberg, Germany with wire covers from Ehret, Emmendingen, Germany (floor area of about 800 cm2) and small amounts of bedding material Pregnant females were provided with nesting material (cellulose wadding) toward the end of gestation. For enrichment wooden gnawing blocks Lignocel® block large (supplied by J. Rettenmaier & Söhne GmbH + Co KG, Rosenberg, Germany) were added. In addition, in Polysulfonate cages large play tunnels (Art. 14153; supplied by PLEXX b.v., Elst, Netherlands) were added.
- Diet: ad libitum.
- Water: ad libitum.
- Acclimation period: 21 days.

ENVIRONMENTAL CONDITIONS
- The cages with the test animals were arranged on the racks in such a way that uniform experimental conditions (ventilation and light) were ensured. The animals were housed in a fully air-conditioned room.
- Temperature (°C): 20-24°C.
- Humidity (%): 45-65% for relative humidity.
- Air changes (per hr): 15 air changes per hour.
- Photoperiod (hrs dark / hrs light): The day/night cycle was 12 hours (12 hours light from 06.00- 18.00 h, 12 hours dark from 18.00-06.00 h).

IN-LIFE DATES: From: 19 Jun 2018 To: 10 Sep 2018.

Route of administration:

oral: gavage

Vehicle:

corn oil

Remarks:

Ph.Eur 8.0

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- The test substance was applied as a solution. To prepare this solution, the appropriate amount of test substance was weighed out depending on the desired concentration. Then, Corn oil Ph.Eur. 8.0 was filled up to the desired volume and intensely mixed with a magnetic stirrer. The test substance preparations were produced weekly, at least.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability of the test substance in corn oil Ph.Eur 8.0 was demonstrated over a period of 7 days at room temperature. As the mixtures were stored no longer than this time period, the stability was guaranteed.
The concentrations of the test substance in corn oil Ph.Eur 8.0 were found to be in the range of 99.5 to 105.4% of the nominal concentrations. These results demonstrated the correctness of the concentrations of the test substance in corn oil Ph.Eur 8.0 being within the specification of the test facility 90-110%.

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: overnight (from about 16.00 h until 06.30 - 09.00 h of the following morning) for a maximum of 2 weeks
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy (gestation day (GD) 0)
- After successful mating each pregnant female was caged (how): Pregnant females were provided with nesting material (cellulose wadding) toward the end of gestation.

Duration of treatment / exposure:

males: 29 days; females: 61 days
The duration of treatment covered a 2-week premating period and mating in both sexes (mating pairs were from the same dose group) as well as entire gestation and lactation period in females up to one day prior to the day of schedule sacrifice of the animals. Additional treated but not mated animals (recovery animals) to groups of 5 male and 5 female animals at nominal doses of 0 and 1000 mg/kg bw/d was maintained for a subsequent period of at least 14 days of no test substance administration in order to observe reversibility of the findings.

Frequency of treatment:

daily

Duration of test:

77 days

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Remarks:

Test group 1

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Remarks:

Test group 2

Dose / conc.:

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

Remarks:

Test group 3

Dose / conc.:

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

Remarks:

Test group 11 (Recovery animals)

No. of animals per sex per dose:

Test group 0, 1, 2, 3: 10 animals per sex per dose
Test group 10, 11 (Recovery animals): 5 animals per sex per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: In accordance with the guideline, three doses were chosen which were applied in two- to four- fold intervals and it was tested up to the limit dose of 1000 mg/kg bw/day.
- Fasting period before blood sampling for clinical biochemistry: The adults were fastened before the blood sampling. A fasting period (withdrawal of food) of about 16 to 20 hours was applied.
- Post-exposure recovery period in satellite groups: yes. Additional treated but not mated animals (recovery animals) to groups of 5 male and 5 female animals at nominal doses of 0 mg/kg bw/d (test group 10) and 1000 mg/kg (test group 11) bw/d were maintained for a subsequent period of at least 14 days of no test substance administration in order to observe reversibility of the findings.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes.
- Time schedule: daily.
- Mortality: A check for moribund and dead animals was made twice daily on working days and once daily on Saturdays, Sundays and public holidays. If animals were in a moribund state, they were sacrificed and necropsied.
- Clinical observations: A cageside examination was conducted at least once daily for any signs of morbidity, pertinent behavioral changes and/or signs of overt toxicity. All animals were checked daily for any abnormal clinical signs before the administration as well as within 2 hours and within 5 hours after the administration. Abnormalities and changes were documented for each animal. The parturition and lactation behavior of the dams was generally be evaluated in the morning in combination with the daily clinical inspection of the dams. Only particular findings (e.g. disability to deliver or umbilical cord not cut) were documented on an individual dam basis. On weekdays (except Saturdays, Sundays 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 to be the 24-hour period from about 15:00 h of one day until about 15:00 h of the following day.

DETAILED CLINICAL OBSERVATIONS: Yes.
- Time schedule: prior to administration and weekly thereafter.
- 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: abnormal behavior in handling, fur, skin, posture, salivation, respiration, activity/arousal level, tremors, convulsions, abnormal movements, gait abnormalities, lacrimation, palpebral closure, exophthalmos, assessment of the feces discharged during the examination (appearance/ consistency), assessment of the urine discharged during the examination, pupil size.

BODY WEIGHT: Yes.
- Time schedule for examinations: before the beginning administration, during administration at day 0 and weekly thereafter.
- Body weight of the male and female parental animals in the main groups as well as the males and females of recovery groups 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 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), PNDs 4, 7, 10 and 13.
-- Females showing no positive evidence of sperm in the vaginal smear were weighed once a week during this mating interval as were the males (for the calculation of the administration volume)
-- Females without litter and after weaning (PND 13) were weighed once a week (for the calculation of the administration volume).

FOOD CONSUMPTION: Yes.
- Time schedule: weekly.
- Generally, food consumption was determined once a week for male and female parental animals of the main groups as well as the males and females of recovery groups, with the following exceptions:
-- Food consumption was not be determined after the 2nd premating week (male parental animals) and during the mating period (male and female parental animals in the main groups).
-- Food consumption of the females with evidence of sperm was determined for GD 0-7, 7-14 and 14-20.
-- Food consumption of the females which gave birth to a litter was determined for PNDs 1-4, 4-7, 7-10 and 10-13.
- 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.

WATER CONSUMPTION: Yes.
- Time schedule for examinations: daily.
- Drinking water consumption was monitored by daily visual inspection of the water bottles for any changes in volume.

POST-MORTEM EXAMINATIONS: Yes.
- Sacrifice on day 62
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.
- Organs examined: The tissues indicated in Table 4 were prepared for microscopic examination and weighed, respectively.

OTHER:

HAEMATOLOGY: Yes.
- Time schedule for collection of blood: for main groups at day 30 (males) and day 50 (females), for recovery groups at day 30/48 (males) at day 62/77 (females).
- Anaesthetic used for blood collection: Yes (isoflurane).
- Animals fasted: Yes.
- How many animals: 5 males and 5 females.
- Parameters checked in table 1 were examined.
- In the morning blood was taken from the retro-bulbar venous plexus from fasted animals. The animals were anaesthetized using isoflurane. The blood sampling procedure and subsequent analysis of blood and serum samples were carried out in a randomized sequence. The assays of blood and serum parameters were performed under internal laboratory quality control conditions with reference controls to assure reliable test results. The results were expressed in International System (SI) units. The parameters listed were examined in the first 5 surviving parental males per group at termination and in the first 5 females with litters (in order of delivery) per group at PND 14 and in the recovery groups at the end of the administration period and at the end of the recovery period.

CLINICAL CHEMISTRY: Yes.
- Time schedule for collection of blood: for main groups at day 30 (males) and day 50 (females), for recovery groups at day 30/48 (males) at day 62/77 (females).
- Animals fasted: Yes.
- How many animals: 5 males and 5 females.
- Parameters checked in table 2 were examined.
- In the morning blood was taken from the retro-bulbar venous plexus from fasted animals. The animals were anaesthetized using isoflurane. The blood sampling procedure and subsequent analysis of blood and serum samples were carried out in a randomized sequence. The assays of blood and serum parameters were performed under internal laboratory quality control conditions with reference controls to assure reliable test results. The results were expressed in International System (SI) units. The parameters listed were examined in the first 5 surviving parental males per group at termination and in the first 5 females with litters (in order of delivery) per group at PND 14 and in the recovery groups at the end of the administration period and at the end of the recovery period.

NEUROBEHAVIOURAL EXAMINATION: Yes; functional observational battery and motor activity assessment.
- Time schedule: FOB and MA at study day 28 (males) and day 57 (females)
FUNCTIONAL OBSERVATIONAL BATTERY:
- A functional observational battery (FOB) was performed in the first five parental male animals per test group and the first five surviving females with litter (in order of delivery) of all main test groups 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.
-Home cage observations: The animals were observed in their closed home cages; during this period 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: Posture, Tremors, Convulsions, Abnormal movements, Gait, Other findings.
- Open field observations: The animals were transferred to a standard arena (50 × 50 cm with sides of 25 cm height) and observed for at least 2 minutes. The following parameters were examined: behavior on removal from the cage, fur, skin, salivation, nasal discharge, lacrimation, eyes/pupil size, posture, palpebral closure, respiration, tremors, convulsions, abnormal movements/stereotypes, gait, activity/arousal level, feces excreted within 2 minutes (appearance/ consistency), urine excreted within 2 minutes (amount/color), rearing within 2 minutes, other findings.
- Sensory motor tests/ reflexes: The animals were then removed from the open field and subjected to following sensory motor or reflex tests: Reaction to an object being moved towards the face (approach response), touch sensitivity (touch response), vision (visual placing response), pupillary reflex, pinna reflex, audition (auditory startle response), coordination of movements (righting response), behavior during handling, vocalization, pain perception (tail pinch), other findings, grip strength of forelimbs, grip strength of hindlimbs, landing foot-splay test.
MOTOR ACTIVITY ASSESSMENT:
Motor activity (MA) was also measured from 14:00 h onwards on the same day as the FOB was performed in the first five parental males and the first five surviving females with litter (in order of delivery) per main group. 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 with a small amount of bedding for the duration of the measurement. Eighteen beams were allocated per cage. The numbers of beam interrupts was counted over 12 intervals of 5 minutes per interval. The sequence in which the animals were placed in the cages was selected at random. On account of the time needed to place the rats in the cages, the starting time was "staggered" for each animal. The measurement period began when the 1st beam was interrupted and finished exactly 1 hour later. No food or water was offered to the animals during these measurements and the measurement room was darkened after the transfer of the last animal. The program required a file name for the measured data to be stored. This name consisted of the reference number and a serial number.

THYROID HORMONES:
- Parameters checked in table 3 were examined.
- Blood samples were taken from all surplus pups per litter at PND 4 as well as one male and one female pup per litter at PND 13 by decapitation under isoflurane anesthesia. Additionally, blood samples from all dams at PND 14 and all males at termination of the main groups as well as all males and females after the administration period and at termination of the recovery groups were taken by puncturing the retrobulbar venous plexus under isoflurane anesthesia. The adults were fastened before the blood sampling. All generated serum samples were frozen at -80°C until measurement. Blood samples from the adult males and the PND 13 pups were assessed for serum levels for thyroid hormones (T4 and TSH). The concentrations of TSH were determined by radioimmunoassay (RIA), using commercially available RIA test kits and a Gamma-Counter (LB 2111, Berthold, Germany). T4 Elisa was measured with a Sunrise MTP-reader, Tecan AG, Maennedorf, Switzerland, and evaluated with the Magellan-Software of the instrument producer.

ESTROUS CYCLE:
- For all females of the pool estrous cycle normality was evaluated before the beginning of the administration. In all parental females in the main groups in the premating phase, estrous cycle length and normality was evaluated by preparing vaginal smears during a minimum of 2 weeks prior to premating, mating and throughout cohabitation until there is evidence of sperm in the vaginal smear. Additionally, on the day of scheduled sacrifice, the estrous status was also be determined in all female F0 main groups rats.

MALE REPRODUCTION DATA:
- 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 (for formulas see "Any other information on materials and methods").

FEMALE REPRODUCTION AND DELIVERY DATA:
- The pairing partners, the number of mating days until vaginal sperm were detected and gestational status were recorded for F0 females.
- For the females, mating, fertility and gestation indices were calculated for F1 litters (for formulas see "Any other information on materials and methods").

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes.
Examinations included:
- Uterus weight: Yes.
- Number of implantations: Yes.

Fetal examinations:

Fetal examinations are not applicable to OECD 422

LITTER/PUB DATA:
- 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. Dead pups were evaluated by the methods, which are described in detail in “Necropsy observations”. The number and percentage of dead pups on the day of birth (PND 0) and of pups dying between PND 1-4, 5-7 and 8-13 were determined. Pups, which died accidentally or had to be sacrificed due to maternal death, were not included in these calculations. The number of live pups/litter was calculated on the day of birth (PND 0), and on lactation days 4, 7 and 13. Furthermore, viability and survival indices were calculated (for formulas see "Any other information on materials and methods").
- 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. Later, during the course of lactation, this initial sex determination was followed up by surveying the external appearance of the anogenital region and the mammary line. The sex of the pups was finally confirmed at necropsy. The sex ratio was calculated at PND 0 and PND 13 (for formulas see "Any other information on materials and methods").
- 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) as well as on PND 4 (before standardization), 7 and 13. 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). In the summary tables 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.
- Nipple/areola anlagen: All surviving male pups were examined for the presence of nipple/areola anlagen on PND 13 of the lactation phase. The number of nipple/areola anlagen was counted.
- Anogenital distance: Anogenital distance (AGD; defined as the distance from the anus [center of the anal opening] to the base of the genital tubercle) measurements was done blind to treatment in a randomized order, using a measuring ocular, on all live male, female and uncertain pups on day 1 after birth. The anogenital index were calculated (for formulas see "Any other information on materials and methods").
- Pup necropsy observations: On PND 4, as a result of standardization, the surplus pups or 2 preferably female pups per litter, respectively, was sacrificed under isoflurane anesthesia by decapitation. Blood was sampled for determination of thyroid hormone concentrations. After sacrifice, the pups were examined externally and eviscerated, and their organs were assessed macroscopically. On PND 13, one selected male and one female pup per litter was sacrificed under isoflurane anesthesia by decapitation. Blood was sampled for determination of thyroid hormone concentrations. Thyroid glands/parathyroid glands were fixed in neutral buffered 4% formaldehyde solution and was transferred to the Pathology Laboratory for possible further processing. The remaining pups were sacrificed under isoflurane anesthesia with CO2. After sacrifice, all pups were examined externally and eviscerated, and their organs were assessed macroscopically. All stillborn pups and all pups that died before day 13 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-bycase basis, depending on the type of finding noted.

Statistics:

see table 5

Indices:

- For the males, mating and fertility indices were calculated for F1 litters (for formulas see "Any other information on materials and methods").
- For the females, mating, fertility and gestation indices were calculated for F1 litters (for formulas see "Any other information on materials and methods").
- For the pubs viability index, survival index (for formulas see "Any other information on materials and methods").

Historical control data:

Historical control data to allow comparison with concurrent controls were provided by the test facility for the following parameters: maternal body weights, mating data, delivery data, litter data, pup weights, pup necropsy observations, anogenital distance and anogenital index, presence of areolas/nipples, clinical pathology, organ weight (epididymides, prostate).

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Males and females and recovery animals:
Salivation within 2 hours after administration was observed from slight to moderate in test group 3 (1000 mg/kg bw/d) in all male and 9 female animals during premating, in all male and 4 female animals during mating phase, in 3 male animals during post-mating phase as well as in all male and female animals of recovery group 11 (1000 mg/kg bw/d). In test group 2 (300 mg/kg bw/d) one male animal during mating period and one male animal during post-mating salivation within 2 hours after administration was observed. The salivation observed only shortly after treatment was assessed as treatment-related but not adverse. It was considered to be related to a slight local reaction to the test substance preparation by a bitter or bad taste. No test substance-related, adverse findings were observed in male and female animals of test groups 1-3 (100, 300, and 1000 mg/kg bw/d) and recovery group 11 (1000 mg/kg bw/d).

Females during gestation:
Salivation shortly after treatment (<2 hours after treatment) was observed from slight to moderate in all female animals of test group 3 (1000 mg/kg bw/d) during gestation. The salivation observed only shortly after treatment was assessed as treatment-related but not adverse. It was considered to be related to a slight local reaction to test substance preparation by a bitter or bad taste. No test substance-related, adverse findings were observed in test groups 1-3 (100, 300, and 1000 mg/kg bw/d) during gestation.

Females during gestation:
Salivation shortly after treatment (<2 hours after treatment) was observed from slight to moderate in all female animals of test group 3 (1000 mg/kg bw/d) during gestation. The salivation observed only shortly after treatment was assessed as treatment-related but not adverse. It was considered to be related to a slight local reaction to test substance preparation by a bitter or bad taste. No test substance-related, adverse findings were observed in test groups 1-3 (100, 300, and 1000 mg/kg bw/d) during gestation.

Clinical observations for females during lactation
Slight salivation shortly after treatment (<2 hours after treatment) was observed in all female animals of test groups 3 (1000 mg/kg bw/d) during lactation. The salivation observed only shortly after treatment was assessed as treatment-related but not adverse. It was considered to be related to a slight local reaction to test substance preparation by a bitter or bad taste. No test substance-related, adverse findings were observed in test groups 1-3 (100, 300, and 1000 mg/kg bw/d) during gestation.

Detailed clinical observations:
In the detailed clinical observations on study days 0, 7, 14, 21 and 28 in parental males and females and additionally, on study days 35, 42, 49 and 56 in parental female animals and in the recovery period of males and females (as well as in the recovery females on study days 63 and 70) no adverse findings were observed.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

No animal died prematurely in the present study.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No test substance-related changes in mean body weights and body weight gain were observed for male and female animals of test groups 1-3 (100, 300 and 1000 mg/kg bw/d) when compared to the control group. Slightly reduced body weight was observed in male animals of recovery group 11 (1000 mg/kg bw/d) only on study day 13 during the in-life phase. Mean body weight gain was significantly decreased in male animals of recovery group 11 (1000 mg/kg bw/d) during the recovery phase between study days 38 to 45 only. Since the body weight of male animals in the recovery group 11 (1000 mg/kg bw/d) was comparable to the current control, the findings were assessed to be related to treatment, but not adverse.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption of the males and females in all dose groups (100, 300 and 1000 mg/kg bw/d) including recovery group was not influenced by the treatment throughout the entire study period.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

no effects observed

Description (incidence and severity):

No test substance-related findings were observed.

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Main groups:
At the end of the administration period in male rats of the main test group 3 (1000 mg/kg bw/d) prothrombin time (Hepatoquick’s test, HQT) was significantly reduced (see table 6). This alteration combined with the cholesterol increase in the same individuals was regarded as treatment-related and adverse.

Recovery group:
At the end of the administration period of the males in the recovery group 11 (1000 mg/kg bw/d) absolute basophil cell counts an in females of the same test group absolute and relative large unstained cell (LUC) counts were significantly increased, but all values were within historical control ranges (males, absolute basophils 0.00-0.02 Giga/L; females, absolute LUC 0.01-0.02 Giga/L, relative LUC 0.2-0.6 %).
At the end of the two-week recovery phase in males of test group 11 (1000 mg/kg bw/d) hemoglobin values were significantly increased, but th values were within the historical control range (males, hemoglobin 8.6-9.5 mmol/L). Therefore, all mentioned changes in rats of test group 11 were regarded as incidental and not treatment-related.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

At the end of the administration period in males of the main test group 3 (1000 mg/kg bw/d) as well as in males of the recovery test group 11 (1000 mg/kg bw/d) cholesterol values were significantly increased (see table 7 and table 8). This change in combination with the reduced prothrombin time was regarded as treatment-related and adverse.
Additionally, after the administration period in males of the recovery test group 11 (1000 mg/kg bw/d) albumin values were significantly increased. The mean was above the historical control range (males, albumin 34.09-37.97 g/L). However, this change was not present in the equally dosed males of the main test group 3 (1000 mg/kg bw/d). Therefore, this change in the recovery males after the administration was regarded as incidental and not treatment-related.
After the two-week recovery period in males of test group 11 (1000 mg/kg bw/d) sodium levels were significantly increased. The values of males in the control group 10 as well as in the test group 11 were above the historical control range (males, sodium 140.0-148.1 mmol/L). Because no electrolyte value change occurred after the administration period, the solely change of sodium after the recovery period was regarded as incidental and not treatment-related. After the recovery, in females of test group 11 (1000 mg/kg bw/d) alkaline phosphatase (ALP) activities were significantly decreased. The ALP means in females of the controls and test group 11 were below the historical control range (females, ALP 0.47-
0.76 μkat/L). The reason for this may be that the individuals of the historical controls were 6 weeks younger compared to the rats of the present study. The most probable cause for lower ALP levels is a growth retardation. However, females of test group 11 did not have significant lower terminal body weights compared to the study controls. Therefore, the statistically significant lower ALP activities in females of test group 11 were regarded as incidental and not treatment-related.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

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, without a dose-response relationship or occurred in single animals only, these observations were considered as incidental. There were no test substance-related findings in male and female animals of all test groups. Following non-significant alterations were assessed as spontaneous in nature and not related to treatment, because of missing dose-dependency: The foot splay test showed decreased results in males of test group 1 (100 mg/kg bw/d; -11%).

Motor activity measurement:
Regarding the individual intervals as well as the overall motor activity, no test substance related deviations were noted for male and female animals.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Main groups:
- Absolute organ weights: When compared to control group 0 (set to 100%), the mean absolute weights of epidymides, prostate and thymus were significantly increased or decreased in one or more test groups (see table 9). All other mean absolute weight parameters did not show significant differences when compared to the control group 0.
- Relative organ weights When compared to control group 0 (set to 100%), the mean relative prostate weights were significantly increased in all treatment groups (see table 10). All other mean relative weight parameters in males and all mean relative weight parameters in females did not show significant differences when compared to the control group 0.
- The mean absolute and relative prostate weights of males in test group 1 (1.13 g, 0.27%), test group 2 (1.19 g, 0.29%), and test group 3 (1.20 g, 0.29%) were within the range of historical control data (1.01 - 1.274 g, 0.244 - 0.315%), whereas the mean absolute and relative prostate weights of control males (0.96 g, 0.24%) were below the historical control range. Furthermore, there were no treatment-related histopathological findings in the prostate. Therefore, the statistical increase of prostate weights in males of all treatment groups can be explained by the comparable low prostate weight in concurrent control males. The mean absolute weight (1.24 g) of epididymides in males of test group 3 was slightly above the historical control range (1.102 – 1.230 g). But the mean relative weight of epididymides (0.30%) was within the historical control range (0.272 – 0.32%) and there were no treatment-related histopathological findings. Therefore, the slightly increased mean absolute weight of epididymides was assessed as incidental. The mean absolute thymus weight was significantly decreased in females of test group 1. As there was no dose-response relationship, this weight decrease was regarded to be incidental.

Recovery group:
- Absolute organ weights: When compared to control group 10 (set to 100%), all mean absolute weight parameters did not show significant differences when compared to the control group 10.
- Relative organ weights/ Recovery group: When compared to control group 0 (set to 100%), the mean relative heart weight was significantly increased in males of test group 11 (see table 11).
- All other mean relative weight parameters in males and all mean relative weight parameters in females did not show significant differences when compared to the control group 10. The slight increase of the relative heart weight in males of test group 11 is related to the slightly but not significantly decreased (-5.1%) terminal body weight in this test group. In addition, there were no significant weight changes of the heart weight in males of the main groups.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Main groups:
- All findings were single or few observations that were considered to be incidental or spontaneous in origin and without any relation to treatment.
- Fertility: The female animal No 129 (test group 2, 300 mg/kg bw/d), which was not pregnant as well as the male mating partner (No. 029) did not show gross lesions.

Recovery group:
- The yellow focus in the vagina in one female of test group 11 (1000 mg/kg bw/d) was a single observation that was considered to be incidental in origin and without any relation to treatment.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Main groups:
- All findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.
- The stages of spermatogenesis in the testes of males of the high dose test group were comparable to those of the controls. In high dose females the different stages of functional bodies in the ovaries were present and comparable to the control animals.
- Fertility: The female animal (No. 129, test group 2, 300 mg/kg bw/d), which was not pregnant as well as the male mating partner (No. 029) did not show relevant histopathological findings.

Recovery group:
- The macroscopically diagnosed yellow focus in the vagina in one female of test group 11 (1000 mg/kg bw/d) correlated with a cyst, histopathologically that was considered to be incidental.
- The vagina of all other females, all other organs of females and all organs of all male animals were not examined, histopathologically.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Number of abortions:

not examined

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

- Postimplantation loss: The postimplantation loss was 5.7% in test group 0 (control), 4.5% in test group 1 (100 mg/kg bw/d), 10.1% in test group 2 (300 mg/kg bw/d) and 3.8% in test group 3 (1000 mg/kg bw/d). These findings 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 (0.0 – 18.1%).

Total litter losses by resorption:

not examined

Early or late resorptions:

not examined

Dead fetuses:

not examined

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

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

Other effects:

no effects observed

Description (incidence and severity):

Estrous cycle:
- Estrous cycle data revealed regular cycles in the rearing F1 females of all test groups including the control. In the different test groups (0-3) the mean number of estrous cycle ranged from 2.5 to 2.8 days and the estrous cycle duration ranged from 3.9 to 4.0 days. Since no treatment-related effect was observed the estrous cycle determination was not carried out in the recovery female animals during the recovery period.

Female reproduction and delivery data:
- Female mating index: The female mating index calculated after the mating period for F1 litter varied between 90% in test group 2 (300 mg/kg bw/d) and 100% in test group 0 (control), 1 (100 mg/kg bw/d) and 3 (1000 mg/kg bw/d). The mean duration until sperm was detected (GD 0) was 2.2 days for test group 0 (control), 3.9 days for test group 1 (100 mg/kg bw/d), 3.3 days for test group 2 (300 mg/kg bw/d), as well as 2.4 days for test groups 3 (1000 mg/kg bw/d). These values 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 (1.6 – 5.8 mating days until day 0 p.c.). This includes also the statistically significant value of 3.3 days in test group 2 (300 mg/kg bw/d).
- Female fertility index: All sperm positive females had implants. Thus, the female fertility index was 100% in all test groups 0-3.
- Live birth indices: The rate live birth indices were 100% in test groups 0 and 1 (100 mg/kg bw/d), 98.1% in test group 2 (300 mg/kg bw/d) and 99.2% in test group 3 (1000 mg/kg bw/d). No treatment related effect was observed.
- Pup number and status at delivery: The mean number of delivered F1 pups per dam was equally distributed among test groups 0, 1, 2 and 3 ranging in mean from 11.7 to 12.1 pups per litter. No significant deviations occurred.
- Pup clinical observations: One male pup (animal no. 139-06) of test group 3 (1000 mg/kg bw/day) and two male pups (animal no. 127-05 and 128-06) of test group 2 (300 mg/kg bw/d) were stillborn. One male pup (animal no. 106-05) of control group was found dead on PND 1. One male pup (animal no. 140-09) of test group 3 (1000 mg/kg bw/day) was cannibalized on PND3. These findings in its observe incidences are within the expected viability and was assessed as spontaneous in nature.

Dose descriptor:

LOAEL

Remarks:

general systemic toxicity

Effect level:

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

Based on:

test mat.

Basis for effect level:

clinical biochemistry

haematology

Dose descriptor:

NOAEL

Remarks:

general systemic toxicity

Effect level:

300 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: no adverse effect observed at this dose

Dose descriptor:

NOAEL

Remarks:

reproductive performance and fertility

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: no adverse effect observed up to the highest tested dose.

Abnormalities:

no effects observed

Fetal body weight changes:

not examined

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

Pup viability index/mortality: The viability index indicating pup mortality during postnatal days (PND) 0-4 varied between 99.3% in test group 0 (control) and test group 3 (1000 mg/kg bw/d) and 100% in test groups 1 (100 mg/kg bw/d) and 2 (300 mg/kg bw/d). No treatment related effect as observed. The survival index indicating pup mortality on PND 4 – 13 was 100% in all test groups.

Changes in sex ratio:

effects observed, non-treatment-related

Description (incidence and severity):

- Sex ratio: The sex distribution and sex ratios of live F1 pups on the day of birth and PND 13 did not show substantial differences between the control and the test substance-treated groups; slight differences were regarded to be spontaneous in nature.
- Anogenital distance and anogenital distance index of male and female pups was not influenced in all test groups.
- The apparent number and percentage of male pups having areolae was not influenced by the test substance when examined on PND 13.

Changes in litter size and weights:

effects observed, non-treatment-related

Description (incidence and severity):

Pup body weight data: Mean pup body weights of all pups in all test groups were comparable to the control group. Two female runts were seen in the control group on PND 1. Three male and 1 female runts were seen in test group 1 (100 mg/kg bw/d) and one male runt was seen in test group 2 (300 mg/kg bw/d) on PND 1. Three male runts were seen in test group 3 (1000 mg/kg bw/d) on PND 1. All values were within the range of the biological variation inherent in the strain of rats used for this study.

Changes in postnatal survival:

no effects observed

Description (incidence and severity):

Pup viability index/mortality: The viability index indicating pup mortality during postnatal days (PND) 0-4 varied between 99.3% in test group 0 (control) and test group 3 (1000 mg/kg bw/d) and 100% in test groups 1 (100 mg/kg bw/d) and 2 (300 mg/kg bw/d). No treatment related effect as observed. The survival index indicating pup mortality on PND 4 – 13 was 100% in all test groups.

External malformations:

no effects observed

Skeletal malformations:

not examined

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

One male pup of test group 3 (1000 mg/kg bw/d) showed an empty stomach and large liver. One male pup of test group 2 (300 mg/kg bw/d) showed a small testis. One male pup of test group 1 (100 mg/kg bw/d) showed a misshapened liver. One male pup of the control group showed an absent liver lobe. These findings occurred without any relation to dosing and were assessed as spontaneous in nature and not related to treatment. All other F1 pups of any test group (0-3) did not show adverse findings during necropsy.

Other effects:

no effects observed

Description (incidence and severity):

Thyroid hormones:
- In male and female pups at PND13 (main test groups 21, 22 and 23; 100, 300 and 1000 mg/kg bw/d), no treatment-related alterations of T4 and TSH levels were observed.

Dose descriptor:

NOAEL

Remarks:

developmental toxicity (F1 progeny)

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed up to the highest tested dose

Remarks on result:

other: fetus analysis is not applicable to OECD TG 422, pubs were investigated instead

Abnormalities:

no effects observed

Developmental effects observed:

no

Table 6: Haematology - males

 

 

		Test Group 0/ M 0 mg/kg bw/d	Test Group 1/ M 100 mg/kg bw/d	Test Group 2/ M 300 mg/kg bw/d	Test Group 3/ M 1000 mg/kg bw/d
RBC	Mean	8.73 k	8.53	8.87	9.03
[tera/L]	S.d.	0.23	0.28	0.38	0.17
day 30	N	5	5	5	5
	Median	8.71	8.50	8.95	9.06
	Deviation Vs Control [%]		-2.29	1.67	3.41
HGB	Mean	9.1 k	9.1	9.0	9.2
[mmol/L]	S.d.	0.1	0.1	0.3	0.3
day 30	N	5	5	5	5
	Median	9.0	9.1	9.2	9.3
	Deviation Vs Control [%]		0.4	-0.7	1.1
HCT	Mean	0.434 k	0.427	0.428	0.437
[L/L]	S.d.	0.002	0.005	0.009	0.008
day 30	N	5	5	5	5
	Median	0.434	0.427	0.426	0.436
	Deviation Vs Control [%]		-1.430	-1.384	0.738
MCV	Mean	49.7 k	50.1	48.2	48.4
[fL]	S.d.	1.3	1.4	2.1	0.4
day 30	N	5	5	5	5
	Median	49.5	50.7	46.9	48.5
	Deviation Vs Control [%]		0.8	-2.9	-2.6
MCH	Mean	1.04 k	1.07	1.02	1.02
[fmol]	S.d.	0.04	0.04	0.06	0.03
day 30	N	5	5	5	5
	Median	1.03	1.07	0.99	1.01
	Deviation Vs Control [%]		2.68	-2.11	-2.30
MCHC	Mean	20.96 k	21.37	21.20	21.11
[mmol/L]	S.d.	0.33	0.26	0.48	0.61
day 30	N	5	5	5	5
	Median	20.80	21.30	21.21	20.85
	Deviation Vs Control [%]		1.97	1.15	0.72
RETA	Mean	149.4 k	160.6	155.2	154.5
[giga/L]	S.d.	25.5	14.5	22.1	30.7
day 30	N	5	5	5	5
	Median	138.5	163.2	165.3	154.0
	Deviation Vs Control [%]		7.5	3.9	3.4
PLT	Mean	594 k	656	697	703
[giga/L]	S.d.	90	85	74	77
day 30	N	5	5	5	5
	Median	590	653	714	698
	Deviation Vs Control [%]		10	17	18
HQT	Mean	35.7 v	35.5	37.3	32.7 **
[sec]	S.d.	0.7	1.5	3.1	1.0
day 30	N	5	5	5	5
	Median	35.6	34.9	36.1	32.7
	Deviation Vs Control [%]		-0.6	4.5	-8.4

 

Statistic Profile = Kruskal-Wallis + Wilcoxon test (two-sided) * p<=0.05, ** p <=0.01, X = Group excluded from statistics k=KRUSKALL-WALLIS; v=KRUSKALL-WALLIS-WILCOX

Table 7: Clinical chemistry - males

 

 

		Test Group 0/ M 0 mg/kg bw/d	Test Group 1/ M 100 mg/kg bw/d	Test Group 2/ M 300 mg/kg bw/d	Test Group 3/ M 1000 mg/kg bw/d
UREA	Mean	4.44 k	4.42	4.43	5.26
[mmol/L]	S.d.	0.36	0.51	0.38	0.32
day 30	N	5	5	5	5
	Median	4.29	4.47	4.27	5.22
	Deviation Vs Control [%]		-0.59	-0.23	18.27
CREA	Mean	21.9 k	22.9	21.3	24.0
[µmol/L]	S.d.	2.6	2.2	1.2	1.5
day 30	N	5	5	5	5
	Median	21.2	22.8	21.5	24.2
	Deviation Vs Control [%]		4.6	-2.8	9.6
GLUC	Mean	7.40 k	6.92	6.21	6.36
[mmol/L]	S.d.	1.15	0.30	0.73	0.67
day 30	N	5	5	5	5
	Median	7.63	6.94	6.00	6.45
	Deviation Vs Control [%]		-6.51	-16.05	-14.05
TBIL C	Mean	1.57 k	1.47	1.33	1.68
[µmol/L]	S.d.	0.31	0.14	0.13	0.29
day 30	N	5	5	5	5
	Median	1.74	1.43	1.34	1.62
	Deviation Vs Control [%]		-6.66	-15.22	6.57
TBA	Mean	14.6 k	12.0	10.6	21.0
[µmol/L]	S.d.	8.5	4.8	6.6	8.4
day 30	N	5	5	5	5
	Median	11.2	12.3	6.8	22.2
	Deviation Vs Control [%]		-17.8	-27.4	43.4
TPROT	Mean	63.07 k	64.56	64.50	65.98
[g/L]	S.d.	1.10	0.62	1.25	2.36
day 30	N	5	5	5	5
	Median	63.36	64.36	64.54	66.52
	Deviation Vs Control [%]		2.37	2.27	4.61
ALB	Mean	36.92 k	37.69	37.26	37.74
[g/L]	S.d.	1.36	0.98	0.51	0.96
day 30	N	5	5	5	5
	Median	36.97	37.53	37.48	37.70
	Deviation Vs Control [%]		2.09	0.93	2.23
GLOB	Mean	26.15 k	26.87	27.24	28.24
[g/L]	S.d.	0.59	0.82	1.14	1.42
day 30	N	5	5	5	5
	Median	26.05	26.83	26.89	28.67
	Deviation Vs Control [%]		2.76	4.17	7.98
CHOL	Mean	1.77 v	2.00	2.17	2.80 **
[mmol/L]	S.d.	0.31	0.32	0.35	0.38
day 30	N	5	5	5	5
	Median	1.68	1.89	2.10	2.79
	Deviation Vs Control [%]		12.51	22.32	58.06
TRIG	Mean	0.77 k	0.83	0.72	0.75
[mmol/L]	S.d.	0.21	0.36	0.15	0.13
day 30	N	5	5	5	5
	Median	0.83	0.62	0.70	0.78
	Deviation Vs Control [%]		7.55	-5.99	-2.86

 

Statistic Profile = Kruskal-Wallis + Wilcoxon test (two-sided) * p<=0.05, ** p <=0.01, X = Group excluded from statistics k=KRUSKALL-WALLIS; v=KRUSKALL-WALLIS-WILCOX

Table 8: Haematology – males, recovery group

 

 

		Test Group 10/ M 0 mg/kg bw/d	Test Group 11/ M 1000 mg/kg bw/d
UREA	Mean	4.81 x	5.50
[mmol/L]	S.d.	0.43	0.59
day 30	N	5	5
	Median	4.68	5.53
	Deviation Vs Control [%]		14.38
CREA	Mean	23.0 x	24.9
[µmol/L]	S.d.	3.1	1.7
day 30	N	5	5
	Median	23.1	24.5
	Deviation Vs Control [%]		8.2
GLUC	Mean	6.80 x	6.22
[mmol/L]	S.d.	0.55	0.51
day 30	N	5	5
	Median	6.84	6.49
	Deviation Vs Control [%]		-8.50
TBIL C	Mean	1.54 x	1.62
[µmol/L]	S.d.	0.27	0.19
day 30	N	5	5
	Median	1.53	1.69
	Deviation Vs Control [%]		5.34
TBA	Mean	22.8 x	24.3
[µmol/L]	S.d.	6.5	10.7
day 30	N	5	5
	Median	20.7	24.4
	Deviation Vs Control [%]		6.5
TPROT	Mean	65.05 x	66.07
[g/L]	S.d.	1.03	1.26
day 30	N	5	5
	Median	64.87	65.51
	Deviation Vs Control [%]		1.57
ALB	Mean	37.37 x	38.21 **
[g/L]	S.d.	0.23	0.21
day 30	N	5	5
	Median	37.42	38.33
	Deviation Vs Control [%]		2.24
GLOB	Mean	27.68 x	27.86
[g/L]	S.d.	0.81	1.42
day 30	N	5	5
	Median	27.45	27.18
	Deviation Vs Control [%]		0.66
CHOL	Mean	1.96 x	2.64 *
[mmol/L]	S.d.	0.30	0.22
day 30	N	5	5
	Median	1.96	2.70
	Deviation Vs Control [%]		34.80
TRIG	Mean	1.12 x	1.19
[mmol/L]	S.d.	0.38	0.41
day 30	N	5	5
	Median	1.25	1.41
	Deviation Vs Control [%]		6.61

 

Statistic Profile = Wilcoxon test (two-sided), *p<=0.05, **p<=0.01, X = Group excluded from statistics

x=WILCOX

Table 9: Absolute organ weights of main groups

 

	Male animals			Female animals
Test group (mg/kg bw/d)	1 (100)	2 (300)	3 (1000)	1 (100)	2 (300)	3 (1000)
Epididymides	+6.04%	+0.88%	+8.32%*
Prostate	+16.8%**	+23.76%**	+24.59%**	%	%	%
Thymus	%	%	%	-21.982%*	+13.453%	-1.141%

*p <= 0.05; **p <= 0.01

 

Table 10: Relative organ weights of main groups

 

	Male animals
Test group (mg/kg bw/d)	1 (100)	2 (300)	3 (1000)
Prostate	+15.1%*	+22.27%*	+22.05%**

*p <= 0.05; **p <= 0.01

 

Table 11: Relative organ weights of the recovery group

 

	Male animals
Test group (mg/kg bw/d)	11 (1000)
Heart	+11.7%*

*p <= 0.05; **p <= 0.01

Table 12: Fertility indices for F0 males

 

 

	Test group 0 (0 mg/kg bw/d)	Test group 1 (100 mg/kg bw/d)	Test group 2 (300 mg/kg bw/d)	Test group 3 (1000 mg/kg bw/d)
Male fertility index [%]	100	100	90	100

 

Table 13: Fertility indices for F0 females

 

 

	Test group 0 (0 mg/kg bw/d)	Test group 1 (100 mg/kg bw/d)	Test group 2 (300 mg/kg bw/d)	Test group 3 (1000 mg/kg bw/d)
Female fertility index [%]	100	100	100	100

 

Table 14: Sex ratio of live F1 pups

 

 

PND 0	Test group 0 (0 mg/kg bw/d)	Test group 1 (100 mg/kg bw/d)	Test group 2 (300 mg/kg bw/d)	Test group 3 (1000 mg/kg bw/d)
Live males [%]	48.3	43.7	57.7	50.7
Live females [%]	51.7	56.3	42.3	49.3
PND 13	Test group 0 (0 mg/kg bw/d)	Test group 1 (100 mg/kg bw/d)	Test group 2 (300 mg/kg bw/d)	Test group 3 (1000 mg/kg bw/d)
Live males [%]	47.5	47.5	52.5	46.2
Live females [%]	52.5	52.5	47.5	53.8

 

Table 15: Summary Mating Report

 

		Test Group 0/ F		Test Group 1/ F	Test Group 2/ F	Test Group 3/ F
		0 mg/kg bw/d		100 mg/kg bw/d	300 mg/kg bw/d	1000 mg/kg bw/d
No. of females mated	N	10		10	10	10
- Inseminated	N	10	f-	10	9	10
Female mating index	%	100.0		100.0	90.0	100.0
-- Pregnant	N	10	f-	10	9	10
Female fertility index	%	100.0		100.0	100.0	100.0
No. of males mated	N	10		10	10	10
- With inseminated females	N	10	f-	10	9	10
Male mating index	%	100.0		100.0	90.0	100.0
- With pregnant females	N	10	f-	10	9	10
Male fertility index	%	100.0		100.0	90.0	100.0
Females with defined Day 0 pc	N	10		10	9	10
Mating days until Day 0 pc	Mean	2.2	x+	3.9	3.3 *	2.4
	S.d.	0.9		3.7	1.1	1.0
	N	10		10	9	10
Days 0 To 4	N	10		9	9	10
	%	100.0		90.0	100.0	100.0
Days 5 To 9	N	0		0	0	0
	%	0.0		0.0	0.0	0.0
Days 10 To 14	N	0		1	0	0
	%	0.0		10.0	0.0	0.0

Statistic Profile = Fisher's exact test (one-sided-), Wilcoxon with Bonferroni-Holm (one-sided+), * p<=0.05, ** p <=0.01, X = Group excluded from statistics f=FISHER-EXACT; x=WILCOX

 

Table 16: Summary Pregnancy Status Report - Reproduction

 

			Test Group 0/F 0 mg/kgbw/d		Test Group 1/F 100 mg/kgbw/d		Test Group 2/F 300 mg/kgbw/d	Test Group 3/F 1000 mg/kgbw/d
No. of females at start	N		10		10		10	10
No. of females mated	N		10		10		10	10
Without evidence of mating	N		0		0		1	0
- Pregnant	N		0		0		0	0
- Not pregnant	N		0		0		1	0
Females with defined Day 0 pc	N		10		10		9	10
Pregnant	N		10		10		9	10
- sacrificed scheduled	N		10		10		9	10
Not pregnant	N		0		0		1	0
- sacrificed scheduled	N		0		0		1	0
Pregnant, not delivering	N		0		0		0	0
Delivering	N		10		10		9	10
-- With liveborn pups	N		10		10		9	10
	%		100.0		100.0		100.0	100.0
-- With all pups stillborn	N		0		0		0	0
	%		0.0		0.0		0.0	0.0

Statistic Profile = Fisher's exact test (one-sided-), Dunnett test (two-sided), Fisher's exact test (one-sided+), * p<=0.05, ** p <=0.01, X = Group excluded from statistics

f=FISHER-EXACT; n=DUNNETT

 

 

Table 17: Summary Delivery Report

 

		Test Group0/F 0 mg/kgbw/d		Test Group1/F 100 mg/kgbw/d		Test Group2/F 300 mg/kgbw/d	Test Group3/F 1000 mg/kgbw/d
No. of females at start	N	10		10		10	10
No. of females mated	N	10	f-	10		10	10
	%	100.0		100.0		100.0	100.0
Pregnant	N	10	f-	10		9	10
	%	100.0		100.0		90.0	100.0
Without delivery	N	0		0		1	0
- Pregnant	N	0		0		0	0
- Not pregnant	N	0		0		1	0
-- Delivering	N	10	f-	10		9	10
	%	100.0		100.0		100.0	100.0
-- With liveborn pups	N	10	f-	10		9	10
Gestation Index	%	100.0		100.0		100.0	100.0
Gestation days	Mean	22.1	n	22.3		22.1	22.0
	S.d.	0.6		0.5		0.6	0.5
	N	10		10		9	10
-- With stillborn pups	N	0		0		2	1
	%	0.0		0.0		22.2	10.0
-- With all pups stillborn	N	0		0		0	0
	%	0.0		0.0		0.0	0.0

 

Statistic Profile = Fisher's exact test (one-sided-), Dunnett test (two-sided), Fisher's exact test (one-sided+), * p<=0.05, ** p <=0.01, X = Group excluded from statistics

f=FISHER-EXACT; n=DUNNETT

Table 18: Summary Litter Report – Pup status I

 

		Test Group 0/ F		Test Group 1/ F	Test Group 2/ F	Test Group 3/ F
		0 mg/kg bw/d		100 mg/kg bw/d	300 mg/kg bw/d	1000 mg/kg bw/d
Total Number of Pregnant Females	N	10		10	9	10
Total number of litters	N	10		10	9	10
With liveborn pups	N	10	f-	10	9	10
	%	100.0		100.0	100.0	100.0
With stillborn pups	N	0	f+	0	2	1
	%	0.0		0.0	22.2	10.0
With all pups stillborn	N	0	f+	0	0	0
	%	0.0		0.0	0.0	0.0
Implantation Sites	N	129		136	119	135
	Mean	12.9	x-	13.6	13.2	13.5
	S.d.	2.1		1.4	2.8	1.5
	N	10		10	9	10
Pups delivered	N	121		130	107	130
	Mean	12.1	x-	13.0	11.9	13.0
	S.d.	2.3		1.6	3.4	1.9
	N	10		10	9	10
Postimplantation Loss	Mean%	5.7	x+	4.5	10.1	3.8
	S.d.	12.6		5.0	16.1	8.1
	N	10		10	9	10

Statistic Profile = Wilcoxon with Bonferroni-Holm (one-sided-), Wilcoxon with Bonferroni-Holm (one-sided+), Wilcoxon test (two-sided), Fisher's exact test (one-sided-),

Fisher's exact test (one-sided+), * p<=0.05, ** p <=0.01, X = Group excluded from statistics

f=FISHER-EXACT; x=WILCOX

 

 

Table 19: Summary Litter Report – Pup status II

 

		Test Group 0/ F		Test Group 1/ F	Test Group 2/ F	Test Group 3/ F
		0 mg/kg bw/d		100 mg/kg bw/d	300 mg/kg bw/d	1000 mg/kg bw/d
Pups liveborn	N	121		130	105	129
	%	100.0		100.0	98.1	99.2
	Mean	12.1	x-	13.0	11.7	12.9
	S.d.	2.3		1.6	3.6	1.9
	N	10		10	9	10
Pups stillborn	N	0		0	2	1
	%	0.0		0.0	1.9	0.8
	Mean	0.0	x+	0.0	0.2	0.1
	S.d.	0.0		0.0	0.4	0.3
	N	10		10	9	10
Perinatal Loss	Mean%	0.0	x+	0.0	2.8	0.8
	S.d.	0.0		0.0	5.9	2.4
	N	10		10	9	10

Statistic Profile = Wilcoxon with Bonferroni-Holm (one-sided-), Wilcoxon with Bonferroni-Holm (one-sided+), Wilcoxon test (two-sided), Fisher's exact test (one-sided-),

Fisher's exact test (one-sided+), * p<=0.05, ** p <=0.01, X = Group excluded from statistics

x=WILCOX

 

 

Table 20: Summary Litter Report - Dead Pups

 

		Test Group0/F 0 mg/kgbw/d	Test Group1/F 100 mg/kgbw/d	Test Group 2/ F 300 mg/kg bw/d	Test Group3/F 1000 mg/kgbw/d
Litters with liveborn pups	N	10	10	9	10
Pups delivered	N	121	130	107	130
found dead [pup] / Dead	N	1	0	0	0
	%	0.8	0.0	0.0	0.0
stillborn / Dead	N	0	0	2	1
	%	0.0	0.0	1.9	0.8
Alive / Alive	N	121	130	105	129
	%	100.0	100.0	98.1	99.2
cannibalized [pup] / Dead	N	0	0	0	1
	%	0.0	0.0	0.0	0.8
sacrificed scheduled [pup] / Dead	N	80	80	68	80
	%	66.1	61.5	63.6	61.5
culled / Dead	N	40	50	37	48
	%	33.1	38.5	34.6	36.9
Litters not surviving Day 13	N	0	0	0	0
	%	0.0	0.0	0.0	0.0

Statistic Profile = Wilcoxon with Bonferroni-Holm (one-sided-), Wilcoxon with Bonferroni-Holm (one-sided+), Wilcoxon test (two-sided), Fisher's exact test (one-sided-),

Fisher's exact test (one-sided+), * p<=0.05, ** p <=0.01, X = Group excluded from statistics

 

 

 

Table 21: Summary Litter Report - Pups Died

 

		Test Group0/F 0 mg/kgbw/d	Test Group1/F 100 mg/kgbw/d	Test Group 2/ F 300 mg/kg bw/d	Test Group3/F 1000 mg/kgbw/d
Litters with liveborn pups	N	10	10	9	10
Pups delivered	N	121	130	107	130
Days 0 To 0	N	0	0	0	0
	%	0	0	0	0
Days 1 To 4	N	1	0	0	1
	%	0.8	0	0	0.8
Days 5 To 7	N	0	0	0	0
	%	0	0	0	0
Days 8 To 13	N	0	0	0	0
	%	0	0	0	0
Pups surviving days 0 To 4	N	120	130	105	128
Viability Index	Mean%	99.3 x-	100.0	100.0	99.3
	S.d.	2.3	0.0	0.0	2.3
	N	10	10	9	10
Pups surviving days 4 To 13	N	80	80	68	80
Survival Index	Mean%	100.0 NA	100.0	100.0	100.0
	S.d.	0.0	0.0	0.0	0.0
	N	10	10	9	10

Statistic Profile = Wilcoxon with Bonferroni-Holm (one-sided-), Wilcoxon with Bonferroni-Holm (one-sided+), Wilcoxon test (two-sided), Fisher's exact test (one-sided-),

Fisher's exact test (one-sided+), * p<=0.05, ** p <=0.01, X = Group excluded from statistics

x=WILCOX; NA=No Test Applicable

 

 

Table 22: Summary Pup Report Body Weights - BW / Body Weights [g] I

 

		Test Group 0/F 0 mg/kgbw/d	Test Group 1/F 100 mg/kgbw/d		Test Group 2/F 300 mg/kgbw/d	Test Group 3/F 1000 mg/kgbw/d
day 1 Runt	Males	0	3		1	3
	Females	2	1		0	0
day 1 Males	Mean	6.8 n	6.6		6.7	6.4
	S.d.	0.9	0.5		0.8	0.4
	N	10	10		9	10
	Deviation Vs Control [%]		-3.4		-1.7	-6.6
day 1 Females	Mean	6.5 n	6.4		6.5	6.2
	S.d.	0.8	0.5		0.9	0.4
	N	10	10		9	10
	Deviation Vs Control [%]		-1.2		0.1	-4.2
day 1 Males + Females	Mean	6.7 n	6.5		6.6	6.3
	S.d.	0.8	0.5		0.9	0.3
	N	10	10		9	10
	Deviation Vs Control [%]		-2.4		-0.2	-5.0
day 4 Males	Mean	10.8 n	10.4		10.5	10.1
	S.d.	1.7	1.0		1.8	0.5
	N	10	10		9	10
	Deviation Vs Control [%]		-3.6		-2.1	-6.4

Statistic Profile = Dunnett test (two-sided), * p<=0.05, ** p <=0.01, X = Group excluded from statistics

n=DUNNETT

 

 

Table 23: Summary Pup Report Body Weights - BW / Body Weights [g] II

 

		Test Group 0/F 0 mg/kgbw/d	Test Group 1/F 100 mg/kgbw/d		Test Group 2/F 300 mg/kgbw/d	Test Group 3/F 1000 mg/kgbw/d
day 4 Females	Mean	10.4 n	10.2		10.3	10.0
	S.d.	1.7	1.0		1.8	0.5
	N	10	10		9	10
	Deviation Vs Control [%]		-2.6		-1.1	-3.7
day 4 Males + Females	Mean	10.6 n	10.3		10.4	10.1
	S.d.	1.7	1.0		1.8	0.5
	N	10	10		9	10
	Deviation Vs Control [%]		-3.2		-1.3	-4.5
day 7 Males	Mean	17.7 n	17.1		17.1	16.8
	S.d.	2.0	1.5		2.1	0.8
	N	10	10		9	10
	Deviation Vs Control [%]		-3.0		-3.0	-5.1
day 7 Females	Mean	17.0 n	17.2		16.7	16.5
	S.d.	2.0	1.5		2.1	0.9
	N	10	10		9	10
	Deviation Vs Control [%]		1.2		-1.9	-3.2
day 7 Males + Females	Mean	17.3 n	17.2		16.9	16.7
	S.d.	1.9	1.5		2.0	0.8
	N	10	10		9	10
	Deviation Vs Control [%]		-0.8		-2.4	-3.8

Statistic Profile = Dunnett test (two-sided), * p<=0.05, ** p <=0.01, X = Group excluded from statistics

n=DUNNETT

 

 

Table 24: Summary Pup Report Body Weights - BW / Body Weights [g] II

 

		Test Group 0/F 0 mg/kgbw/d	Test Group 1/F 100 mg/kgbw/d		Test Group 2/F 300 mg/kgbw/d	Test Group 3/F 1000 mg/kgbw/d
day 13 Males	Mean	33.1 n	32.1		32.4	31.8
	S.d.	2.2	2.3		2.3	1.8
	N	10	10		9	10
	Deviation Vs Control [%]		-2.9		-1.9	-3.9
day 13 Females	Mean	32.3 n	32.1		31.7	31.5
	S.d.	2.3	2.3		2.2	2.0
	N	10	10		9	10
	Deviation Vs Control [%]		-0.7		-1.9	-2.4
day 13 Males + Females	Mean	32.7 n	32.1		32.1	31.7
	S.d.	2.2	2.3		2.2	1.9
	N	10	10		9	10
	Deviation Vs Control [%]		-1.8		-1.9	-3.0

Statistic Profile = Dunnett test (two-sided), * p<=0.05, ** p <=0.01, X = Group excluded from statistics

n=DUNNETT

 

 

Table 25: Summary Pup Report AG Distance + AG Index of males

 

		Test Group 0/F 0 mg/kgbw/d	Test Group 1/F 100 mg/kgbw/d		Test Group 2/F 300 mg/kgbw/d	Test Group 3/F 1000 mg/kgbw/d
AG Distance	Mean	3.06 n	3.08		3.13	3.02
[mm]	S.d.	0.31	0.20		0.27	0.18
day 1 Males	N	10	10		9	10
AG Index Cubic Root	Mean	1.61 n	1.64		1.66	1.63
[---]	S.d.	0.10	0.08		0.13	0.09
day 1 Males	N	10	10		9	10

Statistic Profile = Dunnett test (two-sided), * p<=0.05, ** p <=0.01, X = Group excluded from statistics

n=DUNNETT

 

 

Table 26: Summary Pup Report AG Distance + AG Index of males

 

		Test Group 0/F 0 mg/kgbw/d	Test Group 1/F 100 mg/kgbw/d		Test Group 2/F 300 mg/kgbw/d	Test Group 3/F 1000 mg/kgbw/d
AG Distance	Mean	1.39 n	1.40		1.38	1.37
[mm]	S.d.	0.17	0.12		0.13	0.08
day 1 Females	N	10	10		9	10
AG Index Cubic Root	Mean	0.75 n	0.76		0.74	0.75
[---]	S.d.	0.08	0.05		0.08	0.04
day 1 Females	N	10	10		9	10

Statistic Profile = Dunnett test (two-sided), * p<=0.05, ** p <=0.01, X = Group excluded from statistics

n=DUNNETT

 

 

Table 27: Summary Pup Report Nipple development

 

			Test Group 0/F 0 mg/kgbw/d	Test Group 1/F 100 mg/kgbw/d		Test Group 2/F 300 mg/kgbw/d		Test Group 3/F 1000 mg/kgbw/d
Nipple development [Incidence]	Passed -N		7	5		7		11
day 13 Males	-% Failed -N		18   31	13   33		20   28		30   26
	-%		82	87		80		70
Nipple development [%]	Mean		17.5 x+	12.5		19.4		30.0
[%]	S.d.		23.7	27.0		20.8		19.7
day 13 Males	N		10	10		9		10
Nipple Number	Mean		0.4 x+	0.3		0.4		0.6
[#]	S.d.		0.6	0.7		0.4		0.4
day 13 Males	N		10	10		9		10

Statistic Profile = Wilcoxon with Bonferroni-Holm (one-sided+), * p<=0.05, ** p <=0.01, X = Group excluded from statistics

x=WILCOX

 

 

Table 28: Summary - Pup Necropsy Observation, Males

 

			Test Group 0/M 0 mg/kgbw/d	Test Group 1/M 100 mg/kgbw/d	Test Group 2/M 300 mg/kgbw/d	Test Group 3/M 1000 mg/kgbw/d
day 0 -> 13 With unscheduled data	Animals examined	N	59	57	62	68
	Animals with signs	N	2	1	1	2
		%	3.4	1.8	1.6	2.9
	Liver	N	0	1	0	1
		%	0.0	1.8	0.0	1.5
	large	N	0	0	0	1
		%	0.0	0.0	0.0	1.5
	misshapened	N	0	1	0	0
		%	0.0	1.8	0.0	0.0
	Testis small	N	0	0	1	0
		%	0.0	0.0	1.6	0.0
	Liver Lobe absent	N	1	0	0	0
		%	1.7	0.0	0.0	0.0
	Stomach empty	N	0	0	0	1
		%	0.0	0.0	0.0	1.5
	General	N	1	0	0	1
		%	1.7	0.0	0.0	1.5
	Post mortem autolysis	N	1	0	0	0
		%	1.7	0.0	0.0	0.0
	not assessed	N	0	0	0	1
		%	0.0	0.0	0.0	1.5
	normal NAD	N	57	56	61	66
		%	96.6	98.2	98.4	97.1

 

Table 28:Summary - Pup Necropsy Observation, Females

 

			Test Group 0/F 0 mg/kgbw/d	Test Group 1/F 100 mg/kgbw/d	Test Group 2/F 300 mg/kgbw/d	Test Group 3/F 1000 mg/kgbw/d
day 0 -> 13 With unscheduled data	Animals examined	N	62	73	45	62
	normal NAD	N	62	73	45	62
		%	3.4	1.8	1.6	2.9

 

 

 

Conclusions:

The no observed adverse effect level (NOAEL) of general systemic toxicity was 300 mg/kg bw/d for male and 1000 mg/kg bw/d for female parental animals.
The no observed adverse effect level (NOAEL) for reproductive performance and fertility was the nominal dose level 1000 mg/kg bw/d in both sexes of parental animals.
The NOAEL for developmental toxicity in the F1 progeny was the nominal dose level 1000 mg/kg bw/d.

Executive summary:

The repeated dose toxicity of the test substance was investigated by means of a Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test according to OECD guideline 422 and GLP.

The test substance was administered daily by gavage as a solution to groups of 10 male and 10 female Wistar rats (F0 animals) at nominal doses of 0, 100, 300 and 1000 mg/kg body weight/day (mg/kg bw/d). Control animals were dosed daily with the vehicle only (Corn oil Ph.Eur. 8.0). The duration of treatment covered a 2-week premating period and mating in both sexes (mating pairs were from the same dose group) as well as entire gestation and lactation period in females up to one day prior to the day of schedule sacrifice of the animals. Additional treated but not mated animals (recovery animals) to groups of 5 male and 5 female animals at nominal doses of 0 and 1000 mg/kg bw/d was maintained for a subsequent period of at least 14 days of no test substance administration in order to observe reversibility of the findings.

The parents' and the pups' state of health was checked each day, and parental animals were examined for their mating and reproductive performances. F0 animals were mated 13 days after the beginning of treatment to produce a litter (F1 generation pups). As soon as sperm was detected in the vaginal smear, mating was discontinued. F0 animals were examined for their reproductive performance including determinations of the number of implantations and the calculation of the postimplantation loss in all F0 females. A detailed clinical observation (DCO) was performed in all animals before initial test substance administration and, as a rule, thereafter at weekly intervals. Food consumption of the F0 parents of main groups and recovery groups were determined regularly once weekly before mating period as well as males and females of the recovery groups until sacrifice. In dams during gestation days 0-7,7-14 and 14-20 and lactation days 1-4, 4-7, 7-10 and 10-13 the food consumption was also determined. In general, the body weights of F0 animals were determined once a week. However, during gestation and lactation, F0 females were weighed on gestation days (GD) 0, 7, 14 and 20, and on postnatal days (PND) 0, 4, 7, 10 and 13. The pups were sexed and examined for macroscopically evident changes on PND 0. They were weighed on PNDs 1, 4, 7 and on PND 13 and their viability was recorded. Anogenital distance (defined as the distance from the anus [center of the anal opening] to the base of the genital tubercle) measurements were conducted in a blind randomized fashion, using a measuring ocular on all live male and female pups on PND 1. On PND 4, the individual litters were standardized in such a way that, whenever possible, each litter contains 4 male and 4 female pups (as a rule, the first 4 surviving pups/sex in each litter were taken for further rearing). On PND 13, all male F1 pups were examined for retention of nipples/areolae. The number of nipples/areola anlagen were counted. At necropsy on PND 4, all pups were sacrificed under isoflurane anesthesia by decapitation. Blood was sampled for determination of thyroid hormone concentrations. After sacrifice, the pups were examined externally and eviscerated, and their organs were assessed macroscopically. At necropsy on PND 13, one selected male and one female pup per litter were sacrificed under isoflurane anesthesia by decapitation. Blood was sampled for determination of thyroid hormone concentrations. Thyroid glands/parathyroid glands were fixed in neutral buffered 4% formaldehyde solution and were transferred to the Pathology Laboratory for possible further processing. The remaining pups were sacrificed under isoflurane anesthesia with CO2. After sacrifice, all pups were examined externally and eviscerated, and their organs were assessed macroscopically. Towards the end of the administration period a functional observational battery was performed and motor activity was measured in 5 animals per sex and test group. Clinicochemical and hematological examinations were performed in 5 animals per sex and group towards the end of the administration period as well as towards the end of recovery period. All F0 parental animals were sacrificed by decapitation, under isoflurane anesthesia, and were assessed by gross pathology. Weights of selected organs were recorded and a histopathological examination was performed. The remaining 5 animals per sex of test groups 10 and 11 (recovery animals) were maintained for a 2-week recovery period after the administration period without test substance exposure. Further examinations in these animals were depend on the findings observed in the animals of the main groups. No functional observational battery was performed as well as no motor activity and estrous cycle were determined during the recovery period.

 

The various analyses confirmed

·        the stability of the test substance in corn oil at room temperature over a period of 7 days,

·        and the correctness of the prepared concentrations.

 

The following test substance-related adverse effects/findings were noted:

 

Test group 3/11: 1000 mg/kg bw/d

F0 PARENTAL ANIMALS

Clinical examinations, reproductive performance and pathology

·        no test substance-related adverse findings

Clinical pathology

·        reduced prothrombin time (HQT) in males (test group 3, only)

·        increased cholesterol values in males

F1 PUPS

Clinical examinations/ gross findings

·        no test substance-related adverse findings

 

Test group 2: 300 mg/kg bw/d

F0 PARENTAL ANIMALS

Clinical examinations, reproductive performance, clinical pathology, and pathology

·        no test substance-related adverse findings

F1 PUPS

Clinical examinations/ gross findings

·        no test substance-related adverse findings

 

Test group 1: 100 mg/kg bw/d

F0 PARENTAL ANIMALS

Clinical examinations, reproductive performance, clinical pathology, and pathology

·        no test substance-related adverse findings

F1 PUPS

Clinical examinations/ gross findings

·        no test substance-related adverse findings

 

Under the conditions of this Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, the oral administration of the test substance by gavage to Wistar rats revealed signs of systemic toxicity manifested in clinical pathology parameters of males in the limit dose group (1000 mg/kg bw/d) at the end of the administration period. However, the clinical pathological alterations were no permanent changes, since no treatment-related findings were noted after the two weeks recovery period. No treatment-related adverse findings were observed in males at 100 and 300 mg/kg bw/d as well as in all female parental animals (F0) and all pups (F1).

The no observed adverse effect level (NOAEL) of general systemic toxicity was 300 mg/kgbw/d for male and 1000 mg/kg bw/d for female parental animals. The no observed adverse effect level (NOAEL) for reproductive performance and fertility was the nominal dose level 1000 mg/kg bw/d in both sexes of parental animals. The NOAEL for developmental toxicity in the F1 progeny was the nominal dose level 1000 mg/kg bw/d.

Cross-reference 2

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 Jun 2018 - 14 Feb 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

29 Jul 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: ZH 472 redestilliert.
- Storage stability: until Jan 2020.

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature.
- Stability under test conditions: The stability of the test substance in solution in corn oil over a period of 7 days at room temperature was proven. the mixtures were stored no longer than this time period,
the stability was guaranteed.
- Solubility and stability of the test substance in the solvent/vehicle: The test substance is completely miscible with corn oil Ph.Eur 8.0 and thus a solution. Therefore, the test-substance preparation was considered to be homogenous. Consequently, no further homogeneity analysis was performed.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test substance was applied as a solution. To prepare this solution, the appropriate amount of test substance was weighed out depending on the desired concentration. Then, Corn oil Ph.Eur. 8.0 was filled up to the desired volume and intensely mixed with a magnetic stirrer. The test substance preparations were produced weekly, at least.

FORM AS APPLIED IN THE TEST (if different from that of starting material) : solution in corn oil.

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Crl:WI(Han)

The rat is the preferred animal species for reproduction studies according to the various test guidelines and the Wistar strain was selected. This Wistar rat strain (Crl:WI(Han)) was selected since extensive historical control data were available on these Wistar rats.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH.
- Females (if applicable) nulliparous and non-pregnant: yes.
- Age at study initiation: age at supply about 12 - 13 weeks (male animals), about 11 weeks (female animals).
- Weight at study initiation: mean weight 393 g (males) and 212 g (females).
- Fasting period before study: no.
- Housing: During pre-treatment: Polysulfonate cages Typ 2000P (H-Temp), floor area about 2065 cm2 (610 x 435 x 215 mm); supplied by TECHNIPLAST, Hohenpeißenberg, Germany During premating, mating, gestation, lactation, males after mating and females after weaning: Polycarbonate cages type III. Pregnant animals and their litters were housed together until PND 13 (end of lactation). For motor activity (MA) measurements the animals were housed individually in polycarbonate cages type III supplied by TECNIPLAST, Hohenpeißenberg, Germany with wire covers from Ehret, Emmendingen, Germany (floor area of about 800 cm2) and small amounts of bedding material Pregnant females were provided with nesting material (cellulose wadding) toward the end of gestation. For enrichment wooden gnawing blocks Lignocel® block large (supplied by J. Rettenmaier & Söhne GmbH + Co KG, Rosenberg, Germany) were added. In addition, in Polysulfonate cages large play tunnels (Art. 14153; supplied by PLEXX b.v., Elst, Netherlands) were added.
- Diet: ad libitum.
- Water: ad libitum.
- Acclimation period: 21 days.

ENVIRONMENTAL CONDITIONS
- The cages with the test animals were arranged on the racks in such a way that uniform experimental conditions (ventilation and light) were ensured. The animals were housed in a fully air-conditioned room.
- Temperature (°C): 20-24°C.
- Humidity (%): 45-65% for relative humidity.
- Air changes (per hr): 15 air changes per hour.
- Photoperiod (hrs dark / hrs light): The day/night cycle was 12 hours (12 hours light from 06.00- 18.00 h, 12 hours dark from 18.00-06.00 h).

IN-LIFE DATES: From: 19 Jun 2018 To: 10 Sep 2018.

Route of administration:

oral: gavage

Details on route of administration:

The oral route was selected since administration by gavage has been proven to be appropriate for the detection of a toxicological hazard.

Vehicle:

corn oil

Remarks:

Ph.Eur 8.0

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
- The test substance was applied as a solution. To prepare this solution, the appropriate amount of test substance was weighed out depending on the desired concentration. Then, Corn oil Ph.Eur. 8.0 was filled up to the desired volume and intensely mixed with a magnetic stirrer. The test substance preparations were produced weekly, at least.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability of the test substance in corn oil Ph.Eur 8.0 was demonstrated over a period of 7 days at room temperature. As the mixtures were stored no longer than this time period, the stability was guaranteed.
The concentrations of the test substance in corn oil Ph.Eur 8.0 were found to be in the range of 99.5 to 105.4% of the nominal concentrations. These results demonstrated the correctness of the concentrations of the test substance in corn oil Ph.Eur 8.0 being within the specification of the test facility 90-110%.

Duration of treatment / exposure:

males: 29 days; females: 61 days
The duration of treatment covered a 2-week premating period and mating in both sexes (mating pairs were from the same dose group) as well as entire gestation and lactation period in females up to one day prior to the day of schedule sacrifice of the animals. Additional treated but not mated animals (recovery animals) to groups of 5 male and 5 female animals at nominal doses of 0 and 1000 mg/kg bw/d was maintained for a subsequent period of at least 14 days of no test substance administration in order to observe reversibility of the findings.

Frequency of treatment:

daily

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Remarks:

Test Group 1

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Remarks:

Test Group 2

Dose / conc.:

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

Remarks:

Test Group 3

Dose / conc.:

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

Remarks:

Test Group 11 (Recovery animals)

No. of animals per sex per dose:

Test group 0, 1, 2, 3: 10 animals per sex per dose
Test group 10, 11 (Recovery animals): 5 animals per sex per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: In accordance with the guideline, three doses were chosen which were applied in two- to four- fold intervals and it was tested up to the limit dose of 1000 mg/kg bw/day.
- Fasting period before blood sampling for clinical biochemistry: The adults were fastened before the blood sampling. A fasting period (withdrawal of food) of about 16 to 20 hours was applied.
- Post-exposure recovery period in satellite groups: yes. Additional treated but not mated animals (recovery animals) to groups of 5 male and 5 female animals at nominal doses of 0 mg/kg bw/d (test group 10) and 1000 mg/kg (test group 11) bw/d were maintained for a subsequent period of at least 14 days of no test substance administration in order to observe reversibility of the findings.

Positive control:

no.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes.
- Time schedule: daily.
- Mortality: A check for moribund and dead animals was made twice daily on working days and once daily on Saturdays, Sundays and public holidays. If animals were in a moribund state, they were sacrificed and necropsied.
- Clinical observations: A cageside examination was conducted at least once daily for any signs of morbidity, pertinent behavioral changes and/or signs of overt toxicity. All animals were checked daily for any abnormal clinical signs before the administration as well as within 2 hours and within 5 hours after the administration. Abnormalities and changes were documented for each animal. The parturition and lactation behavior of the dams was generally be evaluated in the morning in combination with the daily clinical inspection of the dams. Only particular findings (e.g. disability to deliver or umbilical cord not cut) were documented on an individual dam basis. On weekdays (except Saturdays, Sundays 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 to be the 24-hour period from about 15:00 h of one day until about 15:00 h of the following day.

DETAILED CLINICAL OBSERVATIONS: Yes.
- Time schedule: prior to administration and weekly thereafter.
- 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: abnormal behavior in handling, fur, skin, posture, salivation, respiration, activity/arousal level, tremors, convulsions, abnormal movements, gait abnormalities, lacrimation, palpebral closure, exophthalmos, assessment of the feces discharged during the examination (appearance/ consistency), assessment of the urine discharged during the examination, pupil size.

BODY WEIGHT: Yes.
- Time schedule for examinations: before the beginning administration, during administration at day 0 and weekly thereafter.
- Body weight of the male and female parental animals in the main groups as well as the males and females of recovery groups 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 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), PNDs 4, 7, 10 and 13.
-- Females showing no positive evidence of sperm in the vaginal smear were weighed once a week during this mating interval as were the males (for the calculation of the administration volume)
-- Females without litter and after weaning (PND 13) were weighed once a week (for the calculation of the administration volume).

FOOD CONSUMPTION: Yes.
- Time schedule: weekly.
- Generally, food consumption was determined once a week for male and female parental animals of the main groups as well as the males and females of recovery groups, with the following exceptions:
-- Food consumption was not be determined after the 2nd premating week (male parental animals) and during the mating period (male and female parental animals in the main groups).
-- Food consumption of the females with evidence of sperm was determined for GD 0-7, 7-14 and 14-20.
-- Food consumption of the females which gave birth to a litter was determined for PNDs 1-4, 4-7, 7-10 and 10-13.
- 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.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No.

WATER CONSUMPTION: Yes.
- Time schedule for examinations: daily.
- Drinking water consumption was monitored by daily visual inspection of the water bottles for any changes in volume.

OPHTHALMOSCOPIC EXAMINATION: No.

HAEMATOLOGY: Yes.
- Time schedule for collection of blood: for main groups at day 30 (males) and day 50 (females), for recovery groups at day 30/48 (males) at day 62/77 (females).
- Anaesthetic used for blood collection: Yes (isoflurane).
- Animals fasted: Yes.
- How many animals: 5 males and 5 females.
- Parameters checked in table 1 were examined.
- In the morning blood was taken from the retro-bulbar venous plexus from fasted animals. The animals were anaesthetized using isoflurane. The blood sampling procedure and subsequent analysis of blood and serum samples were carried out in a randomized sequence. The assays of blood and serum parameters were performed under internal laboratory quality control conditions with reference controls to assure reliable test results. The results were expressed in International System (SI) units. The parameters listed were examined in the first 5 surviving parental males per group at termination and in the first 5 females with litters (in order of delivery) per group at PND 14 and in the recovery groups at the end of the administration period and at the end of the recovery period.

CLINICAL CHEMISTRY: Yes.
- Time schedule for collection of blood: for main groups at day 30 (males) and day 50 (females), for recovery groups at day 30/48 (males) at day 62/77 (females).
- Animals fasted: Yes.
- How many animals: 5 males and 5 females.
- Parameters checked in table 2 were examined.
- In the morning blood was taken from the retro-bulbar venous plexus from fasted animals. The animals were anaesthetized using isoflurane. The blood sampling procedure and subsequent analysis of blood and serum samples were carried out in a randomized sequence. The assays of blood and serum parameters were performed under internal laboratory quality control conditions with reference controls to assure reliable test results. The results were expressed in International System (SI) units. The parameters listed were examined in the first 5 surviving parental males per group at termination and in the first 5 females with litters (in order of delivery) per group at PND 14 and in the recovery groups at the end of the administration period and at the end of the recovery period.

URINALYSIS: No.

NEUROBEHAVIOURAL EXAMINATION: Yes; functional observational battery and motor activity assessment.
- Time schedule: FOB and MA at study day 28 (males) and day 57 (females)
FUNCTIONAL OBSERVATIONAL BATTERY:
- A functional observational battery (FOB) was performed in the first five parental male animals per test group and the first five surviving females with litter (in order of delivery) of all main test groups 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.
-Home cage observations: The animals were observed in their closed home cages; during this period 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: Posture, Tremors, Convulsions, Abnormal movements, Gait, Other findings.
- Open field observations: The animals were transferred to a standard arena (50 × 50 cm with sides of 25 cm height) and observed for at least 2 minutes. The following parameters were examined: behavior on removal from the cage, fur, skin, salivation, nasal discharge, lacrimation, eyes/pupil size, posture, palpebral closure, respiration, tremors, convulsions, abnormal movements/stereotypes, gait, activity/arousal level, feces excreted within 2 minutes (appearance/ consistency), urine excreted within 2 minutes (amount/color), rearing within 2 minutes, other findings.
- Sensory motor tests/ reflexes: The animals were then removed from the open field and subjected to following sensory motor or reflex tests: Reaction to an object being moved towards the face (approach response), touch sensitivity (touch response), vision (visual placing response), pupillary reflex, pinna reflex, audition (auditory startle response), coordination of movements (righting response), behavior during handling, vocalization, pain perception (tail pinch), other findings, grip strength of forelimbs, grip strength of hindlimbs, landing foot-splay test.
MOTOR ACTIVITY ASSESSMENT:
Motor activity (MA) was also measured from 14:00 h onwards on the same day as the FOB was performed in the first five parental males and the first five surviving females with litter (in order of delivery) per main group. 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 with a small amount of bedding for the duration of the measurement. Eighteen beams were allocated per cage. The numbers of beam interrupts was counted over 12 intervals of 5 minutes per interval. The sequence in which the animals were placed in the cages was selected at random. On account of the time needed to place the rats in the cages, the starting time was "staggered" for each animal. The measurement period began when the 1st beam was interrupted and finished exactly 1 hour later. No food or water was offered to the animals during these measurements and the measurement room was darkened after the transfer of the last animal. The program required a file name for the measured data to be stored. This name consisted of the reference number and a serial number.

IMMUNOLOGY: No.

OTHER:

ESTROUS CYCLE:
- For all females of the pool estrous cycle normality was evaluated before the beginning of the administration. In all parental females in the main groups in the premating phase, estrous cycle length and normality was evaluated by preparing vaginal smears during a minimum of 2 weeks prior to premating, mating and throughout cohabitation until there is evidence of sperm in the vaginal smear. Additionally, on the day of scheduled sacrifice, the estrous status was also be determined in all female F0 main groups rats.

MALE REPRODUCTION DATA:
- 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 (for formulas see "Any other information on materials and methods").

FEMALE REPRODUCTION AND DELIVERY DATA:
- The pairing partners, the number of mating days until vaginal sperm were detected and gestational status were recorded for F0 females.
- For the females, mating, fertility and gestation indices were calculated for F1 litters (for formulas see "Any other information on materials and methods").

LITTER/PUB DATA:
- 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. Dead pups were evaluated by the methods, which are described in detail in “Necropsy observations”. The number and percentage of dead pups on the day of birth (PND 0) and of pups dying between PND 1-4, 5-7 and 8-13 were determined. Pups, which died accidentally or had to be sacrificed due to maternal death, were not included in these calculations. The number of live pups/litter was calculated on the day of birth (PND 0), and on lactation days 4, 7 and 13. Furthermore, viability and survival indices were calculated (for formulas see "Any other information on materials and methods").
- 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. Later, during the course of lactation, this initial sex determination was followed up by surveying the external appearance of the anogenital region and the mammary line. The sex of the pups was finally confirmed at necropsy. The sex ratio was calculated at PND 0 and PND 13 (for formulas see "Any other information on materials and methods").
- 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) as well as on PND 4 (before standardization), 7 and 13. 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). In the summary tables 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.
- Nipple/areola anlagen: All surviving male pups were examined for the presence of nipple/areola anlagen on PND 13 of the lactation phase. The number of nipple/areola anlagen was counted.
- Anogenital distance: Anogenital distance (AGD; defined as the distance from the anus [center of the anal opening] to the base of the genital tubercle) measurements was done blind to treatment in a randomized order, using a measuring ocular, on all live male, female and uncertain pups on day 1 after birth. The anogenital index were calculated (for formulas see "Any other information on materials and methods").
- Pup necropsy observations: On PND 4, as a result of standardization, the surplus pups or 2 preferably female pups per litter, respectively, was sacrificed under isoflurane anesthesia by decapitation. Blood was sampled for determination of thyroid hormone concentrations. After sacrifice, the pups were examined externally and eviscerated, and their organs were assessed macroscopically. On PND 13, one selected male and one female pup per litter was sacrificed under isoflurane anesthesia by decapitation. Blood was sampled for determination of thyroid hormone concentrations. Thyroid glands/parathyroid glands were fixed in neutral buffered 4% formaldehyde solution and was transferred to the Pathology Laboratory for possible further processing. The remaining pups were sacrificed under isoflurane anesthesia with CO2. After sacrifice, all pups were examined externally and eviscerated, and their organs were assessed macroscopically. All stillborn pups and all pups that died before day 13 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-bycase basis, depending on the type of finding noted.

THYROID HORMONES:
- Parameters checked in table 3 were examined.
- Blood samples were taken from all surplus pups per litter at PND 4 as well as one male and one female pup per litter at PND 13 by decapitation under isoflurane anesthesia. Additionally, blood samples from all dams at PND 14 and all males at termination of the main groups as well as all males and females after the administration period and at termination of the recovery groups were taken by puncturing the retrobulbar venous plexus under isoflurane anesthesia. The adults were fastened before the blood sampling. All generated serum samples were frozen at -80°C until measurement. Blood samples from the adult males and the PND 13 pups were assessed for serum levels for thyroid hormones (T4 and TSH). The concentrations of TSH were determined by radioimmunoassay (RIA), using commercially available RIA test kits and a Gamma-Counter (LB 2111, Berthold, Germany). T4 Elisa was measured with a Sunrise MTP-reader, Tecan AG, Maennedorf, Switzerland, and evaluated with the Magellan-Software of the instrument producer.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table 4, organ weights).

HISTOPATHOLOGY: Yes (see table 4).

Statistics:

See table 5.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Males and females and recovery animals:
Salivation within 2 hours after administration was observed from slight to moderate in test group 3 (1000 mg/kg bw/d) in all male and 9 female animals during premating, in all male and 4 female animals during mating phase, in 3 male animals during post-mating phase as well as in all male and female animals of recovery group 11 (1000 mg/kg bw/d). In test group 2 (300 mg/kg bw/d) one male animal during mating period and one male animal during post-mating salivation within 2 hours after administration was observed. The salivation observed only shortly after treatment was assessed as treatment-related but not adverse. It was considered to be related to a slight local reaction to the test substance preparation by a bitter or bad taste. No test substance-related, adverse findings were observed in male and female animals of test groups 1-3 (100, 300, and 1000 mg/kg bw/d) and recovery group 11 (1000 mg/kg bw/d).

Females during gestation:
Salivation shortly after treatment (<2 hours after treatment) was observed from slight to moderate in all female animals of test group 3 (1000 mg/kg bw/d) during gestation. The salivation observed only shortly after treatment was assessed as treatment-related but not adverse. It was considered to be related to a slight local reaction to test substance preparation by a bitter or bad taste. No test substance-related, adverse findings were observed in test groups 1-3 (100, 300, and 1000 mg/kg bw/d) during gestation.

Females during gestation:
Salivation shortly after treatment (<2 hours after treatment) was observed from slight to moderate in all female animals of test group 3 (1000 mg/kg bw/d) during gestation. The salivation observed only shortly after treatment was assessed as treatment-related but not adverse. It was considered to be related to a slight local reaction to test substance preparation by a bitter or bad taste. No test substance-related, adverse findings were observed in test groups 1-3 (100, 300, and 1000 mg/kg bw/d) during gestation.

Clinical observations for females during lactation
Slight salivation shortly after treatment (<2 hours after treatment) was observed in all female animals of test groups 3 (1000 mg/kg bw/d) during lactation. The salivation observed only shortly after treatment was assessed as treatment-related but not adverse. It was considered to be related to a slight local reaction to test substance preparation by a bitter or bad taste. No test substance-related, adverse findings were observed in test groups 1-3 (100, 300, and 1000 mg/kg bw/d) during gestation.

Detailed clinical observations:
In the detailed clinical observations on study days 0, 7, 14, 21 and 28 in parental males and females and additionally, on study days 35, 42, 49 and 56 in parental female animals and in the recovery period of males and females (as well as in the recovery females on study days 63 and 70) no adverse findings were observed.

Mortality:

no mortality observed

Description (incidence):

No animal died prematurely in the present study.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

No test substance-related changes in mean body weights and body weight gain were observed for male and female animals of test groups 1-3 (100, 300 and 1000 mg/kg bw/d) when compared to the control group. Slightly reduced body weight was observed in male animals of recovery group 11 (1000 mg/kg bw/d) only on study day 13 during the in-life phase. Mean body weight gain was significantly decreased in male animals of recovery group 11 (1000 mg/kg bw/d) during the recovery phase between study days 38 to 45 only. Since the body weight of male animals in the recovery group 11 (1000 mg/kg bw/d) was comparable to the current control, the findings were assessed to be related to treatment, but not adverse.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption of the males and females in all dose groups (100, 300 and 1000 mg/kg bw/d) including recovery group was not influenced by the treatment throughout the entire study period.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

no effects observed

Description (incidence and severity):

No test substance-related findings were observed.

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Main groups:
At the end of the administration period in male rats of the main test group 3 (1000 mg/kg bw/d) prothrombin time (Hepatoquick’s test, HQT) was significantly reduced (see table 6). This alteration combined with the cholesterol increase in the same individuals was regarded as treatment-related and adverse.

Recovery group:
At the end of the administration period of the males in the recovery group 11 (1000 mg/kg bw/d) absolute basophil cell counts an in females of the same test group absolute and relative large unstained cell (LUC) counts were significantly increased, but all values were within historical control ranges (males, absolute basophils 0.00-0.02 Giga/L; females, absolute LUC 0.01-0.02 Giga/L, relative LUC 0.2-0.6 %).
At the end of the two-week recovery phase in males of test group 11 (1000 mg/kg bw/d) hemoglobin values were significantly increased, but th values were within the historical control range (males, hemoglobin 8.6-9.5 mmol/L). Therefore, all mentioned changes in rats of test group 11 were regarded as incidental and not treatment-related.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

At the end of the administration period in males of the main test group 3 (1000 mg/kg bw/d) as well as in males of the recovery test group 11 (1000 mg/kg bw/d) cholesterol values were significantly increased (see table 7 and table 8). This change in combination with the reduced prothrombin time was regarded as treatment-related and adverse.
Additionally, after the administration period in males of the recovery test group 11 (1000 mg/kg bw/d) albumin values were significantly increased. The mean was above the historical control range (males, albumin 34.09-37.97 g/L). However, this change was not present in the equally dosed males of the main test group 3 (1000 mg/kg bw/d). Therefore, this change in the recovery males after the administration was regarded as incidental and not treatment-related.
After the two-week recovery period in males of test group 11 (1000 mg/kg bw/d) sodium levels were significantly increased. The values of males in the control group 10 as well as in the test group 11 were above the historical control range (males, sodium 140.0-148.1 mmol/L). Because no electrolyte value change occurred after the administration period, the solely change of sodium after the recovery period was regarded as incidental and not treatment-related. After the recovery, in females of test group 11 (1000 mg/kg bw/d) alkaline phosphatase (ALP) activities were significantly decreased. The ALP means in females of the controls and test group 11 were below the historical control range (females, ALP 0.47-
0.76 μkat/L). The reason for this may be that the individuals of the historical controls were 6 weeks younger compared to the rats of the present study. The most probable cause for lower ALP levels is a growth retardation. However, females of test group 11 did not have significant lower terminal body weights compared to the study controls. Therefore, the statistically significant lower ALP activities in females of test group 11 were regarded as incidental and not treatment-related.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

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, without a dose-response relationship or occurred in single animals only, these observations were considered as incidental. There were no test substance-related findings in male and female animals of all test groups. Following non-significant alterations were assessed as spontaneous in nature and not related to treatment, because of missing dose-dependency: The foot splay test showed decreased results in males of test group 1 (100 mg/kg bw/d; -11%).

Motor activity measurement:
Regarding the individual intervals as well as the overall motor activity, no test substance related deviations were noted for male and female animals.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Main groups:
- Absolute organ weights: When compared to control group 0 (set to 100%), the mean absolute weights of epidymides, prostate and thymus were significantly increased or decreased in one or more test groups (see table 9). All other mean absolute weight parameters did not show significant differences when compared to the control group 0.
- Relative organ weights When compared to control group 0 (set to 100%), the mean relative prostate weights were significantly increased in all treatment groups (see table 10). All other mean relative weight parameters in males and all mean relative weight parameters in females did not show significant differences when compared to the control group 0.
- The mean absolute and relative prostate weights of males in test group 1 (1.13 g, 0.27%), test group 2 (1.19 g, 0.29%), and test group 3 (1.20 g, 0.29%) were within the range of historical control data (1.01 - 1.274 g, 0.244 - 0.315%), whereas the mean absolute and relative prostate weights of control males (0.96 g, 0.24%) were below the historical control range. Furthermore, there were no treatment-related histopathological findings in the prostate. Therefore, the statistical increase of prostate weights in males of all treatmentgroups can be explained by the comparable low prostate weight in concurrent control males. The mean absolute weight (1.24 g) of epididymides in males of test group 3 was slightly above the historical control range (1.102 – 1.230 g). But the mean relative weight of epididymides (0.30%) was within the historical control range (0.272 – 0.32%) and there were no treatment-related histopathological findings. Therefore, the slightly increased mean absolute weight of epididymides was assessed as incidental. The mean absolute thymus weight was significantly decreased in females of test group 1. As there was no dose-response relationship, this weight decrease was regarded to be incidental.

Recovery group:
- Absolute organ weights: When compared to control group 10 (set to 100%), all mean absolute weight parameters did not show significant differences when compared to the control group 10.
- Relative organ weights/ Recovery group: When compared to control group 0 (set to 100%), the mean relative heart weight was significantly increased in males of test group 11 (see table 11).
- All other mean relative weight parameters in males and all mean relative weight parameters in females did not show significant differences when compared to the control group 10. The slight increase of the relative heart weight in males of test group 11 is related to the slightly but not significantly decreased (-5.1%) terminal body weight in this test group. In addition, there were no significant weight changes of the heart weight in males of the main groups.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Main groups:
- All findings were single or few observations that were considered to be incidental or spontaneous in origin and without any relation to treatment.
- Fertility: The female animal No 129 (test group 2, 300 mg/kg bw/d), which was not pregnant as well as the male mating partner (No. 029) did not show gross lesions.

Recovery group:
- The yellow focus in the vagina in one female of test group 11 (1000 mg/kg bw/d) was a single observation that was considered to be incidental in origin and without any relation to treatment.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Main groups:
- All findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.
- The stages of spermatogenesis in the testes of males of the high dose test group were comparable to those of the controls. In high dose females the different stages of functional bodies in the ovaries were present and comparable to the control animals.
- Fertility: The female animal (No. 129, test group 2, 300 mg/kg bw/d), which was not pregnant as well as the male mating partner (No. 029) did not show relevant histopathological findings.

Recovery group:
- The macroscopically diagnosed yellow focus in the vagina in one female of test group 11 (1000 mg/kg bw/d) correlated with a cyst, histopathologically that was considered to be incidental.
- The vagina of all other females, all other organs of females and all organs of all male animals were not examined, histopathologically.

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Estrous cycle:
Estrous cycle data revealed regular cycles in the rearing F1 females of all test groups including the control. In the different test groups (0-3) the mean number of estrous cycle ranged from 2.5 to 2.8 days and the estrous cycle duration ranged from 3.9 to 4.0 days. Since no treatment-related effect was observed the estrous cycle determination was not carried out in the recovery female animals during the recovery period.

Male reproduction data:
- Male mating index: The male mating index calculated after the mating period for F1 litter varied between 90% in test group 2 (300 mg/kg bw/d) and 100% in test group 0 (control), 1 (100 mg/kg bw/d) and 3 (1000 mg/kg bw/d).
- Male fertility index: Fertility was proven for most of the F0 parental males within the scheduled mating interval to produce F1 litter. Male animal No. 29 of test group 2 (300 mg/kg bw/d), which was tried to be mated with female No. 129, did neither had a confirmed copulation nor generate F1 pups. Thus, the male fertility index was 90% in test group 2 and 100% in test groups 0, 1 and 3. These values 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 reproduction and delivery data:
- Female mating index: The female mating index calculated after the mating period for F1 litter varied between 90% in test group 2 (300 mg/kg bw/d) and 100% in test group 0 (control), 1 (100 mg/kg bw/d) and 3 (1000 mg/kg bw/d). The mean duration until sperm was detected (GD 0) was 2.2 days for test group 0 (control), 3.9 days for test group 1 (100 mg/kg bw/d), 3.3 days for test group 2 (300 mg/kg bw/d), as well as 2.4 days for test groups 3 (1000 mg/kg bw/d). These values 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 (1.6 – 5.8 mating days until day 0
p.c.). This includes also the statistically significant value of 3.3 days in test group 2 (300 mg/kg bw/d).
- Female fertility index: All sperm positive females had implants. Thus, the female fertility index was 100% in all test groups 0-3.
- Gestation index: The gestation index was 100% in all test groups.
- Live birth indices: The rate live birth indices were 100% in test groups 0 and 1 (100 mg/kg bw/d), 98.1% in test group 2 (300 mg/kg bw/d) and 99.2% in test group 3 (1000 mg/kg bw/d). No treatment related effect was observed.
- Postimplantation loss: The postimplantation loss was 5.7% in test group 0 (control), 4.5% in test group 1 (100 mg/kg bw/d), 10.1% in test group 2 (300 mg/kg bw/d) and 3.8% in test group 3 (1000 mg/kg bw/d). These findings 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 (0.0 – 18.1%).

Pub/Litter data:
- Pup number and status at delivery: The mean number of delivered F1 pups per dam was equally distributed among test groups 0, 1, 2 and 3 ranging in mean from 11.7 to 12.1 pups per litter. No significant deviations occurred.
- Pup viability index/mortality: The viability index indicating pup mortality during postnatal days (PND) 0-4 varied between 99.3% in test group 0 (control) and test group 3 (1000 mg/kg bw/d) and 100% in test groups 1 (100 mg/kg bw/d) and 2 (300 mg/kg bw/d). No treatment related effect as observed. The survival index indicating pup mortality on PND 4 – 13 was 100% in all test groups.
- Sex ratio: The sex distribution and sex ratios of live F1 pups on the day of birth and PND 13 did not show substantial differences between the control and the test substance-treated groups; slight differences were regarded to be spontaneous in nature.
- Pup clinical observations: One male pup (animal no. 139-06) of test group 3 (1000 mg/kg bw/day) and two male pups (animal no. 127-05 and 128-06) of test group 2 (300 mg/kg bw/d) were stillborn. One male pup (animal no. 106-05) of control group was found dead on PND 1. One male pup (animal no. 140-09) of test group 3 (1000 mg/kg bw/day) was cannibalized on PND3. These findings in its observe incidences are within the expected viability and was assessed as spontaneous in nature. All other F1 pups of any test group (0-3) did not show adverse clinical signs up to scheduled sacrifice on PND 4, resp. PND 13.
- Pup body weight data: Mean pup body weights of all pups in all test groups were comparable to the control group. Two female runts were seen in the control group on PND 1. Three male and 1 female runts were seen in test group 1 (100 mg/kg bw/d) and one male runt was seen in test group 2 (300 mg/kg bw/d) on PND 1. Three male runts were seen in test group 3 (1000 mg/kg bw/d) on PND 1. All values were within the range of the biological variation inherent in the strain of rats used for this study.
- Anogenital distance: Anogenital distance and anogenital distance index of male and female pups was not influenced in all test groups.
- Nipple/areola anlagen: The apparent number and percentage of male pups having areolae was not influenced by the test substance when examined on PND 13.
- Pup necropsy observations: One male pup of test group 3 (1000 mg/kg bw/d) showed an empty stomach and large liver. One male pup of test group 2 (300 mg/kg bw/d) showed a small testis. One male pup of test group 1 (100 mg/kg bw/d) showed a misshapened liver. One male pup of the control group showed an absent liver lobe. These findings occurred without any relation to dosing and were assessed as spontaneous in nature and not related to treatment. All other F1 pups of any test group (0-3) did not show adverse findings during necropsy.

Thyroid hormones:
- In parental males (main test groups 1, 2 and 3; 100, 300 and 1000 mg/kg bw/d) and in male and female pups at PND13 (main test groups 21, 22 and 23; 100, 300 and 1000 mg/kg bw/d), no treatment-related alterations of T4 and TSH levels were observed.

Details on results:

Regarding clinical examinations of parental animals, no treatment-related, adverse effects were observed in the main and recovery test groups investigating up to the nominal dose of the test substance 1000 mg/kg bw/d. In the subsequent investigations including the functional observational battery (FOB) and measurement of motor activity (MA) no treatment related, adverse differences to control
were observed at any dose level. Regarding clinical pathology the males of the high dose test groups 3 and 11 (1000 mg/kg bw/d) showed increased cholesterol levels indicating an altered liver cell metabolism. This is confirmed by significantly reduced prothrombin time (HQT). However, both alterations recovered totally after two weeks. Regarding pathology, there were no treatment-related organ weight changes, gross lesions, and histopathological findings in male and female Wistar rats in the main groups (F1) and the recovery groups (R1).
Regarding fertility and reproductive performance, no signs of toxicity were observed in male or female parental animals of all test groups investigating up to the nominal dose of the test substance 1000 mg/kg bw/d. Regarding developmental toxicity, no signs of toxicity were observed in male or female pups of all test groups investigating up to the nominal dose of the test substance 1000 mg/kg
bw/d. All findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Dose descriptor:

LOAEL

Remarks:

general systemic toxicity

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical biochemistry

haematology

Dose descriptor:

NOAEL

Remarks:

general systemic toxicity

Effect level:

300 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effect observed at this dose

Dose descriptor:

NOAEL

Remarks:

reproductive performance and fertility

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effect observed up to the highest tested dose.

Dose descriptor:

NOAEL

Remarks:

developmental toxicity (F1 progeny)

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effect observed up to the highest tested dose.

Critical effects observed:

no

Table 6: Haematology - males

 

 

		Test Group 0/ M 0 mg/kg bw/d	Test Group 1/ M 100 mg/kg bw/d	Test Group 2/ M 300 mg/kg bw/d	Test Group 3/ M 1000 mg/kg bw/d
RBC	Mean	8.73 k	8.53	8.87	9.03
[tera/L]	S.d.	0.23	0.28	0.38	0.17
day 30	N	5	5	5	5
	Median	8.71	8.50	8.95	9.06
	Deviation Vs Control [%]		-2.29	1.67	3.41
HGB	Mean	9.1 k	9.1	9.0	9.2
[mmol/L]	S.d.	0.1	0.1	0.3	0.3
day 30	N	5	5	5	5
	Median	9.0	9.1	9.2	9.3
	Deviation Vs Control [%]		0.4	-0.7	1.1
HCT	Mean	0.434 k	0.427	0.428	0.437
[L/L]	S.d.	0.002	0.005	0.009	0.008
day 30	N	5	5	5	5
	Median	0.434	0.427	0.426	0.436
	Deviation Vs Control [%]		-1.430	-1.384	0.738
MCV	Mean	49.7 k	50.1	48.2	48.4
[fL]	S.d.	1.3	1.4	2.1	0.4
day 30	N	5	5	5	5
	Median	49.5	50.7	46.9	48.5
	Deviation Vs Control [%]		0.8	-2.9	-2.6
MCH	Mean	1.04 k	1.07	1.02	1.02
[fmol]	S.d.	0.04	0.04	0.06	0.03
day 30	N	5	5	5	5
	Median	1.03	1.07	0.99	1.01
	Deviation Vs Control [%]		2.68	-2.11	-2.30
MCHC	Mean	20.96 k	21.37	21.20	21.11
[mmol/L]	S.d.	0.33	0.26	0.48	0.61
day 30	N	5	5	5	5
	Median	20.80	21.30	21.21	20.85
	Deviation Vs Control [%]		1.97	1.15	0.72
RETA	Mean	149.4 k	160.6	155.2	154.5
[giga/L]	S.d.	25.5	14.5	22.1	30.7
day 30	N	5	5	5	5
	Median	138.5	163.2	165.3	154.0
	Deviation Vs Control [%]		7.5	3.9	3.4
PLT	Mean	594 k	656	697	703
[giga/L]	S.d.	90	85	74	77
day 30	N	5	5	5	5
	Median	590	653	714	698
	Deviation Vs Control [%]		10	17	18
HQT	Mean	35.7 v	35.5	37.3	32.7 **
[sec]	S.d.	0.7	1.5	3.1	1.0
day 30	N	5	5	5	5
	Median	35.6	34.9	36.1	32.7
	Deviation Vs Control [%]		-0.6	4.5	-8.4

 

Statistic Profile = Kruskal-Wallis + Wilcoxon test (two-sided) * p<=0.05, ** p <=0.01, X = Group excluded from statistics k=KRUSKALL-WALLIS; v=KRUSKALL-WALLIS-WILCOX

Table 7: Clinical chemistry - males

 

 

		Test Group 0/ M 0 mg/kg bw/d	Test Group 1/ M 100 mg/kg bw/d	Test Group 2/ M 300 mg/kg bw/d	Test Group 3/ M 1000 mg/kg bw/d
UREA	Mean	4.44 k	4.42	4.43	5.26
[mmol/L]	S.d.	0.36	0.51	0.38	0.32
day 30	N	5	5	5	5
	Median	4.29	4.47	4.27	5.22
	Deviation Vs Control [%]		-0.59	-0.23	18.27
CREA	Mean	21.9 k	22.9	21.3	24.0
[µmol/L]	S.d.	2.6	2.2	1.2	1.5
day 30	N	5	5	5	5
	Median	21.2	22.8	21.5	24.2
	Deviation Vs Control [%]		4.6	-2.8	9.6
GLUC	Mean	7.40 k	6.92	6.21	6.36
[mmol/L]	S.d.	1.15	0.30	0.73	0.67
day 30	N	5	5	5	5
	Median	7.63	6.94	6.00	6.45
	Deviation Vs Control [%]		-6.51	-16.05	-14.05
TBIL C	Mean	1.57 k	1.47	1.33	1.68
[µmol/L]	S.d.	0.31	0.14	0.13	0.29
day 30	N	5	5	5	5
	Median	1.74	1.43	1.34	1.62
	Deviation Vs Control [%]		-6.66	-15.22	6.57
TBA	Mean	14.6 k	12.0	10.6	21.0
[µmol/L]	S.d.	8.5	4.8	6.6	8.4
day 30	N	5	5	5	5
	Median	11.2	12.3	6.8	22.2
	Deviation Vs Control [%]		-17.8	-27.4	43.4
TPROT	Mean	63.07 k	64.56	64.50	65.98
[g/L]	S.d.	1.10	0.62	1.25	2.36
day 30	N	5	5	5	5
	Median	63.36	64.36	64.54	66.52
	Deviation Vs Control [%]		2.37	2.27	4.61
ALB	Mean	36.92 k	37.69	37.26	37.74
[g/L]	S.d.	1.36	0.98	0.51	0.96
day 30	N	5	5	5	5
	Median	36.97	37.53	37.48	37.70
	Deviation Vs Control [%]		2.09	0.93	2.23
GLOB	Mean	26.15 k	26.87	27.24	28.24
[g/L]	S.d.	0.59	0.82	1.14	1.42
day 30	N	5	5	5	5
	Median	26.05	26.83	26.89	28.67
	Deviation Vs Control [%]		2.76	4.17	7.98
CHOL	Mean	1.77 v	2.00	2.17	2.80 **
[mmol/L]	S.d.	0.31	0.32	0.35	0.38
day 30	N	5	5	5	5
	Median	1.68	1.89	2.10	2.79
	Deviation Vs Control [%]		12.51	22.32	58.06
TRIG	Mean	0.77 k	0.83	0.72	0.75
[mmol/L]	S.d.	0.21	0.36	0.15	0.13
day 30	N	5	5	5	5
	Median	0.83	0.62	0.70	0.78
	Deviation Vs Control [%]		7.55	-5.99	-2.86

 

Statistic Profile = Kruskal-Wallis + Wilcoxon test (two-sided) * p<=0.05, ** p <=0.01, X = Group excluded from statistics k=KRUSKALL-WALLIS; v=KRUSKALL-WALLIS-WILCOX

Table 8: Haematology – males, recovery group

 

 

		Test Group 10/ M 0 mg/kg bw/d	Test Group 11/ M 1000 mg/kg bw/d
UREA	Mean	4.81 x	5.50
[mmol/L]	S.d.	0.43	0.59
day 30	N	5	5
	Median	4.68	5.53
	Deviation Vs Control [%]		14.38
CREA	Mean	23.0 x	24.9
[µmol/L]	S.d.	3.1	1.7
day 30	N	5	5
	Median	23.1	24.5
	Deviation Vs Control [%]		8.2
GLUC	Mean	6.80 x	6.22
[mmol/L]	S.d.	0.55	0.51
day 30	N	5	5
	Median	6.84	6.49
	Deviation Vs Control [%]		-8.50
TBIL C	Mean	1.54 x	1.62
[µmol/L]	S.d.	0.27	0.19
day 30	N	5	5
	Median	1.53	1.69
	Deviation Vs Control [%]		5.34
TBA	Mean	22.8 x	24.3
[µmol/L]	S.d.	6.5	10.7
day 30	N	5	5
	Median	20.7	24.4
	Deviation Vs Control [%]		6.5
TPROT	Mean	65.05 x	66.07
[g/L]	S.d.	1.03	1.26
day 30	N	5	5
	Median	64.87	65.51
	Deviation Vs Control [%]		1.57
ALB	Mean	37.37 x	38.21 **
[g/L]	S.d.	0.23	0.21
day 30	N	5	5
	Median	37.42	38.33
	Deviation Vs Control [%]		2.24
GLOB	Mean	27.68 x	27.86
[g/L]	S.d.	0.81	1.42
day 30	N	5	5
	Median	27.45	27.18
	Deviation Vs Control [%]		0.66
CHOL	Mean	1.96 x	2.64 *
[mmol/L]	S.d.	0.30	0.22
day 30	N	5	5
	Median	1.96	2.70
	Deviation Vs Control [%]		34.80
TRIG	Mean	1.12 x	1.19
[mmol/L]	S.d.	0.38	0.41
day 30	N	5	5
	Median	1.25	1.41
	Deviation Vs Control [%]		6.61

 

Statistic Profile = Wilcoxon test (two-sided), *p<=0.05, **p<=0.01, X = Group excluded from statistics

x=WILCOX

Table 9: Absolute organ weights of main groups

 

	Male animals			Female animals
Test group (mg/kg bw/d)	1 (100)	2 (300)	3 (1000)	1 (100)	2 (300)	3 (1000)
Epididymides	+6.04%	+0.88%	+8.32%*
Prostate	+16.8%**	+23.76%**	+24.59%**	%	%	%
Thymus	%	%	%	-21.982%*	+13.453%	-1.141%

*p <= 0.05; **p <= 0.01

 

Table 10: Relative organ weights of main groups

 

	Male animals
Test group (mg/kg bw/d)	1 (100)	2 (300)	3 (1000)
Prostate	+15.1%*	+22.27%*	+22.05%**

*p <= 0.05; **p <= 0.01

 

Table 11: Relative organ weights of the recovery group

 

	Male animals
Test group (mg/kg bw/d)	11 (1000)
Heart	+11.7%*

*p <= 0.05; **p <= 0.01

 

Conclusions:

The no observed adverse effect level (NOAEL) of general systemic toxicity was 300 mg/kg bw/d for male and 1000 mg/kg bw/d for female parental animals.

Executive summary:

The repeated dose toxicity of the test substance was investigated by means of a Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test according to OECD guideline 422 and GLP.

The test substance was administered daily by gavage as a solution to groups of 10 male and 10 female Wistar rats (F0 animals) at nominal doses of 0, 100, 300 and 1000 mg/kg body weight/day (mg/kg bw/d). Control animals were dosed daily with the vehicle only (Corn oil Ph.Eur. 8.0). The duration of treatment covered a 2-week premating period and mating in both sexes (mating pairs were from the same dose group) as well as entire gestation and lactation period in females up to one day prior to the day of schedule sacrifice of the animals. Additional treated but not mated animals (recovery animals) to groups of 5 male and 5 female animals at nominal doses of 0 and 1000 mg/kg bw/d was maintained for a subsequent period of at least 14 days of no test substance administration in order to observe reversibility of the findings.

The parents' and the pups' state of health was checked each day, and parental animals were examined for their mating and reproductive performances. F0 animals were mated 13 days after the beginning of treatment to produce a litter (F1 generation pups). As soon as sperm was detected in the vaginal smear, mating was discontinued. F0 animals were examined for their reproductive performance including determinations of the number of implantations and the calculation of the postimplantation loss in all F0 females. A detailed clinical observation (DCO) was performed in all animals before initial test substance administration and, as a rule, thereafter at weekly intervals. Food consumption of the F0 parents of main groups and recovery groups were determined regularly once weekly before mating period as well as males and females of the recovery groups until sacrifice. In dams during gestation days 0-7,7-14 and 14-20 and lactation days 1-4, 4-7, 7-10 and 10-13 the food consumption was also determined. In general, the body weights of F0 animals were determined once a week. However, during gestation and lactation, F0 females were weighed on gestation days (GD) 0, 7, 14 and 20, and on postnatal days (PND) 0, 4, 7, 10 and 13. The pups were sexed and examined for macroscopically evident changes on PND 0. They were weighed on PNDs 1, 4, 7 and on PND 13 and their viability was recorded. Anogenital distance (defined as the distance from the anus [center of the anal opening] to the base of the genital tubercle) measurements were conducted in a blind randomized fashion, using a measuring ocular on all live male and female pups on PND 1. On PND 4, the individual litters were standardized in such a way that, whenever possible, each litter contains 4 male and 4 female pups (as a rule, the first 4 surviving pups/sex in each litter were taken for further rearing). On PND 13, all male F1 pups were examined for retention of nipples/areolae. The number of nipples/areola anlagen were counted. At necropsy on PND 4, all pups were sacrificed under isoflurane anesthesia by decapitation. Blood was sampled for determination of thyroid hormone concentrations. After sacrifice, the pups were examined externally and eviscerated, and their organs were assessed macroscopically. At necropsy on PND 13, one selected male and one female pup per litter were sacrificed under isoflurane anesthesia by decapitation. Blood was sampled for determination of thyroid hormone concentrations. Thyroid glands/parathyroid glands were fixed in neutral buffered 4% formaldehyde solution and were transferred to the Pathology Laboratory for possible further processing. The remaining pups were sacrificed under isoflurane anesthesia with CO2. After sacrifice, all pups were examined externally and eviscerated, and their organs were assessed macroscopically. Towards the end of the administration period a functional observational battery was performed and motor activity was measured in 5 animals per sex and test group. Clinicochemical and hematological examinations were performed in 5 animals per sex and group towards the end of the administration period as well as towards the end of recovery period. All F0 parental animals were sacrificed by decapitation, under isoflurane anesthesia, and were assessed by gross pathology. Weights of selected organs were recorded and a histopathological examination was performed. The remaining 5 animals per sex of test groups 10 and 11 (recovery animals) were maintained for a 2-week recovery period after the administration period without test substance exposure. Further examinations in these animals were depend on the findings observed in the animals of the main groups. No functional observational battery was performed as well as no motor activity and estrous cycle were determined during the recovery period.

 

The various analyses confirmed

·        the stability of the test substance in corn oil at room temperature over a period of 7 days,

·        and the correctness of the prepared concentrations.

 

The following test substance-related adverse effects/findings were noted:

 

Test group 3/11: 1000 mg/kg bw/d

F0 PARENTAL ANIMALS

Clinical examinations, reproductive performance and pathology

·        no test substance-related adverse findings

Clinical pathology

·        reduced prothrombin time (HQT) in males (test group 3, only)

·        increased cholesterol values in males

F1 PUPS

Clinical examinations/ gross findings

·        no test substance-related adverse findings

 

Test group 2: 300 mg/kg bw/d

F0 PARENTAL ANIMALS

Clinical examinations, reproductive performance, clinical pathology, and pathology

·        no test substance-related adverse findings

F1 PUPS

Clinical examinations/ gross findings

·        no test substance-related adverse findings

 

Test group 1: 100 mg/kg bw/d

F0 PARENTAL ANIMALS

Clinical examinations, reproductive performance, clinical pathology, and pathology

·        no test substance-related adverse findings

F1 PUPS

Clinical examinations/ gross findings

·        no test substance-related adverse findings

 

Under the conditions of this Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, the oral administration of the test substance by gavage to Wistar rats revealed signs of systemic toxicity manifested in clinical pathology parameters of males in the limit dose group (1000 mg/kg bw/d) at the end of the administration period. However, the clinical pathological alterations were no permanent changes, since no treatment-related findings were noted after the two weeks recovery period. No treatment-related adverse findings were observed in males at 100 and 300 mg/kg bw/d as well as in all female parental animals (F0) and all pups (F1).

The no observed adverse effect level (NOAEL) of general systemic toxicity was 300 mg/kg bw/d for male and 1000 mg/kg bw/d for female parental animals.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

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)

Version / remarks:

29 Jul 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

3-(acetyloxy)-2,2-dimethylpropyl acetate

EC Number:

826-122-1

Cas Number:

13431-57-7

Molecular formula:

C9 H16 O4

IUPAC Name:

3-(acetyloxy)-2,2-dimethylpropyl acetate

Test material form:

liquid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Lot/batch No.of test material:
ZH 472 redestilliert.
- Storage stability: until Jan 2020.

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:
room temperature.
- Stability under test conditions:
The stability of the test substance in solution in corn oil over a period of 7 days at room temperature was proven. the mixtures were stored no longer than this time period,
the stability was guaranteed.
- Solubility and stability of the test substance in the solvent/vehicle: The test substance is completely miscible with corn oil Ph.Eur 8.0 and thus a solution. Therefore, the test-substance preparation was considered to be homogenous. Consequently, no further homogeneity analysis was performed.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:
The test substance was applied as a solution. To prepare this solution, the appropriate amount of test substance was weighed out depending on the desired concentration. Then, Corn oil Ph.Eur. 8.0 was filled up to the desired volume and intensely mixed with a magnetic stirrer. The test substance preparations were produced weekly, at least.

FORM AS APPLIED IN THE TEST (if different from that of starting material)
: solution in corn oil.

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Crl:WI(Han)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH.
- Females (if applicable) nulliparous and non-pregnant: yes.
- Age at study initiation: age at supply about 12 - 13 weeks (male animals), about 11 weeks (female animals).
- Weight at study initiation: mean weight 393 g (males) and 212 g (females).
- Fasting period before study: no.
- Housing: During pre-treatment: Polysulfonate cages Typ 2000P (H-Temp), floor area about 2065 cm2 (610 x 435 x 215 mm); supplied by TECHNIPLAST, Hohenpeißenberg, Germany During premating, mating, gestation, lactation, males after mating and females after weaning: Polycarbonate cages type III. Pregnant animals and their litters were housed together until PND 13 (end of lactation). For motor activity (MA) measurements the animals were housed individually in polycarbonate cages type III supplied by TECNIPLAST, Hohenpeißenberg, Germany with wire covers from Ehret, Emmendingen, Germany (floor area of about 800 cm2) and small amounts of bedding material Pregnant females were provided with nesting material (cellulose wadding) toward the end of gestation. For enrichment wooden gnawing blocks Lignocel® block large (supplied by J. Rettenmaier & Söhne GmbH + Co KG, Rosenberg, Germany) were added. In addition, in Polysulfonate cages large play tunnels (Art. 14153; supplied by PLEXX b.v., Elst, Netherlands) were added.
- Diet: ad libitum.
- Water: ad libitum.
- Acclimation period: 21 days.

ENVIRONMENTAL CONDITIONS
- The cages with the test animals were arranged on the racks in such a way that uniform experimental conditions (ventilation and light) were ensured. The animals were housed in a fully air-conditioned room.
- Temperature (°C): 20-24°C.
- Humidity (%): 45-65% for relative humidity.
- Air changes (per hr): 15 air changes per hour.
- Photoperiod (hrs dark / hrs light): The day/night cycle was 12 hours (12 hours light from 06.00- 18.00 h, 12 hours dark from 18.00-06.00 h).

IN-LIFE DATES: From: 19 Jun 2018 To: 10 Sep 2018.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Remarks:

Ph.Eur 8.0

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- The test substance was applied as a solution. To prepare this solution, the appropriate amount of test substance was weighed out depending on the desired concentration. Then, Corn oil Ph.Eur. 8.0 was filled up to the desired volume and intensely mixed with a magnetic stirrer. The test substance preparations were produced weekly, at least.

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: overnight (from about 16.00 h until 06.30 - 09.00 h of the following morning) for a maximum of 2 weeks
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy (gestation day (GD) 0)
- After successful mating each pregnant female was caged (how): Pregnant females were provided with nesting material (cellulose wadding) toward the end of gestation.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability of the test substance in corn oil Ph.Eur 8.0 was demonstrated over a period of 7 days at room temperature. As the mixtures were stored no longer than this time period, the stability was guaranteed.
The concentrations of the test substance in corn oil Ph.Eur 8.0 were found to be in the range of 99.5 to 105.4% of the nominal concentrations. These results demonstrated the correctness of the concentrations of the test substance in corn oil Ph.Eur 8.0 being within the specification of the test facility 90-110%.

Duration of treatment / exposure:

males: 29 days; females: 61 days
The duration of treatment covered a 2-week premating period and mating in both sexes (mating pairs were from the same dose group) as well as entire gestation and lactation period in females up to one day prior to the day of schedule sacrifice of the animals. Additional treated but not mated animals (recovery animals) to groups of 5 male and 5 female animals at nominal doses of 0 and 1000 mg/kg bw/d was maintained for a subsequent period of at least 14 days of no test substance administration in order to observe reversibility of the findings.

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Remarks:

Test group 1

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Remarks:

Test group 2

Doses / concentrations 3

Dose / conc.:

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

Remarks:

Test group 3

Doses / concentrations 4

Dose / conc.:

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

Remarks:

Test group 11 (Recovery animals)

No. of animals per sex per dose:

Test group 0, 1, 2, 3: 10 animals per sex per dose
Test group 10, 11 (Recovery animals): 5 animals per sex per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: In accordance with the guideline, three doses were chosen which were applied in two- to four- fold intervals and it was tested up to the limit dose of 1000 mg/kg bw/day.
- Fasting period before blood sampling for clinical biochemistry: The adults were fastened before the blood sampling. A fasting period (withdrawal of food) of about 16 to 20 hours was applied.
- Post-exposure recovery period in satellite groups: yes. Additional treated but not mated animals (recovery animals) to groups of 5 male and 5 female animals at nominal doses of 0 mg/kg bw/d (test group 10) and 1000 mg/kg (test group 11) bw/d were maintained for a subsequent period of at least 14 days of no test substance administration in order to observe reversibility of the findings.

Positive control:

no.

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes.
- Time schedule: daily.
- Mortality: A check for moribund and dead animals was made twice daily on working days and once daily on Saturdays, Sundays and public holidays. If animals were in a moribund state, they were sacrificed and necropsied.
- Clinical observations: A cageside examination was conducted at least once daily for any signs of morbidity, pertinent behavioral changes and/or signs of overt toxicity. All animals were checked daily for any abnormal clinical signs before the administration as well as within 2 hours and within 5 hours after the administration. Abnormalities and changes were documented for each animal. The parturition and lactation behavior of the dams was generally be evaluated in the morning in combination with the daily clinical inspection of the dams. Only particular findings (e.g. disability to deliver or umbilical cord not cut) were documented on an individual dam basis. On weekdays (except Saturdays, Sundays 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 to be the 24-hour period from about 15:00 h of one day until about 15:00 h of the following day.

DETAILED CLINICAL OBSERVATIONS: Yes.
- Time schedule: prior to administration and weekly thereafter.
- 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: abnormal behavior in handling, fur, skin, posture, salivation, respiration, activity/arousal level, tremors, convulsions, abnormal movements, gait abnormalities, lacrimation, palpebral closure, exophthalmos, assessment of the feces discharged during the examination (appearance/ consistency), assessment of the urine discharged during the examination, pupil size.

BODY WEIGHT: Yes.
- Time schedule for examinations: before the beginning administration, during administration at day 0 and weekly thereafter.
- Body weight of the male and female parental animals in the main groups as well as the males and females of recovery groups 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 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), PNDs 4, 7, 10 and 13.
-- Females showing no positive evidence of sperm in the vaginal smear were weighed once a week during this mating interval as were the males (for the calculation of the administration volume)
-- Females without litter and after weaning (PND 13) were weighed once a week (for the calculation of the administration volume).

FOOD CONSUMPTION: Yes.
- Time schedule: weekly.
- Generally, food consumption was determined once a week for male and female parental animals of the main groups as well as the males and females of recovery groups, with the following exceptions:
-- Food consumption was not be determined after the 2nd premating week (male parental animals) and during the mating period (male and female parental animals in the main groups).
-- Food consumption of the females with evidence of sperm was determined for GD 0-7, 7-14 and 14-20.
-- Food consumption of the females which gave birth to a litter was determined for PNDs 1-4, 4-7, 7-10 and 10-13.
- 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.

WATER CONSUMPTION: Yes.
- Time schedule for examinations: daily.
- Drinking water consumption was monitored by daily visual inspection of the water bottles for any changes in volume.


OTHER:

HAEMATOLOGY: Yes.
- Time schedule for collection of blood: for main groups at day 30 (males) and day 50 (females), for recovery groups at day 30/48 (males) at day 62/77 (females).
- Anaesthetic used for blood collection: Yes (isoflurane).
- Animals fasted: Yes.
- How many animals: 5 males and 5 females.
- Parameters checked in table 1 were examined.
- In the morning blood was taken from the retro-bulbar venous plexus from fasted animals. The animals were anaesthetized using isoflurane. The blood sampling procedure and subsequent analysis of blood and serum samples were carried out in a randomized sequence. The assays of blood and serum parameters were performed under internal laboratory quality control conditions with reference controls to assure reliable test results. The results were expressed in International System (SI) units. The parameters listed were examined in the first 5 surviving parental males per group at termination and in the first 5 females with litters (in order of delivery) per group at PND 14 and in the recovery groups at the end of the administration period and at the end of the recovery period.

CLINICAL CHEMISTRY: Yes.
- Time schedule for collection of blood: for main groups at day 30 (males) and day 50 (females), for recovery groups at day 30/48 (males) at day 62/77 (females).
- Animals fasted: Yes.
- How many animals: 5 males and 5 females.
- Parameters checked in table 2 were examined.
- In the morning blood was taken from the retro-bulbar venous plexus from fasted animals. The animals were anaesthetized using isoflurane. The blood sampling procedure and subsequent analysis of blood and serum samples were carried out in a randomized sequence. The assays of blood and serum parameters were performed under internal laboratory quality control conditions with reference controls to assure reliable test results. The results were expressed in International System (SI) units. The parameters listed were examined in the first 5 surviving parental males per group at termination and in the first 5 females with litters (in order of delivery) per group at PND 14 and in the recovery groups at the end of the administration period and at the end of the recovery period.

NEUROBEHAVIOURAL EXAMINATION: Yes; functional observational battery and motor activity assessment.
- Time schedule: FOB and MA at study day 28 (males) and day 57 (females)
FUNCTIONAL OBSERVATIONAL BATTERY:
- A functional observational battery (FOB) was performed in the first five parental male animals per test group and the first five surviving females with litter (in order of delivery) of all main test groups 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.
-Home cage observations: The animals were observed in their closed home cages; during this period 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: Posture, Tremors, Convulsions, Abnormal movements, Gait, Other findings.
- Open field observations: The animals were transferred to a standard arena (50 × 50 cm with sides of 25 cm height) and observed for at least 2 minutes. The following parameters were examined: behavior on removal from the cage, fur, skin, salivation, nasal discharge, lacrimation, eyes/pupil size, posture, palpebral closure, respiration, tremors, convulsions, abnormal movements/stereotypes, gait, activity/arousal level, feces excreted within 2 minutes (appearance/ consistency), urine excreted within 2 minutes (amount/color), rearing within 2 minutes, other findings.
- Sensory motor tests/ reflexes: The animals were then removed from the open field and subjected to following sensory motor or reflex tests: Reaction to an object being moved towards the face (approach response), touch sensitivity (touch response), vision (visual placing response), pupillary reflex, pinna reflex, audition (auditory startle response), coordination of movements (righting response), behavior during handling, vocalization, pain perception (tail pinch), other findings, grip strength of forelimbs, grip strength of hindlimbs, landing foot-splay test.
MOTOR ACTIVITY ASSESSMENT:
Motor activity (MA) was also measured from 14:00 h onwards on the same day as the FOB was performed in the first five parental males and the first five surviving females with litter (in order of delivery) per main group. 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 with a small amount of bedding for the duration of the measurement. Eighteen beams were allocated per cage. The numbers of beam interrupts was counted over 12 intervals of 5 minutes per interval. The sequence in which the animals were placed in the cages was selected at random. On account of the time needed to place the rats in the cages, the starting time was "staggered" for each animal. The measurement period began when the 1st beam was interrupted and finished exactly 1 hour later. No food or water was offered to the animals during these measurements and the measurement room was darkened after the transfer of the last animal. The program required a file name for the measured data to be stored. This name consisted of the reference number and a serial number.

THYROID HORMONES:
- Parameters checked in table 3 were examined.
- Blood samples were taken from all surplus pups per litter at PND 4 as well as one male and one female pup per litter at PND 13 by decapitation under isoflurane anesthesia. Additionally, blood samples from all dams at PND 14 and all males at termination of the main groups as well as all males and females after the administration period and at termination of the recovery groups were taken by puncturing the retrobulbar venous plexus under isoflurane anesthesia. The adults were fastened before the blood sampling. All generated serum samples were frozen at -80°C until measurement. Blood samples from the adult males and the PND 13 pups were assessed for serum levels for thyroid hormones (T4 and TSH). The concentrations of TSH were determined by radioimmunoassay (RIA), using commercially available RIA test kits and a Gamma-Counter (LB 2111, Berthold, Germany). T4 Elisa was measured with a Sunrise MTP-reader, Tecan AG, Maennedorf, Switzerland, and evaluated with the Magellan-Software of the instrument producer.

Oestrous cyclicity (parental animals):

ESTROUS CYCLE:
- For all females of the pool estrous cycle normality was evaluated before the beginning of the administration. In all parental females in the main groups in the premating phase, estrous cycle length and normality was evaluated by preparing vaginal smears during a minimum of 2 weeks prior to premating, mating and throughout cohabitation until there is evidence of sperm in the vaginal smear. Additionally, on the day of scheduled sacrifice, the estrous status was also be determined in all female F0 main groups rats.

Sperm parameters (parental animals):

Parameters examined in parental males:
testis weight, epididymides weight, prostate weight, weight of seminal vesicles with coagulating glands; histopathological examination of epididymides, prostate gland, seminal vesicles, testes; spermatogenic staging profiles.

Litter observations:

LITTER/PUB DATA:
- 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. Dead pups were evaluated by the methods, which are described in detail in “Necropsy observations”. The number and percentage of dead pups on the day of birth (PND 0) and of pups dying between PND 1-4, 5-7 and 8-13 were determined. Pups, which died accidentally or had to be sacrificed due to maternal death, were not included in these calculations. The number of live pups/litter was calculated on the day of birth (PND 0), and on lactation days 4, 7 and 13. Furthermore, viability and survival indices were calculated (for formulas see "Any other information on materials and methods").
- 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. Later, during the course of lactation, this initial sex determination was followed up by surveying the external appearance of the anogenital region and the mammary line. The sex of the pups was finally confirmed at necropsy. The sex ratio was calculated at PND 0 and PND 13 (for formulas see "Any other information on materials and methods").
- 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) as well as on PND 4 (before standardization), 7 and 13. 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). In the summary tables 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.
- Nipple/areola anlagen: All surviving male pups were examined for the presence of nipple/areola anlagen on PND 13 of the lactation phase. The number of nipple/areola anlagen was counted.
- Anogenital distance: Anogenital distance (AGD; defined as the distance from the anus [center of the anal opening] to the base of the genital tubercle) measurements was done blind to treatment in a randomized order, using a measuring ocular, on all live male, female and uncertain pups on day 1 after birth. The anogenital index were calculated (for formulas see "Any other information on materials and methods").
- Pup necropsy observations: On PND 4, as a result of standardization, the surplus pups or 2 preferably female pups per litter, respectively, was sacrificed under isoflurane anesthesia by decapitation. Blood was sampled for determination of thyroid hormone concentrations. After sacrifice, the pups were examined externally and eviscerated, and their organs were assessed macroscopically. On PND 13, one selected male and one female pup per litter was sacrificed under isoflurane anesthesia by decapitation. Blood was sampled for determination of thyroid hormone concentrations. Thyroid glands/parathyroid glands were fixed in neutral buffered 4% formaldehyde solution and was transferred to the Pathology Laboratory for possible further processing. The remaining pups were sacrificed under isoflurane anesthesia with CO2. After sacrifice, all pups were examined externally and eviscerated, and their organs were assessed macroscopically. All stillborn pups and all pups that died before day 13 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-bycase basis, depending on the type of finding noted.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals of the main groups on study day 30, animals of the recovery group on day 48.
- Maternal animals: All surviving animals of the main groups on study day 61, animals of the recovery group on day 77.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table 4 were prepared for microscopic examination and weighed, respectively.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring were sacrificed at 4 (surplus pups after litter standardization) and 13 (remaining pups after litter standardization) days of age.
- These animals were subjected to postmortem examinations (macroscopic and microscopic (if required) as follows: the pups were examined externally and eviscerated, and the organs were assessed macroscopically).

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY
- Thyroid glands/parathyroid glands of one male and one female pup per litter at 13 days of age were fixed in neutral buffered 4% formaldehyde solution for possible further processing.

Statistics:

See table 5.

Reproductive indices:

MALE REPRODUCTION DATA:
- 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 (for formulas see "Any other information on materials and methods").

FEMALE REPRODUCTION AND DELIVERY DATA:
- The pairing partners, the number of mating days until vaginal sperm were detected and gestational status were recorded for F0 females.
- For the females, mating, fertility and gestation indices were calculated for F1 litters (for formulas see "Any other information on materials and methods").

Offspring viability indices:

- viability index, survival index (for formulas see "Any other information on materials and methods").

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Males and females and recovery animals:
Salivation within 2 hours after administration was observed from slight to moderate in test group 3 (1000 mg/kg bw/d) in all male and 9 female animals during premating, in all male and 4 female animals during mating phase, in 3 male animals during post-mating phase as well as in all male and female animals of recovery group 11 (1000 mg/kg bw/d). In test group 2 (300 mg/kg bw/d) one male animal during mating period and one male animal during post-mating salivation within 2 hours after administration was observed. The salivation observed only shortly after treatment was assessed as treatment-related but not adverse. It was considered to be related to a slight local reaction to the test substance preparation by a bitter or bad taste. No test substance-related, adverse findings were observed in male and female animals of test groups 1-3 (100, 300, and 1000 mg/kg bw/d) and recovery group 11 (1000 mg/kg bw/d).

Females during gestation:
Salivation shortly after treatment (<2 hours after treatment) was observed from slight to moderate in all female animals of test group 3 (1000 mg/kg bw/d) during gestation. The salivation observed only shortly after treatment was assessed as treatment-related but not adverse. It was considered to be related to a slight local reaction to test substance preparation by a bitter or bad taste. No test substance-related, adverse findings were observed in test groups 1-3 (100, 300, and 1000 mg/kg bw/d) during gestation.

Females during gestation:
Salivation shortly after treatment (<2 hours after treatment) was observed from slight to moderate in all female animals of test group 3 (1000 mg/kg bw/d) during gestation. The salivation observed only shortly after treatment was assessed as treatment-related but not adverse. It was considered to be related to a slight local reaction to test substance preparation by a bitter or bad taste. No test substance-related, adverse findings were observed in test groups 1-3 (100, 300, and 1000 mg/kg bw/d) during gestation.

Clinical observations for females during lactation
Slight salivation shortly after treatment (<2 hours after treatment) was observed in all female animals of test groups 3 (1000 mg/kg bw/d) during lactation. The salivation observed only shortly after treatment was assessed as treatment-related but not adverse. It was considered to be related to a slight local reaction to test substance preparation by a bitter or bad taste. No test substance-related, adverse findings were observed in test groups 1-3 (100, 300, and 1000 mg/kg bw/d) during gestation.

Detailed clinical observations:
In the detailed clinical observations on study days 0, 7, 14, 21 and 28 in parental males and females and additionally, on study days 35, 42, 49 and 56 in parental female animals and in the recovery period of males and females (as well as in the recovery females on study days 63 and 70) no adverse findings were observed.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

No animal died prematurely in the present study.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No test substance-related changes in mean body weights and body weight gain were observed for male and female animals of test groups 1-3 (100, 300 and 1000 mg/kg bw/d) when compared to the control group. Slightly reduced body weight was observed in male animals of recovery group 11 (1000 mg/kg bw/d) only on study day 13 during the in-life phase. Mean body weight gain was significantly decreased in male animals of recovery group 11 (1000 mg/kg bw/d) during the recovery phase between study days 38 to 45 only. Since the body weight of male animals in the recovery group 11 (1000 mg/kg bw/d) was comparable to the current control, the findings were assessed to be related to treatment, but not adverse.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption of the males and females in all dose groups (100, 300 and 1000 mg/kg bw/d) including recovery group was not influenced by the treatment throughout the entire study period.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

no effects observed

Description (incidence and severity):

No test substance-related findings were observed.

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Main groups:
At the end of the administration period in male rats of the main test group 3 (1000 mg/kg bw/d) prothrombin time (Hepatoquick’s test, HQT) was significantly reduced (see table 6). This alteration combined with the cholesterol increase in the same individuals was regarded as treatment-related and adverse.

Recovery group:
At the end of the administration period of the males in the recovery group 11 (1000 mg/kg bw/d) absolute basophil cell counts an in females of the same test group absolute and relative large unstained cell (LUC) counts were significantly increased, but all values were within historical control ranges (males, absolute basophils 0.00-0.02 Giga/L; females, absolute LUC 0.01-0.02 Giga/L, relative LUC 0.2-0.6 %).
At the end of the two-week recovery phase in males of test group 11 (1000 mg/kg bw/d) hemoglobin values were significantly increased, but th values were within the historical control range (males, hemoglobin 8.6-9.5 mmol/L). Therefore, all mentioned changes in rats of test group 11 were regarded as incidental and not treatment-related.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

At the end of the administration period in males of the main test group 3 (1000 mg/kg bw/d) as well as in males of the recovery test group 11 (1000 mg/kg bw/d) cholesterol values were significantly increased (see table 7 and table 8). This change in combination with the reduced prothrombin time was regarded as treatment-related and adverse.
Additionally, after the administration period in males of the recovery test group 11 (1000 mg/kg bw/d) albumin values were significantly increased. The mean was above the historical control range (males, albumin 34.09-37.97 g/L). However, this change was not present in the equally dosed males of the main test group 3 (1000 mg/kg bw/d). Therefore, this change in the recovery males after the administration was regarded as incidental and not treatment-related.
After the two-week recovery period in males of test group 11 (1000 mg/kg bw/d) sodium levels were significantly increased. The values of males in the control group 10 as well as in the test group 11 were above the historical control range (males, sodium 140.0-148.1 mmol/L). Because no electrolyte value change occurred after the administration period, the solely change of sodium after the recovery period was regarded as incidental and not treatment-related. After the recovery, in females of test group 11 (1000 mg/kg bw/d) alkaline phosphatase (ALP) activities were significantly decreased. The ALP means in females of the controls and test group 11 were below the historical control range (females, ALP 0.47-
0.76 μkat/L). The reason for this may be that the individuals of the historical controls were 6 weeks younger compared to the rats of the present study. The most probable cause for lower ALP levels is a growth retardation. However, females of test group 11 did not have significant lower terminal body weights compared to the study controls. Therefore, the statistically significant lower ALP activities in females of test group 11 were regarded as incidental and not treatment-related.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

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, without a dose-response relationship or occurred in single animals only, these observations were considered as incidental. There were no test substance-related findings in male and female animals of all test groups. Following non-significant alterations were assessed as spontaneous in nature and not related to treatment, because of missing dose-dependency: The foot splay test showed decreased results in males of test group 1 (100 mg/kg bw/d; -11%).

Motor activity measurement:
Regarding the individual intervals as well as the overall motor activity, no test substance related deviations were noted for male and female animals.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Main groups:
- All findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.
- The stages of spermatogenesis in the testes of males of the high dose test group were comparable to those of the controls. In high dose females the different stages of functional bodies in the ovaries were present and comparable to the control animals.
- Fertility: The female animal (No. 129, test group 2, 300 mg/kg bw/d), which was not pregnant as well as the male mating partner (No. 029) did not show relevant histopathological findings.

Recovery group:
- The macroscopically diagnosed yellow focus in the vagina in one female of test group 11 (1000 mg/kg bw/d) correlated with a cyst, histopathologically that was considered to be incidental.
- The vagina of all other females, all other organs of females and all organs of all male animals were not examined, histopathologically.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Thyroid hormones:
- In parental males (main test groups 1, 2 and 3; 100, 300 and 1000 mg/kg bw/d), no treatment-related alterations of T4 and TSH levels were observed.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Estrous cycle data revealed regular cycles in the rearing F1 females of all test groups including the control. In the different test groups (0-3) the mean number of estrous cycle ranged from 2.5 to 2.8 days and the estrous cycle duration ranged from 3.9 to 4.0 days. Since no treatment-related effect was observed the estrous cycle determination was not carried out in the recovery female animals during the recovery period.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

The stages of spermatogenesis in the testes of males of the high dose test group were comparable to those of the controls.

Reproductive performance:

no effects observed

Description (incidence and severity):

Male reproduction data:
- Male mating index: The male mating index calculated after the mating period for F1 litter varied between 90% in test group 2 (300 mg/kg bw/d) and 100% in test group 0 (control), 1 (100 mg/kg bw/d) and 3 (1000 mg/kg bw/d).
- Male fertility index: Fertility was proven for most of the F0 parental males within the scheduled mating interval to produce F1 litter. Male animal No. 29 of test group 2 (300 mg/kg bw/d), which was tried to be mated with female No. 129, did neither had a confirmed copulation nor generate F1 pups. Thus, the male fertility index was 90% in test group 2 and 100% in test groups 0, 1 and 3. These values 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 reproduction and delivery data:
- Female mating index: The female mating index calculated after the mating period for F1 litter varied between 90% in test group 2 (300 mg/kg bw/d) and 100% in test group 0 (control), 1 (100 mg/kg bw/d) and 3 (1000 mg/kg bw/d). The mean duration until sperm was detected (GD 0) was 2.2 days for test group 0 (control), 3.9 days for test group 1 (100 mg/kg bw/d), 3.3 days for test group 2 (300 mg/kg bw/d), as well as 2.4 days for test groups 3 (1000 mg/kg bw/d). These values 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 (1.6 – 5.8 mating days until day 0
p.c.). This includes also the statistically significant value of 3.3 days in test group 2 (300 mg/kg bw/d).
- Female fertility index: All sperm positive females had implants. Thus, the female fertility index was 100% in all test groups 0-3.
- Gestation index: The gestation index was 100% in all test groups.
- Live birth indices: The rate live birth indices were 100% in test groups 0 and 1 (100 mg/kg bw/d), 98.1% in test group 2 (300 mg/kg bw/d) and 99.2% in test group 3 (1000 mg/kg bw/d). No treatment related effect was observed.
- Postimplantation loss: The postimplantation loss was 5.7% in test group 0 (control), 4.5% in test group 1 (100 mg/kg bw/d), 10.1% in test group 2 (300 mg/kg bw/d) and 3.8% in test group 3 (1000 mg/kg bw/d). These findings 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 (0.0 – 18.1%).
- Pup number and status at delivery: The mean number of delivered F1 pups per dam was equally distributed among test groups 0, 1, 2 and 3 ranging in mean from 11.7 to 12.1 pups per litter. No significant deviations occurred.
- Pup clinical observations: One male pup (animal no. 139-06) of test group 3 (1000 mg/kg bw/day) and two male pups (animal no. 127-05 and 128-06) of test group 2 (300 mg/kg bw/d) were stillborn. One male pup (animal no. 106-05) of control group was found dead on PND 1. One male pup (animal no. 140-09) of test group 3 (1000 mg/kg bw/day) was cannibalized on PND3. These findings in its observe incidences are within the expected viability and was assessed as spontaneous in nature

Effect levels (P0)

1

Dose descriptor:

LOAEL

Remarks:

general systemic toxicity

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

haematology

clinical biochemistry

2

Dose descriptor:

NOAEL

Remarks:

general systemic toxicity

Effect level:

300 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effect observed at this dose

3

Dose descriptor:

NOAEL

Remarks:

reproductive performance and fertility

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effect observed up to the highest tested dose.

Target system / organ toxicity (P0)

Critical effects observed:

no

Results: P1 (second parental generation)

Effect levels (P1)

Remarks on result:

other: not applicable to OECD TG 422

Results: F1 generation

General toxicity (F1)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Pup clinical observations: One male pup (animal no. 139-06) of test group 3 (1000 mg/kg bw/day) and two male pups (animal no. 127-05 and 128-06) of test group 2 (300 mg/kg bw/d) were stillborn. One male pup (animal no. 106-05) of control group was found dead on PND 1. One male pup (animal no. 140-09) of test group 3 (1000 mg/kg bw/day) was cannibalized on PND3. These findings in its observe incidences are within the expected viability and was assessed as spontaneous in nature. All other F1 pups of any test group (0-3) did not show adverse clinical signs up to scheduled sacrifice on PND 4, resp. PND 13.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

Pup viability index/mortality: The viability index indicating pup mortality during postnatal days (PND) 0-4 varied between 99.3% in test group 0 (control) and test group 3 (1000 mg/kg bw/d) and 100% in test groups 1 (100 mg/kg bw/d) and 2 (300 mg/kg bw/d). No treatment related effect as observed. The survival index indicating pup mortality on PND 4 – 13 was 100% in all test groups.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Pup body weight data: Mean pup body weights of all pups in all test groups were comparable to the control group. Two female runts were seen in the control group on PND 1. Three male and 1 female runts were seen in test group 1 (100 mg/kg bw/d) and one male runt was seen in test group 2 (300 mg/kg bw/d) on PND 1. Three male runts were seen in test group 3 (1000 mg/kg bw/d) on PND 1. All values were within the range of the biological variation inherent in the strain of rats used for this study.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

no effects observed

Description (incidence and severity):

- Sex ratio: The sex distribution and sex ratios of live F1 pups on the day of birth and PND 13 did not show substantial differences between the control and the test substance-treated groups; slight differences were regarded to be spontaneous in nature.

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

Anogenital distance and anogenital distance index of male and female pups was not influenced in all test groups.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

The apparent number and percentage of male pups having areolae was not influenced by the test substance when examined on PND 13.

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

One male pup of test group 3 (1000 mg/kg bw/d) showed an empty stomach and large liver. One male pup of test group 2 (300 mg/kg bw/d) showed a small testis. One male pup of test group 1 (100 mg/kg bw/d) showed a misshapened liver. One male pup of the control group showed an absent liver lobe. These findings occurred without any relation to dosing and were assessed as spontaneous in nature and not related to treatment. All other F1 pups of any test group (0-3) did not show adverse findings during necropsy.

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Thyroid hormones:
- In male and female pups at PND13 (main test groups 21, 22 and 23; 100, 300 and 1000 mg/kg bw/d), no treatment-related alterations of T4 and TSH levels were observed.

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

Dose descriptor:

NOAEL

Remarks:

developmental toxicity (F1 progeny)

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effect observed up to the highest tested dose.

Target system / organ toxicity (F1)

Critical effects observed:

no

Results: F2 generation

Effect levels (F2)

Remarks on result:

other: not applicable to OECD TG 422

Overall reproductive toxicity

Reproductive effects observed:

no

Any other information on results incl. tables

Table 6: Haematology - males

 

 

		Test Group 0/ M 0 mg/kg bw/d	Test Group 1/ M 100 mg/kg bw/d	Test Group 2/ M 300 mg/kg bw/d	Test Group 3/ M 1000 mg/kg bw/d
RBC	Mean	8.73 k	8.53	8.87	9.03
[tera/L]	S.d.	0.23	0.28	0.38	0.17
day 30	N	5	5	5	5
	Median	8.71	8.50	8.95	9.06
	Deviation Vs Control [%]		-2.29	1.67	3.41
HGB	Mean	9.1 k	9.1	9.0	9.2
[mmol/L]	S.d.	0.1	0.1	0.3	0.3
day 30	N	5	5	5	5
	Median	9.0	9.1	9.2	9.3
	Deviation Vs Control [%]		0.4	-0.7	1.1
HCT	Mean	0.434 k	0.427	0.428	0.437
[L/L]	S.d.	0.002	0.005	0.009	0.008
day 30	N	5	5	5	5
	Median	0.434	0.427	0.426	0.436
	Deviation Vs Control [%]		-1.430	-1.384	0.738
MCV	Mean	49.7 k	50.1	48.2	48.4
[fL]	S.d.	1.3	1.4	2.1	0.4
day 30	N	5	5	5	5
	Median	49.5	50.7	46.9	48.5
	Deviation Vs Control [%]		0.8	-2.9	-2.6
MCH	Mean	1.04 k	1.07	1.02	1.02
[fmol]	S.d.	0.04	0.04	0.06	0.03
day 30	N	5	5	5	5
	Median	1.03	1.07	0.99	1.01
	Deviation Vs Control [%]		2.68	-2.11	-2.30
MCHC	Mean	20.96 k	21.37	21.20	21.11
[mmol/L]	S.d.	0.33	0.26	0.48	0.61
day 30	N	5	5	5	5
	Median	20.80	21.30	21.21	20.85
	Deviation Vs Control [%]		1.97	1.15	0.72
RETA	Mean	149.4 k	160.6	155.2	154.5
[giga/L]	S.d.	25.5	14.5	22.1	30.7
day 30	N	5	5	5	5
	Median	138.5	163.2	165.3	154.0
	Deviation Vs Control [%]		7.5	3.9	3.4
PLT	Mean	594 k	656	697	703
[giga/L]	S.d.	90	85	74	77
day 30	N	5	5	5	5
	Median	590	653	714	698
	Deviation Vs Control [%]		10	17	18
HQT	Mean	35.7 v	35.5	37.3	32.7 **
[sec]	S.d.	0.7	1.5	3.1	1.0
day 30	N	5	5	5	5
	Median	35.6	34.9	36.1	32.7
	Deviation Vs Control [%]		-0.6	4.5	-8.4

 

Statistic Profile = Kruskal-Wallis + Wilcoxon test (two-sided) * p<=0.05, ** p <=0.01, X = Group excluded from statistics k=KRUSKALL-WALLIS; v=KRUSKALL-WALLIS-WILCOX

Table 7: Clinical chemistry - males

 

 

		Test Group 0/ M 0 mg/kg bw/d	Test Group 1/ M 100 mg/kg bw/d	Test Group 2/ M 300 mg/kg bw/d	Test Group 3/ M 1000 mg/kg bw/d
UREA	Mean	4.44 k	4.42	4.43	5.26
[mmol/L]	S.d.	0.36	0.51	0.38	0.32
day 30	N	5	5	5	5
	Median	4.29	4.47	4.27	5.22
	Deviation Vs Control [%]		-0.59	-0.23	18.27
CREA	Mean	21.9 k	22.9	21.3	24.0
[µmol/L]	S.d.	2.6	2.2	1.2	1.5
day 30	N	5	5	5	5
	Median	21.2	22.8	21.5	24.2
	Deviation Vs Control [%]		4.6	-2.8	9.6
GLUC	Mean	7.40 k	6.92	6.21	6.36
[mmol/L]	S.d.	1.15	0.30	0.73	0.67
day 30	N	5	5	5	5
	Median	7.63	6.94	6.00	6.45
	Deviation Vs Control [%]		-6.51	-16.05	-14.05
TBIL C	Mean	1.57 k	1.47	1.33	1.68
[µmol/L]	S.d.	0.31	0.14	0.13	0.29
day 30	N	5	5	5	5
	Median	1.74	1.43	1.34	1.62
	Deviation Vs Control [%]		-6.66	-15.22	6.57
TBA	Mean	14.6 k	12.0	10.6	21.0
[µmol/L]	S.d.	8.5	4.8	6.6	8.4
day 30	N	5	5	5	5
	Median	11.2	12.3	6.8	22.2
	Deviation Vs Control [%]		-17.8	-27.4	43.4
TPROT	Mean	63.07 k	64.56	64.50	65.98
[g/L]	S.d.	1.10	0.62	1.25	2.36
day 30	N	5	5	5	5
	Median	63.36	64.36	64.54	66.52
	Deviation Vs Control [%]		2.37	2.27	4.61
ALB	Mean	36.92 k	37.69	37.26	37.74
[g/L]	S.d.	1.36	0.98	0.51	0.96
day 30	N	5	5	5	5
	Median	36.97	37.53	37.48	37.70
	Deviation Vs Control [%]		2.09	0.93	2.23
GLOB	Mean	26.15 k	26.87	27.24	28.24
[g/L]	S.d.	0.59	0.82	1.14	1.42
day 30	N	5	5	5	5
	Median	26.05	26.83	26.89	28.67
	Deviation Vs Control [%]		2.76	4.17	7.98
CHOL	Mean	1.77 v	2.00	2.17	2.80 **
[mmol/L]	S.d.	0.31	0.32	0.35	0.38
day 30	N	5	5	5	5
	Median	1.68	1.89	2.10	2.79
	Deviation Vs Control [%]		12.51	22.32	58.06
TRIG	Mean	0.77 k	0.83	0.72	0.75
[mmol/L]	S.d.	0.21	0.36	0.15	0.13
day 30	N	5	5	5	5
	Median	0.83	0.62	0.70	0.78
	Deviation Vs Control [%]		7.55	-5.99	-2.86

 

Statistic Profile = Kruskal-Wallis + Wilcoxon test (two-sided) * p<=0.05, ** p <=0.01, X = Group excluded from statistics k=KRUSKALL-WALLIS; v=KRUSKALL-WALLIS-WILCOX

Table 8: Haematology – males, recovery group

 

 

		Test Group 10/ M 0 mg/kg bw/d	Test Group 11/ M 1000 mg/kg bw/d
UREA	Mean	4.81 x	5.50
[mmol/L]	S.d.	0.43	0.59
day 30	N	5	5
	Median	4.68	5.53
	Deviation Vs Control [%]		14.38
CREA	Mean	23.0 x	24.9
[µmol/L]	S.d.	3.1	1.7
day 30	N	5	5
	Median	23.1	24.5
	Deviation Vs Control [%]		8.2
GLUC	Mean	6.80 x	6.22
[mmol/L]	S.d.	0.55	0.51
day 30	N	5	5
	Median	6.84	6.49
	Deviation Vs Control [%]		-8.50
TBIL C	Mean	1.54 x	1.62
[µmol/L]	S.d.	0.27	0.19
day 30	N	5	5
	Median	1.53	1.69
	Deviation Vs Control [%]		5.34
TBA	Mean	22.8 x	24.3
[µmol/L]	S.d.	6.5	10.7
day 30	N	5	5
	Median	20.7	24.4
	Deviation Vs Control [%]		6.5
TPROT	Mean	65.05 x	66.07
[g/L]	S.d.	1.03	1.26
day 30	N	5	5
	Median	64.87	65.51
	Deviation Vs Control [%]		1.57
ALB	Mean	37.37 x	38.21 **
[g/L]	S.d.	0.23	0.21
day 30	N	5	5
	Median	37.42	38.33
	Deviation Vs Control [%]		2.24
GLOB	Mean	27.68 x	27.86
[g/L]	S.d.	0.81	1.42
day 30	N	5	5
	Median	27.45	27.18
	Deviation Vs Control [%]		0.66
CHOL	Mean	1.96 x	2.64 *
[mmol/L]	S.d.	0.30	0.22
day 30	N	5	5
	Median	1.96	2.70
	Deviation Vs Control [%]		34.80
TRIG	Mean	1.12 x	1.19
[mmol/L]	S.d.	0.38	0.41
day 30	N	5	5
	Median	1.25	1.41
	Deviation Vs Control [%]		6.61

 

Statistic Profile = Wilcoxon test (two-sided), *p<=0.05, **p<=0.01, X = Group excluded from statistics

x=WILCOX

Table 9: Absolute organ weights of main groups

 

	Male animals			Female animals
Test group (mg/kg bw/d)	1 (100)	2 (300)	3 (1000)	1 (100)	2 (300)	3 (1000)
Epididymides	+6.04%	+0.88%	+8.32%*
Prostate	+16.8%**	+23.76%**	+24.59%**	%	%	%
Thymus	%	%	%	-21.982%*	+13.453%	-1.141%

*p <= 0.05; **p <= 0.01

 

Table 10: Relative organ weights of main groups

 

	Male animals
Test group (mg/kg bw/d)	1 (100)	2 (300)	3 (1000)
Prostate	+15.1%*	+22.27%*	+22.05%**

*p <= 0.05; **p <= 0.01

 

Table 11: Relative organ weights of the recovery group

 

	Male animals
Test group (mg/kg bw/d)	11 (1000)
Heart	+11.7%*

*p <= 0.05; **p <= 0.01

Table 12: Fertility indices for F0 males

 

 

	Test group 0 (0 mg/kg bw/d)	Test group 1 (100 mg/kg bw/d)	Test group 2 (300 mg/kg bw/d)	Test group 3 (1000 mg/kg bw/d)
Male fertility index [%]	100	100	90	100

 

Table 13: Fertility indices for F0 females

 

 

	Test group 0 (0 mg/kg bw/d)	Test group 1 (100 mg/kg bw/d)	Test group 2 (300 mg/kg bw/d)	Test group 3 (1000 mg/kg bw/d)
Female fertility index [%]	100	100	100	100

 

Table 14: Sex ratio of live F1 pups

 

 

PND 0	Test group 0 (0 mg/kg bw/d)	Test group 1 (100 mg/kg bw/d)	Test group 2 (300 mg/kg bw/d)	Test group 3 (1000 mg/kg bw/d)
Live males [%]	48.3	43.7	57.7	50.7
Live females [%]	51.7	56.3	42.3	49.3
PND 13	Test group 0 (0 mg/kg bw/d)	Test group 1 (100 mg/kg bw/d)	Test group 2 (300 mg/kg bw/d)	Test group 3 (1000 mg/kg bw/d)
Live males [%]	47.5	47.5	52.5	46.2
Live females [%]	52.5	52.5	47.5	53.8

 

Table 15: Summary Mating Report

 

		Test Group 0/ F		Test Group 1/ F	Test Group 2/ F	Test Group 3/ F
		0 mg/kg bw/d		100 mg/kg bw/d	300 mg/kg bw/d	1000 mg/kg bw/d
No. of females mated	N	10		10	10	10
- Inseminated	N	10	f-	10	9	10
Female mating index	%	100.0		100.0	90.0	100.0
-- Pregnant	N	10	f-	10	9	10
Female fertility index	%	100.0		100.0	100.0	100.0
No. of males mated	N	10		10	10	10
- With inseminated females	N	10	f-	10	9	10
Male mating index	%	100.0		100.0	90.0	100.0
- With pregnant females	N	10	f-	10	9	10
Male fertility index	%	100.0		100.0	90.0	100.0
Females with defined Day 0 pc	N	10		10	9	10
Mating days until Day 0 pc	Mean	2.2	x+	3.9	3.3 *	2.4
	S.d.	0.9		3.7	1.1	1.0
	N	10		10	9	10
Days 0 To 4	N	10		9	9	10
	%	100.0		90.0	100.0	100.0
Days 5 To 9	N	0		0	0	0
	%	0.0		0.0	0.0	0.0
Days 10 To 14	N	0		1	0	0
	%	0.0		10.0	0.0	0.0

Statistic Profile = Fisher's exact test (one-sided-), Wilcoxon with Bonferroni-Holm (one-sided+), * p<=0.05, ** p <=0.01, X = Group excluded from statistics f=FISHER-EXACT; x=WILCOX

 

Table 16: Summary Pregnancy Status Report - Reproduction

 

			Test Group 0/F 0 mg/kgbw/d		Test Group 1/F 100 mg/kgbw/d		Test Group 2/F 300 mg/kgbw/d	Test Group 3/F 1000 mg/kgbw/d
No. of females at start	N		10		10		10	10
No. of females mated	N		10		10		10	10
Without evidence of mating	N		0		0		1	0
- Pregnant	N		0		0		0	0
- Not pregnant	N		0		0		1	0
Females with defined Day 0 pc	N		10		10		9	10
Pregnant	N		10		10		9	10
- sacrificed scheduled	N		10		10		9	10
Not pregnant	N		0		0		1	0
- sacrificed scheduled	N		0		0		1	0
Pregnant, not delivering	N		0		0		0	0
Delivering	N		10		10		9	10
-- With liveborn pups	N		10		10		9	10
	%		100.0		100.0		100.0	100.0
-- With all pups stillborn	N		0		0		0	0
	%		0.0		0.0		0.0	0.0

Statistic Profile = Fisher's exact test (one-sided-), Dunnett test (two-sided), Fisher's exact test (one-sided+), * p<=0.05, ** p <=0.01, X = Group excluded from statistics

f=FISHER-EXACT; n=DUNNETT

 

 

Table 17: Summary Delivery Report

 

		Test Group0/F 0 mg/kgbw/d		Test Group1/F 100 mg/kgbw/d		Test Group2/F 300 mg/kgbw/d	Test Group3/F 1000 mg/kgbw/d
No. of females at start	N	10		10		10	10
No. of females mated	N	10	f-	10		10	10
	%	100.0		100.0		100.0	100.0
Pregnant	N	10	f-	10		9	10
	%	100.0		100.0		90.0	100.0
Without delivery	N	0		0		1	0
- Pregnant	N	0		0		0	0
- Not pregnant	N	0		0		1	0
-- Delivering	N	10	f-	10		9	10
	%	100.0		100.0		100.0	100.0
-- With liveborn pups	N	10	f-	10		9	10
Gestation Index	%	100.0		100.0		100.0	100.0
Gestation days	Mean	22.1	n	22.3		22.1	22.0
	S.d.	0.6		0.5		0.6	0.5
	N	10		10		9	10
-- With stillborn pups	N	0		0		2	1
	%	0.0		0.0		22.2	10.0
-- With all pups stillborn	N	0		0		0	0
	%	0.0		0.0		0.0	0.0

 

Statistic Profile = Fisher's exact test (one-sided-), Dunnett test (two-sided), Fisher's exact test (one-sided+), * p<=0.05, ** p <=0.01, X = Group excluded from statistics

f=FISHER-EXACT; n=DUNNETT

Table 18: Summary Litter Report – Pup status I

 

		Test Group 0/ F		Test Group 1/ F	Test Group 2/ F	Test Group 3/ F
		0 mg/kg bw/d		100 mg/kg bw/d	300 mg/kg bw/d	1000 mg/kg bw/d
Total Number of Pregnant Females	N	10		10	9	10
Total number of litters	N	10		10	9	10
With liveborn pups	N	10	f-	10	9	10
	%	100.0		100.0	100.0	100.0
With stillborn pups	N	0	f+	0	2	1
	%	0.0		0.0	22.2	10.0
With all pups stillborn	N	0	f+	0	0	0
	%	0.0		0.0	0.0	0.0
Implantation Sites	N	129		136	119	135
	Mean	12.9	x-	13.6	13.2	13.5
	S.d.	2.1		1.4	2.8	1.5
	N	10		10	9	10
Pups delivered	N	121		130	107	130
	Mean	12.1	x-	13.0	11.9	13.0
	S.d.	2.3		1.6	3.4	1.9
	N	10		10	9	10
Postimplantation Loss	Mean%	5.7	x+	4.5	10.1	3.8
	S.d.	12.6		5.0	16.1	8.1
	N	10		10	9	10

Statistic Profile = Wilcoxon with Bonferroni-Holm (one-sided-), Wilcoxon with Bonferroni-Holm (one-sided+), Wilcoxon test (two-sided), Fisher's exact test (one-sided-),

Fisher's exact test (one-sided+), * p<=0.05, ** p <=0.01, X = Group excluded from statistics

f=FISHER-EXACT; x=WILCOX

 

 

Table 19: Summary Litter Report – Pup status II

 

		Test Group 0/ F		Test Group 1/ F	Test Group 2/ F	Test Group 3/ F
		0 mg/kg bw/d		100 mg/kg bw/d	300 mg/kg bw/d	1000 mg/kg bw/d
Pups liveborn	N	121		130	105	129
	%	100.0		100.0	98.1	99.2
	Mean	12.1	x-	13.0	11.7	12.9
	S.d.	2.3		1.6	3.6	1.9
	N	10		10	9	10
Pups stillborn	N	0		0	2	1
	%	0.0		0.0	1.9	0.8
	Mean	0.0	x+	0.0	0.2	0.1
	S.d.	0.0		0.0	0.4	0.3
	N	10		10	9	10
Perinatal Loss	Mean%	0.0	x+	0.0	2.8	0.8
	S.d.	0.0		0.0	5.9	2.4
	N	10		10	9	10

Statistic Profile = Wilcoxon with Bonferroni-Holm (one-sided-), Wilcoxon with Bonferroni-Holm (one-sided+), Wilcoxon test (two-sided), Fisher's exact test (one-sided-),

Fisher's exact test (one-sided+), * p<=0.05, ** p <=0.01, X = Group excluded from statistics

x=WILCOX

 

 

Table 20: Summary Litter Report - Dead Pups

 

		Test Group0/F 0 mg/kgbw/d	Test Group1/F 100 mg/kgbw/d	Test Group 2/ F 300 mg/kg bw/d	Test Group3/F 1000 mg/kgbw/d
Litters with liveborn pups	N	10	10	9	10
Pups delivered	N	121	130	107	130
found dead [pup] / Dead	N	1	0	0	0
	%	0.8	0.0	0.0	0.0
stillborn / Dead	N	0	0	2	1
	%	0.0	0.0	1.9	0.8
Alive / Alive	N	121	130	105	129
	%	100.0	100.0	98.1	99.2
cannibalized [pup] / Dead	N	0	0	0	1
	%	0.0	0.0	0.0	0.8
sacrificed scheduled [pup] / Dead	N	80	80	68	80
	%	66.1	61.5	63.6	61.5
culled / Dead	N	40	50	37	48
	%	33.1	38.5	34.6	36.9
Litters not surviving Day 13	N	0	0	0	0
	%	0.0	0.0	0.0	0.0

Statistic Profile = Wilcoxon with Bonferroni-Holm (one-sided-), Wilcoxon with Bonferroni-Holm (one-sided+), Wilcoxon test (two-sided), Fisher's exact test (one-sided-),

Fisher's exact test (one-sided+), * p<=0.05, ** p <=0.01, X = Group excluded from statistics

 

 

 

Table 21: Summary Litter Report - Pups Died

 

		Test Group0/F 0 mg/kgbw/d	Test Group1/F 100 mg/kgbw/d	Test Group 2/ F 300 mg/kg bw/d	Test Group3/F 1000 mg/kgbw/d
Litters with liveborn pups	N	10	10	9	10
Pups delivered	N	121	130	107	130
Days 0 To 0	N	0	0	0	0
	%	0	0	0	0
Days 1 To 4	N	1	0	0	1
	%	0.8	0	0	0.8
Days 5 To 7	N	0	0	0	0
	%	0	0	0	0
Days 8 To 13	N	0	0	0	0
	%	0	0	0	0
Pups surviving days 0 To 4	N	120	130	105	128
Viability Index	Mean%	99.3 x-	100.0	100.0	99.3
	S.d.	2.3	0.0	0.0	2.3
	N	10	10	9	10
Pups surviving days 4 To 13	N	80	80	68	80
Survival Index	Mean%	100.0 NA	100.0	100.0	100.0
	S.d.	0.0	0.0	0.0	0.0
	N	10	10	9	10

Statistic Profile = Wilcoxon with Bonferroni-Holm (one-sided-), Wilcoxon with Bonferroni-Holm (one-sided+), Wilcoxon test (two-sided), Fisher's exact test (one-sided-),

Fisher's exact test (one-sided+), * p<=0.05, ** p <=0.01, X = Group excluded from statistics

x=WILCOX; NA=No Test Applicable

 

 

Table 22: Summary Pup Report Body Weights - BW / Body Weights [g] I

 

		Test Group 0/F 0 mg/kgbw/d	Test Group 1/F 100 mg/kgbw/d		Test Group 2/F 300 mg/kgbw/d	Test Group 3/F 1000 mg/kgbw/d
day 1 Runt	Males	0	3		1	3
	Females	2	1		0	0
day 1 Males	Mean	6.8 n	6.6		6.7	6.4
	S.d.	0.9	0.5		0.8	0.4
	N	10	10		9	10
	Deviation Vs Control [%]		-3.4		-1.7	-6.6
day 1 Females	Mean	6.5 n	6.4		6.5	6.2
	S.d.	0.8	0.5		0.9	0.4
	N	10	10		9	10
	Deviation Vs Control [%]		-1.2		0.1	-4.2
day 1 Males + Females	Mean	6.7 n	6.5		6.6	6.3
	S.d.	0.8	0.5		0.9	0.3
	N	10	10		9	10
	Deviation Vs Control [%]		-2.4		-0.2	-5.0
day 4 Males	Mean	10.8 n	10.4		10.5	10.1
	S.d.	1.7	1.0		1.8	0.5
	N	10	10		9	10
	Deviation Vs Control [%]		-3.6		-2.1	-6.4

Statistic Profile = Dunnett test (two-sided), * p<=0.05, ** p <=0.01, X = Group excluded from statistics

n=DUNNETT

 

 

Table 23: Summary Pup Report Body Weights - BW / Body Weights [g] II

 

		Test Group 0/F 0 mg/kgbw/d	Test Group 1/F 100 mg/kgbw/d		Test Group 2/F 300 mg/kgbw/d	Test Group 3/F 1000 mg/kgbw/d
day 4 Females	Mean	10.4 n	10.2		10.3	10.0
	S.d.	1.7	1.0		1.8	0.5
	N	10	10		9	10
	Deviation Vs Control [%]		-2.6		-1.1	-3.7
day 4 Males + Females	Mean	10.6 n	10.3		10.4	10.1
	S.d.	1.7	1.0		1.8	0.5
	N	10	10		9	10
	Deviation Vs Control [%]		-3.2		-1.3	-4.5
day 7 Males	Mean	17.7 n	17.1		17.1	16.8
	S.d.	2.0	1.5		2.1	0.8
	N	10	10		9	10
	Deviation Vs Control [%]		-3.0		-3.0	-5.1
day 7 Females	Mean	17.0 n	17.2		16.7	16.5
	S.d.	2.0	1.5		2.1	0.9
	N	10	10		9	10
	Deviation Vs Control [%]		1.2		-1.9	-3.2
day 7 Males + Females	Mean	17.3 n	17.2		16.9	16.7
	S.d.	1.9	1.5		2.0	0.8
	N	10	10		9	10
	Deviation Vs Control [%]		-0.8		-2.4	-3.8

Statistic Profile = Dunnett test (two-sided), * p<=0.05, ** p <=0.01, X = Group excluded from statistics

n=DUNNETT

 

 

Table 24: Summary Pup Report Body Weights - BW / Body Weights [g] II

 

		Test Group 0/F 0 mg/kgbw/d	Test Group 1/F 100 mg/kgbw/d		Test Group 2/F 300 mg/kgbw/d	Test Group 3/F 1000 mg/kgbw/d
day 13 Males	Mean	33.1 n	32.1		32.4	31.8
	S.d.	2.2	2.3		2.3	1.8
	N	10	10		9	10
	Deviation Vs Control [%]		-2.9		-1.9	-3.9
day 13 Females	Mean	32.3 n	32.1		31.7	31.5
	S.d.	2.3	2.3		2.2	2.0
	N	10	10		9	10
	Deviation Vs Control [%]		-0.7		-1.9	-2.4
day 13 Males + Females	Mean	32.7 n	32.1		32.1	31.7
	S.d.	2.2	2.3		2.2	1.9
	N	10	10		9	10
	Deviation Vs Control [%]		-1.8		-1.9	-3.0

Statistic Profile = Dunnett test (two-sided), * p<=0.05, ** p <=0.01, X = Group excluded from statistics

n=DUNNETT

 

 

Table 25: Summary Pup Report AG Distance + AG Index of males

 

		Test Group 0/F 0 mg/kgbw/d	Test Group 1/F 100 mg/kgbw/d		Test Group 2/F 300 mg/kgbw/d	Test Group 3/F 1000 mg/kgbw/d
AG Distance	Mean	3.06 n	3.08		3.13	3.02
[mm]	S.d.	0.31	0.20		0.27	0.18
day 1 Males	N	10	10		9	10
AG Index Cubic Root	Mean	1.61 n	1.64		1.66	1.63
[---]	S.d.	0.10	0.08		0.13	0.09
day 1 Males	N	10	10		9	10

Statistic Profile = Dunnett test (two-sided), * p<=0.05, ** p <=0.01, X = Group excluded from statistics

n=DUNNETT

 

 

Table 26: Summary Pup Report AG Distance + AG Index of males

 

		Test Group 0/F 0 mg/kgbw/d	Test Group 1/F 100 mg/kgbw/d		Test Group 2/F 300 mg/kgbw/d	Test Group 3/F 1000 mg/kgbw/d
AG Distance	Mean	1.39 n	1.40		1.38	1.37
[mm]	S.d.	0.17	0.12		0.13	0.08
day 1 Females	N	10	10		9	10
AG Index Cubic Root	Mean	0.75 n	0.76		0.74	0.75
[---]	S.d.	0.08	0.05		0.08	0.04
day 1 Females	N	10	10		9	10

Statistic Profile = Dunnett test (two-sided), * p<=0.05, ** p <=0.01, X = Group excluded from statistics

n=DUNNETT

 

 

Table 27: Summary Pup Report Nipple development

 

			Test Group 0/F 0 mg/kgbw/d	Test Group 1/F 100 mg/kgbw/d		Test Group 2/F 300 mg/kgbw/d		Test Group 3/F 1000 mg/kgbw/d
Nipple development [Incidence]	Passed -N		7	5		7		11
day 13 Males	-% Failed -N		18   31	13   33		20   28		30   26
	-%		82	87		80		70
Nipple development [%]	Mean		17.5 x+	12.5		19.4		30.0
[%]	S.d.		23.7	27.0		20.8		19.7
day 13 Males	N		10	10		9		10
Nipple Number	Mean		0.4 x+	0.3		0.4		0.6
[#]	S.d.		0.6	0.7		0.4		0.4
day 13 Males	N		10	10		9		10

Statistic Profile = Wilcoxon with Bonferroni-Holm (one-sided+), * p<=0.05, ** p <=0.01, X = Group excluded from statistics

x=WILCOX

 

 

Table 28: Summary - Pup Necropsy Observation, Males

 

			Test Group 0/M 0 mg/kgbw/d	Test Group 1/M 100 mg/kgbw/d	Test Group 2/M 300 mg/kgbw/d	Test Group 3/M 1000 mg/kgbw/d
day 0 -> 13 With unscheduled data	Animals examined	N	59	57	62	68
	Animals with signs	N	2	1	1	2
		%	3.4	1.8	1.6	2.9
	Liver	N	0	1	0	1
		%	0.0	1.8	0.0	1.5
	large	N	0	0	0	1
		%	0.0	0.0	0.0	1.5
	misshapened	N	0	1	0	0
		%	0.0	1.8	0.0	0.0
	Testis small	N	0	0	1	0
		%	0.0	0.0	1.6	0.0
	Liver Lobe absent	N	1	0	0	0
		%	1.7	0.0	0.0	0.0
	Stomach empty	N	0	0	0	1
		%	0.0	0.0	0.0	1.5
	General	N	1	0	0	1
		%	1.7	0.0	0.0	1.5
	Post mortem autolysis	N	1	0	0	0
		%	1.7	0.0	0.0	0.0
	not assessed	N	0	0	0	1
		%	0.0	0.0	0.0	1.5
	normal NAD	N	57	56	61	66
		%	96.6	98.2	98.4	97.1

 

Table 28:Summary - Pup Necropsy Observation, Females

 

			Test Group 0/F 0 mg/kgbw/d	Test Group 1/F 100 mg/kgbw/d	Test Group 2/F 300 mg/kgbw/d	Test Group 3/F 1000 mg/kgbw/d
day 0 -> 13 With unscheduled data	Animals examined	N	62	73	45	62
	normal NAD	N	62	73	45	62
		%	3.4	1.8	1.6	2.9

 

 

 

Applicant's summary and conclusion

Conclusions:

The no observed adverse effect level (NOAEL) of general systemic toxicity was 300 mg/kg bw/d for male and 1000 mg/kg bw/d for female parental animals.
The no observed adverse effect level (NOAEL) for reproductive performance and fertility was the nominal dose level 1000 mg/kg bw/d in both sexes of parental animals.
The NOAEL for developmental toxicity in the F1 progeny was the nominal dose level 1000 mg/kg bw/d.

Executive summary:

The repeated dose toxicity of the test substance was investigated by means of a Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test according to OECD guideline 422 and GLP.

The test substance was administered daily by gavage as a solution to groups of 10 male and 10 female Wistar rats (F0 animals) at nominal doses of 0, 100, 300 and 1000 mg/kg body weight/day (mg/kg bw/d). Control animals were dosed daily with the vehicle only (Corn oil Ph.Eur. 8.0). The duration of treatment covered a 2-week premating period and mating in both sexes (mating pairs were from the same dose group) as well as entire gestation and lactation period in females up to one day prior to the day of schedule sacrifice of the animals. Additional treated but not mated animals (recovery animals) to groups of 5 male and 5 female animals at nominal doses of 0 and 1000 mg/kg bw/d was maintained for a subsequent period of at least 14 days of no test substance administration in order to observe reversibility of the findings.

The parents' and the pups' state of health was checked each day, and parental animals were examined for their mating and reproductive performances. F0 animals were mated 13 days after the beginning of treatment to produce a litter (F1 generation pups). As soon as sperm was detected in the vaginal smear, mating was discontinued. F0 animals were examined for their reproductive performance including determinations of the number of implantations and the calculation of the postimplantation loss in all F0 females. A detailed clinical observation (DCO) was performed in all animals before initial test substance administration and, as a rule, thereafter at weekly intervals. Food consumption of the F0 parents of main groups and recovery groups were determined regularly once weekly before mating period as well as males and females of the recovery groups until sacrifice. In dams during gestation days 0-7,7-14 and 14-20 and lactation days 1-4, 4-7, 7-10 and 10-13 the food consumption was also determined. In general, the body weights of F0 animals were determined once a week. However, during gestation and lactation, F0 females were weighed on gestation days (GD) 0, 7, 14 and 20, and on postnatal days (PND) 0, 4, 7, 10 and 13. The pups were sexed and examined for macroscopically evident changes on PND 0. They were weighed on PNDs 1, 4, 7 and on PND 13 and their viability was recorded. Anogenital distance (defined as the distance from the anus [center of the anal opening] to the base of the genital tubercle) measurements were conducted in a blind randomized fashion, using a measuring ocular on all live male and female pups on PND 1. On PND 4, the individual litters were standardized in such a way that, whenever possible, each litter contains 4 male and 4 female pups (as a rule, the first 4 surviving pups/sex in each litter were taken for further rearing). On PND 13, all male F1 pups were examined for retention of nipples/areolae. The number of nipples/areola anlagen were counted. At necropsy on PND 4, all pups were sacrificed under isoflurane anesthesia by decapitation. Blood was sampled for determination of thyroid hormone concentrations. After sacrifice, the pups were examined externally and eviscerated, and their organs were assessed macroscopically. At necropsy on PND 13, one selected male and one female pup per litter were sacrificed under isoflurane anesthesia by decapitation. Blood was sampled for determination of thyroid hormone concentrations. Thyroid glands/parathyroid glands were fixed in neutral buffered 4% formaldehyde solution and were transferred to the Pathology Laboratory for possible further processing. The remaining pups were sacrificed under isoflurane anesthesia with CO2. After sacrifice, all pups were examined externally and eviscerated, and their organs were assessed macroscopically. Towards the end of the administration period a functional observational battery was performed and motor activity was measured in 5 animals per sex and test group. Clinicochemical and hematological examinations were performed in 5 animals per sex and group towards the end of the administration period as well as towards the end of recovery period. All F0 parental animals were sacrificed by decapitation, under isoflurane anesthesia, and were assessed by gross pathology. Weights of selected organs were recorded and a histopathological examination was performed. The remaining 5 animals per sex of test groups 10 and 11 (recovery animals) were maintained for a 2-week recovery period after the administration period without test substance exposure. Further examinations in these animals were depend on the findings observed in the animals of the main groups. No functional observational battery was performed as well as no motor activity and estrous cycle were determined during the recovery period.

 

The various analyses confirmed

·        the stability of the test substance in corn oil at room temperature over a period of 7 days,

·        and the correctness of the prepared concentrations.

 

The following test substance-related adverse effects/findings were noted:

 

Test group 3/11: 1000 mg/kg bw/d

F0 PARENTAL ANIMALS

Clinical examinations, reproductive performance and pathology

·        no test substance-related adverse findings

Clinical pathology

·        reduced prothrombin time (HQT) in males (test group 3, only)

·        increased cholesterol values in males

F1 PUPS

Clinical examinations/ gross findings

·        no test substance-related adverse findings

 

Test group 2: 300 mg/kg bw/d

F0 PARENTAL ANIMALS

Clinical examinations, reproductive performance, clinical pathology, and pathology

·        no test substance-related adverse findings

F1 PUPS

Clinical examinations/ gross findings

·        no test substance-related adverse findings

 

Test group 1: 100 mg/kg bw/d

F0 PARENTAL ANIMALS

Clinical examinations, reproductive performance, clinical pathology, and pathology

·        no test substance-related adverse findings

F1 PUPS

Clinical examinations/ gross findings

·        no test substance-related adverse findings

 

Under the conditions of this Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, the oral administration of the test substance by gavage to Wistar rats revealed signs of systemic toxicity manifested in clinical pathology parameters of males in the limit dose group (1000 mg/kg bw/d) at the end of the administration period. However, the clinical pathological alterations were no permanent changes, since no treatment-related findings were noted after the two weeks recovery period. No treatment-related adverse findings were observed in males at 100 and 300 mg/kg bw/d as well as in all female parental animals (F0) and all pups (F1).

The no observed adverse effect level (NOAEL) of general systemic toxicity was 300 mg/kgbw/d for male and 1000 mg/kg bw/d for female parental animals. The no observed adverse effect level (NOAEL) for reproductive performance and fertility was the nominal dose level 1000 mg/kg bw/d in both sexes of parental animals. The NOAEL for developmental toxicity in the F1 progeny was the nominal dose level 1000 mg/kg bw/d.