2-Oxazolidinone, 3-ethenyl-5-methyl-

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

21 Jul 2015 - 10 Jun 2016

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

Remarks:

see also chapter 7.5.1

Cross-reference 2

Reason / purpose for cross-reference:

reference to same study

Remarks:

see also chapter 7.8.2

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

22 mar 1996

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3650: Combined Repeated Dose Toxicity Study With the Reproduction/Developmental Toxicity Screening Test

Version / remarks:

Jul 2000

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

1

Reference substance name:

3-ethenyl-5-methyl-1,3-oxazolidin-2-one

EC Number:

809-852-5

Cas Number:

3395-98-0

Molecular formula:

C6H9NO2

IUPAC Name:

3-ethenyl-5-methyl-1,3-oxazolidin-2-one

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Test substanc No.: 14/0031-3
- Cas No.: 3395-98-0
- Source and lot/batch No.of test material: DEIMLIB-00070
- Expiration date of the lot/batch: 26 Jan 2017
- Purity: assumed 99.5%


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability under test conditions: The stability of the test substance under storage conditions over the test period was guaranteed by the sponsor, and
the sponsor holds this responsibility.
- Solubility and stability of the test substance in the solvent/vehicle:
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium:


OTHER SPECIFICS:
- Physical state/appearance; liquid, colorless, clear

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services Germany GmbH
- Age at study initiation: 10-11 weels
- Housing: During the study period, the rats were housed individually in polycarbonate cages type III
(floor area of about 800 cm²) with the following exceptions:
• During overnight matings, male and female mating partners were housed together in
polycarbonate cages type III.
• Dams and their litters were housed together until PND 4 (end of lactation).
• For motor activity (MA) measurements the animals were housed individually in polycarbonate cages supplied by TECNIPLAST, Hohenpeißenberg, Germany with
wire covers from Ehret, Emmendingen, Germany (floor area of about 800 cm2) and small amounts of bedding material
- Diet (e.g. ad libitum): ground The food used was ground Kliba maintenance diet mouse-rat “GLP”, meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland.
- Water: ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24°C
- Humidity (%): 30 - 70%
- Air changes (per hr): 15 per hour
- Photoperiod (hrs dark / hrs light): 12 hours (12 hours light from 06.00- 18.00h, 12 hours dark from 18.00-06.00h

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
5-Methyl-3-vinyloxazolidin-2-on was applied as solutions. To prepare this solution, the appropriate amount of test substance was weighed out depending on the desired concentration. Then, corn oil was filled up to the desired volume, subsequently mixed with a magnetic stirrer. The test substance preparations were produced at least once a week and were stored at room temperature. The administration volume was 4 mL/kg body weight

VEHICLE
- Justification for use and choice of vehicle (if other than water):
- Concentration in vehicle: 0.375, 1.250 3.750 g/100ml
- Amount of vehicle (if gavage): 4 ml/kg bw

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: overnight, for a maximum of 2 weeks.
- The animals were paired by placing the female in the cage of the male mating partner from
about 16.00 h until 06.30 - 09.00 h of the following morning. Deviations from the specified
times were possible on weekends and public holidays and were reported in the raw data. A
vaginal smear was prepared after each mating and examined for the presence of sperm. If
sperm was detected, pairing of the animals was discontinued. The day on which sperm was
detected was denoted gestation day (GD) 0 and the following day "GD 1".

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The analyses of the test substance preparations were carried out at the Analytical Chemistry Laboratory of Experimental Toxicology and Ecology of BASF SE, Ludwigshafen, Germany.
The stability of 5-Methyl-3-vinyloxazolidin-2-on in corn oil over a period of 7 days at room temperature was proven before the start of the study.
Samples of the test substance preparations were sent to the analytical laboratory for verification of the concentrations. The samples taken for the concentration control analyses
were also used to verify the homogeneity of the samples of the low- and high-concentrations. Three samples (one from the top, middle and bottom) were taken from the preparation
vessels with the magnetic stirrer running. Of each sample, one additional reserve sample was retained. Details of the sampling schedule were recorded with the raw data.

Duration of treatment / exposure:

Males:
- 14 days premating
- up to 14 days mating
- sacrifice minimum 4 days after littering
The exposure duration was at least 30 days

Females:
-14 days premating
- up to 14 days mating
- gestation about 22 days
- sacrifice minimum 4 days after littering
The exposure duration was at least 52 days

Frequency of treatment:

daily

Doses / concentrations

Remarks:

Doses/Concentrations:
0, 15, 50, 150 mg/kg bw/day

No. of animals per sex per dose:

10 males/10 females

Control animals:

yes, concurrent vehicle

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
A cageside examination was conducted once daily for any signs of morbidity, pertinent behavioral changes and signs of overt toxicity before the administration. Furthermore, all animals were checked for any abnormal clinically signs within 2 hours and within 5 hours after the administration. Abnormalities and changes were documented daily for each affected animal. If signs occurred the animals were examined several times daily.
The littering and lactation behavior of the dams was generally evaluated in the mornings in combination with the daily clinical inspection of the dams. Only particular findings (e.g. inability to deliver) were documented on an individual dam basis. On weekdays (except 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 littering was considered the 24-hour period from about 15.00 h of one day until about 15.00 h of the following day.

DETAILED CLINICAL OBSERVATIONS: Yes
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).

BODY WEIGHT: Yes
Body weight was determined before the start of the administration period in order to randomize the animals. During the administration period body weight was determined on
study day 0 (start of the administration period) and thereafter once a week at the same time of the day (in the morning).The body weight change of the animals was calculated from
these results.
The following exceptions are notable for the female animals:
• During the mating period the parental females were weighed on the day of positive evidence of sperm (GD 0) and on GD 7, 14 and 20.
• Females with litter were weighed on the day of parturition (PND 0) and on PND 4.
• Females without a litter and without positive evidence of sperm in the vaginal smear or
waiting for necropsy were weighed weekly. These body weight data were solely used for the calculations of the dose volume.

FOOD CONSUMPTION:
Generally, food consumption was determined once a week for male and female parental animals, with the following exceptions:
• Food consumption was not determined during the mating period (male and female F0 animals).
• Food consumption of the F0 females with evidence of sperm was determined on GD 0 - 7, 7 - 14 and 14 - 20.
• Food consumption of F0 females, which gave birth to a litter was determined for PND 1 - 4.
Food consumption was not determined in females without positive evidence of sperm (during the mating period of dams) and females without litter (during the lactation period of
dams) and in males after the premating period.

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

HEAMATOLOGY
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 randomizedsequence. For urinalysis the individual animals were transferred to metabolism cages
(withdrawal of food and water) and urine was collected overnight. Urine samples wereevaluated in a randomized sequence
The assays of blood and serum parameters were performed under internal laboratory qualitycontrol conditions with reference controls to assure reliable test results
The following examinations were carried out in the first 5 surviving parental males and the first 5 surviving females with litter (in order of delivery) per group.
Leukocyte count (WBC), Erythrocyte count (RBC), Hemoglobin (HGB), Hematocrit (HCT), Mean corpuscular volume (MCV), Mean corpuscular hemoglobin (MCH)
Mean corpuscular hemoglobin concentration (MCHC), Platelet count (PLT), Differential blood, Reticulocytes (RET)

CLINICAL CHEMISTRY
Alanine aminotransferase; Aspartate aminotransferase; Alkaline phosphatase; γ-Glutamyltransferase
Sodium, Potassium, Chloride, Inorganic phosphate, Calcium, Urea, Creatine, Glucose, Total bilirubin, Total protein, Albumin, Globlulins, Triglycerides, Cholesterol, Bile acids,

URINALYSIS
pH, Protein, Glucose, Ketones, Urobilnogen, Bilirubin, Blood, Specific gravity, Sediment, Color turbitdity; Volume

FUNCTIONAL OBSERVATION BATTERY
A functional observational battery was performed in the first five surviving male animals per test group and the first five surviving females with litter (in order of delivery) of all test groups
at the end of the administration period starting in the morning. 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.

MOTOR ACTIVITY ASSESSMENT
Motor activity (MA) was also measured in the early afternoon 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 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 animals 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.

Litter observations:

Pup number and status at delivery
All pups delivered from the F0 parents (F1 litter) were examined as soon as possible on the day of birth to determine the total number of pups, the sex and the number of liveborn and
stillborn pups in each litter. At the same time, the pups were also being examined for macroscopically evident changes. Pups, which died before this initial examination, were
defined as stillborn pups.

Pup viability/mortality
In general, a check was made for any dead or moribund pups twice daily on workdays (once in the morning and once in the afternoon) or as a rule, only in the morning on Saturdays, Sundays or
public holidays. The number and percentage of dead pups on the day of birth (PND 0) and of pups dying between PND 1 - 4 (lactation period) were determined. Pups which died accidentally or were sacrificed due to
maternal death were not included in these calculations. The number of live pups/litter was calculated on the day after birth, and on lactation day 4. The viability index was calculated
according to the following formula:
Viablity index(%)=(number of live pups on day 4 after birth/number of live pups on the day of birth)x100

Sex ratio
On the day of birth (PND 0) the sex of the pups was determined by observing the distance between the anus and the base of the genital tubercle; normally, the anogenital distance is considerably
greater in male than in female pups. The sex of the pups was finally confirmed at necropsy. The sex ratio was calculated at day 0 and day 4 after birth according to the following formula:
Sex ratio = (number of live male or female pups on day 0/4 / number of live male and female pups on day 0/4)x100


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

Pup body weight data
The pups were weighed on the day after birth (PND 1) and on PND 4. Pups' body weight change was calculated from these results. The individual weights were always determined at about the same time
of the day (in the morning). 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.

Postmortem examinations (parental animals):

Necropsy
All parental animals were sacrificed by decapitation under isoflurane anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology, special attention
being given to the reproductive organs. One female animal of the control group (No. 109) which died spontaneously after blood sampling, was necropsied and assessed by gross
pathology as soon as possible.

Organ weights
The following weights were determined in all animals sacrificed on schedule:
Anesthetized animals, epididymides, Testes
The following weights were determined in 5 animals/sex and test group (females with litters, same animals as used for clinical pathology examinations):
Adrenal glands, Brain, Heart, Kidneys, Liver, Spleen, Thymus

Histopathology
All gross lesions , Adrenal cortex ,Adrenal medulla, Bone marrow (femur), Brain,Cecum, Cervix, Coagulating glands, Colon, Duodenum, Epididymides, Heart, leum, Jejunum, Kidneys, Liver, Lung,
Lymph nodes, Ovaries, Oviducts, Peyers patches, Prostate, Rectum, Sciatic nerve, Seminal vesicles, spinal cord, Spleen, Stomach, Testes, Thymus , Thyroid glands, Trachea, Urinary bladder, Uterus, Vagina.

Postmortem examinations (offspring):

All pups with scheduled sacrifice on PND 4 were sacrificed under isoflurane anesthesia with CO2. All pups were examined externally and eviscerated; their organs were assessed
macroscopically. All stillborn pups and all pups that died before PND 4 were examined externally, eviscerated and their organs were assessed macroscopically.
All pups without notable findings or abnormalities were discarded after their macroscopic evaluation. Animals with notable findings or abnormalities were evaluated on a case-bycase
basis, depending on the type of finding noted.

Statistics:

Food consumption (parental animals), body weight and body weight change (parental animals and pups; for the pup weights, the litter means were used), gestation days: DUNNETT test (two-sided)
Male and female mating indices, male and female fertility indices, gestation index (females with liveborn pups), females delivering,females with stillborn pups, females with all stillborn pups, female pregnant: FISHER'S EXACT test (onesided)
Mating days until day 0 pc,%postimplantation loss, pups stillborn, %perinatal loss: WILCOXON test (one-sided+) with BONFERRONI-HOLM adjustment
Implantation sites, pups delivered, pups liveborn, live pups day x, viability Index:WILCOXON test (one-sided+) with BONFERRONI-HOLM adjustment
% live male day x, %live female day x: WILCOXON test (two-sided)
Rearing, grip strength of forelimbs and hindlimbs, landing foot-splay test, motor activity: WILCOXON test (two-sided)

Reproductive indices:

Reproductive indices
Male mating index
Male mating index (%) = (number of males with confirmed mating*/number of males placed with females) x 100
* defined by a female with vaginal sperm or with implants in utero
Male fertility index
Male fertility index (%) = (number of males proving their fertility*/number of males placed with females)x100
* defined by a female with implants in utero
Female mating index
Female mating index (%) = (number of females mated*/number of females placed with males) x 100
* defined as the number of females with vaginal sperm or with implants in utero
Female fertility index
Female fertility index (%) = (number of females pregnant*/number of females mated**) x 100
* defined as the number of females with implants in utero
** defined as the number of females with vaginal sperm or with implants in utero
Gestation index
Gestation index (%) = (number of females with live pups on the day of birth/number of females pregnant*) x 100
* defined as the number of females with implants in utero
Live birth index
Live birth index (%) = (number of liveborn pups at birth/total number of pups born) x 100

Offspring viability indices:

Pup viability/mortality
In general, a check was made for any dead or moribund pups twice daily on workdays (once in the morning and once in the afternoon) or as a rule, only in the morning on Saturdays, Sundays or public
holidays.
The number and percentage of dead pups on the day of birth (PND 0) and of pups dying between PND 1´- 4 (lactation period) were determined. Pups which died accidentally or were sacrificed due to maternal
death were not included in these calculations. The number of live pups/litter was calculated on the day after birth, and on lactation day 4. The viability index was calculated according to the following formula:
Viability index (%) = (number of live pups on day 4 after birth/number of live pups on the day of birth) x 100

Results and discussion

Results: P0 (first parental generation)

Details on results (P0)

In the detailed clinical observations on study days 0, 7, 14, 21 and 28 in males and females and
additionally, on study days 35, 42 and 49 in female animals only no adverse findings were observed.
In test group 3 (150 mg/kg bw/d) slight to severe salivation within 2 hours after treatment had been observed intermittent during pre-mating in 1 male and
4 females, during mating in 8 males and 7 females and during post-mating in 3 males.
In test group 2 (50 mg/kg bw/d) slight and moderate salivation within 2 hours after treatment had been observed intermittent during mating in 4 males and
3 females and during postmating in 3 males.
In test group 1 (15 mg/kg bw/d) slight and moderate salivation within 2 hours after treatment had been observed intermittent during mating in 1 male.
These findings were considered to be related to treatment, most likely caused by bad taste of the test compound, and assessed as being not adverse.
Clinical observations for females during gestation
In all animals of test group 3 (150 mg/kg bw/d) slight to severe intermittent salivation and in 6 animals
of test group 2 (50 mg/kg bw/d) slight and moderate intermittent salivation was observed during gestation.
The findings were considered to be related to treatment, most likely caused by bad taste of the test
compound, and therefore assessed as being not adverse.
Clinical observations for females during lactation
On lactation day (LD) 15 (study day 52) one female (No. 109) of test group 0 (0 mg/kg bw/d) had an sudden death immediately before sacrifice. The animal
died after anesthesia for blood sampling. No corresponding macroscopic finding was observed in necropsy to explain
the death. This isolated finding was considered to be incidental and not treatment related.
During lactation in all animals of test group 3 (150 mg/kg bw/d) slight to severe intermittent salivation and in 9 animals of test group 2 (50 mg/kg bw/d) slight to severe intermittent salivation was observed. The findings were considered to be related to treatment, most likely caused by bad taste of the test compound,
and therefore assessed as being not adverse.

FOOD CONSUMPTION
During pre-mating the food consumption was significantly decreased in male animals of test group 3 (150 mg/kg bw/d) -21.6% between study days 0 to 7 and -8.4% between study days
7 to13. This resulted in a significant decrease of food consumption in this test group over the whole pre-mating period (-15.5%). In female animals of test group 3 the food consumption was decreased during pre-mating between study day 0 and 7 (-29.0%) and over the whole pre-mating period (-18.1%). The observed significant decreased food consumptions in males and females were assessed to be treatment related. In consideration of given or missing body weights effect in this dose group the decreased food consumption was assessed as adverse in males but not adverse in females. No significant deviations
from control in males and females were observed in food consumption during pre-mating in test groups 1 and 2 (15 and 50 mg/kg bw/d). Furthermore, no significant deviations from control in female animals were observed in food consumption during gestation and lactation.

WATER CONSUMPTION
No test substance-related changes were observed.

BODY WEIGHT DATA
Body weights were significantly decreased in male animals of test group 3 (150 mg/kg bw/d)
during pre-mating from study day 7 onwards as well as during mating and post-mating up to
-5.7%. This finding was assessed as treatment related and adverse.
In female animals of this test group no significant difference was observed for body weight
values in comparison to control, however, the animals showed a slight body weight loss
between pre-mating days 0 and 7. Considering the decreased food consumption at these
study days and the transient character this finding, the alteration was assessed as
treatment-related but not adverse.
In all animals of test groups 1 and 2 (15 and 50 mg/kg bw/d) no deviations were detected in
body weight values.
Body weight changes were significantly decreased in male animals of test group 3
(150 mg/kg bw/d) during the first 7 days of pre-mating (-79.9%) and thus also in overall value
of pre-mating phase (-48.7%). This finding was considered to be treatment related.
In male animals of test group 2 (50 mg/kg bw/d) body weight changes were decreased
significantly (-33.6%) between day 0 and 7 of pre-mating. Based on the transient character
of this finding, the alteration was assessed as treatment-related but not adverse.
The body weight change was significantly decreased in female animals of test group 3
(150 mg/kg bw/d) during the first 7 days of pre-mating (-115.9%), however, this decrease
was almost compensated in the following days.
Body weight changes were not significantly decreased in females of test groups 2 (50 mg/kg
bw/d) and of test group 1 (15 mg/kg bw/d) during pre-mating.
No significant alteration of body weights and body weight changes were determined during
gestation and lactation. The significant increase of body weight gain (31.6%) in females of
test group 1 (15 mg/kg bw/d) in gestation was considered as incidental and not treatment related.

FUNCTIONAL OBSERVATION 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 doseresponse
relationship or occurred in single rats only, these observations were consideredas incidental.
The following examinations were performed during FOB and are assessed individually:
Home Cage Observation
No test substance-related effects were observed.
Open field observations
In one male animal of test group1 (15 mg/kg bw/d) piloerection was seen during open
field observation. Since no dose depending effect can be seen, this single isolated finding
was assessed as spontaneous in nature and not treatment related.
No test substance-related effects were observed neither in males nor in females.
Sensorimotor tests/ reflexes
No test substance-related effects were observed.
Quantitative Parameters
No test substance-related effects were observed.
Motor activity measurement
Regarding the overall motor activity as well as single intervals, no test substance-related
deviations were noted for male and female rats.

REPRODUCTIVE PERFORMANCE
Male reproduction data
Male mating index
For F0 parental males, which were placed with females to generate F1 pups, mating was
confirmed. The male mating index was 100% in all test groups (0-3; 0, 15, 50 and 150 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.
One male of control group (No. 5 mated with female No. 105) did not generate implants in utero.
The male fertility index was 100% in dose groups 1 to 3 (15, 50 and 150 mg/kg bw/d) and 90% in control group.

Female reproduction and delivery data
Female mating index
The female mating index calculated after the mating period for F1 litter was 100% in all test groups.
The mean duration until sperm was detected (GD 0) was 2.8, 2.6, 2.7, and 2.5 days in test
groups 0 - 3 (0, 15, 50 and 150 mg/kg bw/d). This finding reflected the normal range of
biological variation inherent in the strain of rats used for this study as all respective values
were within the range of the historical control data.
Female fertility index
All sperm positive rats got pregnant with one exception in control group.One female animal
which was mated did get sperm in vaginal smear but showed no implants.
The female fertility index was 100% in dose groups 1 to 3 (15, 50 and 150 mg/kg bw/d) and
90% in control group
The gestation index was 100% in all test groups (0-3; 0, 15, 50 and 150 mg/kg bw/d).
The rate live birth indices were 100% in all test groups (0-3; 0, 15, 50 and 150 mg/kg bw/d).
The postimplantation loss was 0.9% in test group 0 (0 mg/kg bw/d), 10.9% in test group 1
(15 mg/kg bw/d), 2.4% in test group 2 (50 mg/kg bw/d) and 6.0% in test group 3 (150 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 (0.7% - 18.3%) of the historical control data

HEMATOLOGY
At the end of the administration period in males of test group 3 (150 mg/kg bw/d) absolute
neutrophil, monocyte and large unstained cell (LUC) counts were increased. Additionally, in
males of test group 2 (50 mg/kg bw/d) absolute neutrophil counts and in males of test group
1 (15 mg/kg bw/d) absolute monocyte counts were higher compared to controls. Although
absolute neutrophil counts in test groups 2 and 3 were above the historical control range
(absolute neutrophils 0.73-1.30 giga/L), the values were not dose-dependently changed.
The monocyte and LUC counts in the mentioned test groups as well as total white blood cell
(WBC) and absolute lymphocyte counts in all test groups were within historical control
ranges (absolute monocytes 0.07-0.16 giga/L; absolute LUC 0.01-0.05 giga/L; WBC 4.15-
7.95 giga/L; absolute lymphocytes 2.69-5.96 giga/L). Therefore, the mentioned differential
blood cell alterations including absolute neutrophil cell count increases in males of test
groups 2 and 3 were regarded as incidental and not treatment-related.
In contrast, in females of test group 3 (150 mg/kg bw/d) at the end of the administration
period, total white blood cell (WBC; mean +53% compared to controls), absolute neutrophil
(mean +45% compared to controls) and lymphocyte counts (mean +59% compared to
controls) were increased, although not statistically significantly. All mentioned parameters
were above historical control ranges (WBC 2.47-4.66 giga/L; absolute neutrophils 0.45-
1.12 giga/L; absolute lymphocytes 1.64-3.51 giga/L).
In males of test group 3 (150 mg/kg bw/d) platelet counts were higher compared to controls,
but the mean was within the historical control range (platelets 641-889 giga/L) and therefore,
this alteration was regarded as incidental and not treatment-related.

CLINICAL CHEMISTRY
No treatment-related changes among clinical chemistry parameters were observed.
In females of test group 3 (150 mg/kg bw/d) globulin values were lower compared to controls.
However, the mean was within the historical control range (globulins 21.91-26.93 g/L).
Therefore, this change was regarded as incidental and not treatment-related.
URINALYSES
No treatment-related changes among urinalysis parameters were observed.
In males of test group 2 (50 mg/kg bw/d) more triple phosphate crystals were found in the urine
sediment compared to controls. This finding was not dose-dependent and therefore it was regarded
as incidental and not treatment-related.

ORGAN WEIGHTS
Absolute organ weights
Only the terminal body weight in males of test group 3 (150 mg/kg bw/d) was statistically
significantly decreased (320.69 g) and beneath the historical control values
(342.47 – 420.58 g). This change was regarded as treatment-related.

RELATIVE ORGAN WEIGHTS
When compared to control group 0 (set to 100%), the mean relative weights showed no
statistically significant changes.

GROSS PATHOLOGY
Only one finding occurred individually, and it was considered to be incidental or spontaneous
in origin and without any relation to treatment. One animal, which died spontaneously after
blood sampling, showed no macroscopic changes.
Fertility:
One female animal, which was not pregnant as well as its male mating partner did not show
relevant gross lesions.

HISTOPATHOLOGY
In test group 3 (150 mg/kg bw/d), 7 out of 10 females showed a minimal centrilobular
hepatocellular hypertrophy, which was regarded as treatment-related but not adverse.
All other findings occurred either individually or were biologically equally distributed over
control and treatment groups. They were considered to be incidental or spontaneous in
origin and without any relation to treatment.
One animal, which died spontaneously after blood sampling, showed no relevant
microscopical changes.
The stages of spermatogenesis in the testes of males of the high dose (150 mg/kg bw/d)
were comparable to those of the controls.
Fertility
One female animal, which was not pregnant as well as its male mating partner did not
show relevant histopathological findings.

Effect levels (P0)

Dose descriptor:

NOAEL

Effect level:

150 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: no reproductive effects were noted

Results: F1 generation

Details on results (F1)

NUMBER AND STATUS AT DELIVERY
The mean number of delivered F1 pups per dam and the rates of liveborn, stillborn,
cannibalized and dead F1 pups were evenly distributed about test groups 0 - 3. The
respective values reflect the normal range of biological variation inherent in the strain used
in this study, as they are in the historical control range

VIABILIY/MORTALITY
The viability index indicating pup mortality during lactation (PND 0 - 4) was 100.0% in test
groups 0 and 2 (0 and 50 mg/kg bw/d), 98.4% in test group 1 (15 mg/kg bw/d) based on one
pup was found dead as well as one pup was missing (cannibalized) and 99.2% in test
group 3 (150 mg/kg bw/d) based on one pup was found dead. 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:

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

CLINICAL OBSERVATION
The surviving F1 pups of any test group did not show adverse clinical signs up to scheduled
sacrifice on PND 4. However, in test group 1 (15 mg/kg bw/d) one male pup could be
assessed until PND 0 only, because it was found dead on PND 1 and one female pup could
be assessed until PND 3 only, because it was missed (cannibalized). Furthermore, in test
group 3 (150 mg/kg bw/d) one female pup could be assessed until PND 1 only, because it
was found dead on PND 1.

BODY WEIGHT
Mean pup body weights/ pup body weight changes of all pups in all test groups were
comparable to the control group. The body weight change of pups was significantly
decreased (-18.2%) between PND 1 and 4 in the sum of male and female pups of test
group 2 (50 mg/kg bw/d). Since, no alterations of body weight change of pups were
determined in test groups 1 and 3 (15 and 150 mg/kg bw/d) this finding had not dosedependency,
was assessed as spontaneous in nature and not adverse.
Three male and one female runts were seen in test group 1 (15 mg/kg bw/d). One male runt
was seen in test group 2 (50 mg/kg bw/d). One male and 3 female runts were seen in test
group 3 (150 mg/kg bw/d). These values were within the range of the biological variation
inherent in the strain of rats used for this study.

NECROPSY OBSERVATIONS
In test group 1 (15 mg/kg bw/d) one male pup showed post mortem autolysis and one female
pup could not be assessed because it was missing (cannibalized). In test group 2 (50 mg/kg
bw/d) two male pups showed a discolored liver. Furthermore, in test group 3 (150 mg/kg
bw/d) one female pup showed a discolored liver. These findings were assessed as being
signs occurred post mortem or spontaneous in nature and thereby not related to treatment.

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

150 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: no developmental effects were noted

Overall reproductive toxicity

Reproductive effects observed:

not specified