Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
04 Jul 2017 to 26 Sep 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose for cross-reference:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
28 Jul 2015
Qualifier:
according to guideline
Guideline:
other: EPA, Health Effects Test Guidelines; OPPTS 870.3650: Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test
Version / remarks:
Jul 2000
GLP compliance:
yes (incl. QA statement)
Remarks:
BASF SE, Experimental Toxicology and Ecology, 67056 Ludwigshafen, Germany
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Glycerides, castor-oil mono-, di- and tri-
EC Number:
294-589-3
EC Name:
Glycerides, castor-oil mono-, di- and tri-
Cas Number:
91744-27-3
Molecular formula:
C21H40O5 – C57H104O9
IUPAC Name:
Glycerides, castor-oil mono-, di- and tri-
Test material form:
liquid
Specific details on test material used for the study:
- Name of test substance used in the report: Sovermol 320
- Test substance No.: 16/0552-1
- Batch identification.: 0012756105
- Content: > 97g / 100g
- Homogeneity: Given
- Stability: The stability of the test substance under storage conditions over the test period was guaranteed by the sponsor, and the sponsor holds this responsibility (expiry date: 01 Jul 2018)
- Date of production: 27 Oct 2014
- Physical state/ appearance: Liquid, viscous/ light brownish, clear
- Storage conditions: Ambient (room temperature)

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Crl:WI(Han)
Details on species / strain selection:
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, Sulzfeld, Germany GmbH
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: about 10 - 11 weeks (males) and about 9 weeks (females)
- Weight at study initiation: 351.3 – 409.6 g (males) and 199.0 – 229.3 g (females)
- Fasting period before study: no
- 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 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 pre-mating, mating, gestation, lactation, males after mating and females after weaning: Polycarbonate cages type III
* 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. Dust-free wooden bedding was used in this study (the present supplier is documented in the raw data). Wooden gnawing blocks (Type NGM E-022) supplied by Abedd® Lab. and Vet. Service GmbH, Vienna, Austria, was added for environmental enrichment. The cages with the test animals were arranged on the racks in such a way that uniform experimental conditions (ventilation and light) were ensured.
- Diet: ground Kliba maintenance diet mouse-rat “GLP”, meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland, ad libitum.
- Drinking water: from water bottles, ad libitum.
- Acclimation period: 28 days

DETAILS OF FOOD AND WATER QUALITY: Diet, water, bedding and cage enrichment evaluation for contaminants and/or nutrients was performed according to facility standard procedures. There were no findings that could interfere with the study.

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



Administration / exposure

Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
0.5% sodium carboxymethyl cellulose in deionized water
Details on exposure:
TEST SUBSTANCE PREPARATIONS
The test substance was applied as a suspension. To prepare this suspension, the appropriate amount of test substance was weighed out depending on the desired concentration. Then, 0.5% sodium carboxymethyl cellulose in deionized water was filled up to the desired volume and subsequently released with a magnetic stirrer. The test substance preparations were produced weekly, at least.

VEHICLE
- 0.5% sodium carboxymethyl cellulose in deionized water
- Amount of vehicle: 10 mL/kg bw/day
Details on mating procedure:
- Each of the male and female animals was mated overnight in a 1:1 ratio for a maximum of 2 weeks. Throughout the mating period, each female animal was paired with a predetermined male animal from the same test group. Sibling mating was avoided as all male and female animals were delivered from different litters according to the breeder.
- A vaginal smear was prepared after each mating and examined for the presence of sperm. If sperm was detected, pairing of the animals was discontinued. The day on which sperm was detected was denoted gestation day (GD) 0 and the following day "GD 1".
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The analytical investigations of the test substance preparations were carried out as a separate study at the test facility Competence Center Analytics of BASF SE, 67056 Ludwigshafen, Germany under the responsibility of the Study Director of this test facility. The study was carried out in compliance with the Principles of Good Laboratory Practice. The stability of the test substance in 0.5% sodium carboxymethyl cellulose in drinking water over a period of 7 days was proven. At the beginning (during pre-mating), twice during gestation and once during lactation of the study each 3 samples were taken from the lowest and highest concentration for potential homogeneity analyses. These samples were used as a concentration control at the same time. At the time points mentioned above, one sample from the mid concentration was additionally taken for concentration control analysis. The samples collected at the beginning of the administration period and during the lactation period were analyzed.
Duration of treatment / exposure:
Exposure for the male animals was 29 days. This included 2 weeks prior to mating, 2 weeks of mating procedure and the day of sacrifice. Exposure for the female animals was 41 to 55 days. This included 2 weeks prior to mating, 2 weeks mating period, the entire gestation and lactation period, and the day of sacrifice.
Frequency of treatment:
Once a day for 7 days per week
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
low-dose level
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Remarks:
mid-dose level
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
high-dose level
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: at the request of the sponsor
- Route of administration selection: the oral route was selected since this was proven to be suitable for the detection of a toxicological hazard.
Positive control:
No

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATION:
- 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 cage side examination was conducted at least once daily for any signs of morbidity, pertinent behavioural 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 behaviour of the dams was generally 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 behaviour 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:
- Time schedule: prior to the administration period and thereafter at weekly intervals
- Parameters examined: abnormal behaviour in handling; fur; skin; posture; salivation; respiration; activity/arousal level; tremors; convulsions; abnormal movements; gait abnormalities; lacrimation; palpebral closure; exophthalmos; assessment of the faeces discharged during the examination (appearance/ consistency); assessment of the urine discharged during the examination; pupil size

BODY WEIGHT:
- 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 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:
- Food consumption was determined, as g food/kg body weight/day, once a week for male and female animals, with the following exceptions:
* Food consumption was not determined after the 2nd premating week (male parental animals) and during the mating period (male and female parental animals).
* Food consumption of the females with evidence of sperm was determined for GD 7, 14 and 20.
* Food consumption of the females which gave birth to a litter was determined for PNDs 4, 7, 10 and 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:
Drinking water consumption was monitored by daily visual inspection of the water bottles for any changes in volume.

NEUROBEHAVIOURAL EXAMINATION: Functional observational battery (FOB):
- 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 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 behaviour 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: behaviour on removal from the cage; fur; skin; salivation; nose discharge; lacrimation; eyes/pupil size; posture; palpebral closure; respiration; tremors; convulsions; abnormal movements/stereotypes; gait abnormalities; activity/arousal level; urine excreted within 2 minutes (amount/colour); 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); behaviour during handling; vocalization; pain perception (tail pinch); grip strength of forelimbs; grip strength of hindlimbs; landing foot-splay test; other findings

NEUROBEHAVIOURAL EXAMINATION: Motor activity (MA) assessment:
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 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 were 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.

HAEMATOLOGY:
- Time schedule for collection of blood: in the morning
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: the first 5 surviving parental males per group at termination and the first 5 females with litters (in order of delivery) per group at PND 14
- The following parameters were determined in blood with EDTA K3 as anticoagulant using a particle counter (Advia 120 model; Bayer, Fernwald, Germany): leukocyte count (WBC); erythrocyte count (RBC); haemoglobin (HGB); haematocrit (HCT); mean corpuscular volume (MCV); mean corpuscular haemoglobin (MCH); mean corpuscular haemoglobin concentration (MCHC); platelet count (PLT); differential blood count; reticulocytes (RETA).
- Blood smears were prepared and stained according to WRIGHT without being evaluated, because of non-ambiguous results of the differential blood cell counts measured by the automated instrument. (reference: Haematology: Principles and Procedures, 6th Edition, Brown AB, Lea & Febiger, Philadelphia, 1993, page 101). Only evaluated blood smears were archived.
- Clotting tests were carried out using a ball coagulometer (AMAX destiny plus model; Trinity biotech, Lemgo, Germany): prothrombin time (Hepato Quick’s test) (HQT)

CLINICAL CHEMISTRY:
- Time schedule for collection of blood: in the morning
- Animals fasted: Yes
- How many animals: the first 5 surviving parental males per group at termination and the first 5 females with litters (in order of delivery) per group at PND 14
- An automatic analyser (Cobas c501; Roche, Mannheim, Germany) was used to examine the clinicochemical parameters: alanine aminotransferase (ALT) (L-alanine: 2-oxoglutarate aminotransferase; EC 2.6.1.2.); aspartate aminotransferase (AST) (L-aspartate: 2-oxoglutarate aminotransferase; EC 2.6.1.1.); alkaline phosphatase (ALP) (orthophosphoric acid monoester phosphohydrolase; EC 3.1.3.1.); gamma-Glutamyltransferase (GGT) (gamma -glutamyl) peptide: aminoacid-gamma-glutamyl-transferase; EC 2.3.2.2.); sodium (NA); potassium (K); chloride (CL); inorganic phosphate (INP); calcium (CA); urea (UREA); creatinine (CREA); glucose (GLUC); total bilirubin (TBIL); total protein (TPROT); albumin (ALB); globulins (GLOB); triglycerides (TRIG); cholesterol (CHOL); bile acids (TBA).

THYROID HORMONES:
- Blood samples from all dams at PND 14 and all males at termination were taken by puncturing the retrobulbar venous plexus under isoflurane anaesthesia. The animals were fastened before the blood sampling.
- All generated serum samples were frozen at -80°C until measurement.
- Blood samples from the adult males were assessed for serum levels for thyroid hormones (total thyroxine T4 and thyroid stimulating hormone TSH).

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.

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.
Oestrous cyclicity (parental animals):
For all females of the pool estrous cycle normality was evaluated before the beginning of the administration.
in all parental females 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 determined in all female F0 rats.
Sperm parameters (parental animals):
- Parameters examined in male parental generation: testis weight, epididymis weight
- Special attention was given to the stages of spermatogenesis and histopathology of interstitial testicular cell structure during histopathology
Litter observations:
STANDARDISATION OF LITTERS
On PND 4, the individual litters were standardized in such a way that, where possible, each litter contained 4 male and 4 female pups (always the first 4 pups/sex and litter were taken for further rearing). If individual litters did not have 4 pups/sex, the litters were processed in such a way that the most evenly distributed 8 pups per litter were present for further rearing (e.g., 5 male and 3 female pups). Surplus pups or 2 preferably female pups per litter were sacrificed. Standardization of litters was not performed in litters with ≤ 8 pups.

PARAMETERS EXAMINED
- Total number of pups, the sex and the number of liveborn and stillborn pups in each litter was determined on the day of birth (PND 0).
- Mortality/viability: 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, 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.
- Clinical signs: examined daily for clinical symptoms (including gross-morphological findings) during the clinical inspection of the dams
- Body weights: measured on the day after birth (PND 1) as well as on PNDs 4, 7 and 13
- Sex : On PND 0, the sex of the pups was determined by observing the distance between the anus and the base of the genital tubercle. 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.
- Anogenital distance: 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.
- 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.
Postmortem examinations (parental animals):
NECROPSY:
All parental animals were sacrificed by decapitation under isoflurane anaesthesia. The exsanguinated animals were necropsied and assessed by gross pathology, special attention being given to the reproductive organs.

ORGAN WEIGHTS:
- The following weights were determined in all animals sacrificed on schedule: anesthetized animals; epididymides; ovaries; prostate; seminal vesicles with coagulating glands; testes; thyroid glands (fixed); uterus (with cervix)
- The following weights were determined in 5 animals per sex/test group sacrificed on schedule (females with litters only, same animals as used for clinical pathological examinations): adrenal glands; brain; heart; kidneys; liver; spleen; thymus

ORGAN/TISSUE FIXATION:
- The following organs or tissues of all parental animals were fixed in in 4% neutral-buffered formaldehyde or in modified Davidson’s solution: all gross lesions; adrenal glands; aorta; bone marrow (femur); brain; cecum; cervix; coagulating glands; colon; duodenum; oesophagus; extra orbital lacrimal glands; epididymides (modified Davidson’s solution); eyes with optic nerve (modified Davidson’s solution); femur with knee joint; heart; ileum; jejunum (with Peyer’s patches); kidneys; larynx; liver; lungs; lymph nodes (axillary and mesenteric); mammary gland (male and female); nose (nasal cavity); ovaries (modified Davidson’s solution); oviducts; pancreas; parathyroid glands; pharynx; pituitary gland; prostate gland; rectum; salivary glands (mandibular and sublingual); sciatic nerve; seminal vesicles; skeletal muscle; spinal cord (cervical, thoracic and lumbar cord); spleen; sternum with marrow; stomach (forestomach and glandular stomach); testes (modified Davidson’s solution); thymus; thyroid glands; trachea; urinary bladder; uterus (uteri of all apparently nonpregnant animals or empty uterus horns were stained according to Salewski E [1964]); vagina.

HISTOPATHOLOGY
Fixation was followed by histotechnical processing, examination by light microscopy. The following organs were assessed in the following groups:
- all animals / test group (control group and high dose group): cervix; coagulating glands; epididymides; kidneys; liver; ovaries; oviducts; prostate gland; seminal vesicles; testes; uterus; vagina
- all animals affected / test group: all gross lesions
- mating pairs suspected of reduced fertility (low dose group and mid dose group): cervix; coagulating glands; epididymides; ovaries; oviducts; prostate gland; seminal vesicles; testes; uterus; vagina
- 5 animals per sex/group, females with litters only, same animals as used for haematology and clinical chemistry examinations (control group and high dose group): adrenal glands; brain; cecum; colon; duodenum; eyes with optic nerve; heart; ileum; jejunum; lungs; lymph nodes (axillary and mesenteric); Peyer’s patches; rectum; sciatic nerves; skeletal muscle; spinal cord (cervical, thoracic, lumbar); spleen; sternum with marrow; stomach (forestomach and glandular stomach); thymus; thyroid glands; trachea; urinary bladder
Postmortem examinations (offspring):
- On post-natal day (PND) 4, as a result of standardization, surplus pups, respectively, were sacrificed under isoflurane anesthesia by decapitation. Blood was sampled for determination of thyroid hormone (total thyroxine T4 and thyroid stimulating hormone TSH) concentrations. After sacrifice, these pups were examined externally, 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 (total thyroxine T4 and thyroid stimulating hormone TSH) concentrations. Thyroid glands/parathyroid glands were fixed in neutral buffered 4% formaldehyde solution and were transferred to the Pathology Laboratory for possible further processing. After sacrifice, the 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 any notable findings or abnormalities were discarded after their macroscopic evaluation. Animals with notable findings or abnormalities were further evaluated on a case-by-case basis (e.g., histopathological evaluation or special staining), depending on the findings noted.
Statistics:
See details about statistics used in this study, in the "Any other information on materials and methods incl.tables" section.
Reproductive indices:
Male mating index (%) = number of males with confirmed mating* / number of males placed with females x 100
*defined by a female with vaginal sperm or with implants in utero

Male fertility index (%) = number of males proving their fertility* / number of males placed with females x 100
* defined by a female with implants in utero

Female mating index (%) = number of females mated* / number of females placed with males x 100
* defined as the number of females with vaginal sperm or with implants in utero

Female fertility index (%) = number of females pregnant* / number of females mated** x 100
* defined as the number of females with implants in utero
** defined as the number of females with vaginal sperm or with implants in utero

Gestation index (%) = number of females with live pups on the day of birth / number of females pregnant* x 100
* defined as the number of females with implants in utero

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 (%) = number of liveborn pups at birth / total number of pups born x 100

Postimplantation loss (%) = (number of implantations – number of pups delivered) / number of implantations x 100
Offspring viability indices:
Viability index (%) = number of live pups on day 4* after birth / number of live pups on the day of birth x 100
* before standardization of litters (i.e. before culling)

Survival index (%) = number of live pups on day 13 after birth / number of live pups on day 4* after birth x 100
* after standardization of litters (i.e. after culling)

Sex ratio = number of live male or female pups on PND 0 and 13 / number of live male and female pups on PND 0 and 13 x 100

Anogenital index = anogenital distance [mm] / cubic root of pup weight [g]

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
- In the pre-mating period, salivation was observed in all male and 9 of 10 female animals of test group 3 (1000 mg/kg bw/d), starting on pre-mating days 7 and 8 in males and females, respectively. In both sexes, the finding also occurred in the mating period as well as in males in the post-mating phase.
- From the temporary, short appearance immediately after dosing (or shortly before) it was concluded that both kind of findings were induced by a bad taste of the test substance or local affection of the upper digestive tract. The effect was related to the test substance but assessed as being non-adverse as no lesions in the upper digestive tract were observed in male and female animals during pathological examinations.
- One male animal of test group 2 (300 mg/kg bw/d) showed a swollen paw and an injury at the left forelimb from mating day 10 onwards. During post-mating, the injured left forelimb with the swollen paw was still observed until sacrifice. Both findings were considered to be incidental and spontaneous in origin and without any relation to treatment.
- Salivation was observed in all females of test group 3 (1000 mg/kg bw/d) between gestation days 0 and 22. The effect was related to the test substance but assessed as being non-adverse.
- Salivation was observed in all females of test group 3 (1000 mg/kg bw/d) between lactation days 0 and 19. The effect was related to the test substance but assessed as being non-adverse. One female of test group 2 (300 mg/kg bw/d) showed piloerection on lactation day 0 and lost its complete litter on lactation day 1. One female of test group 1 (100 mg/kg bw/d) showed unsteady gait and semiclosed eyelids on lactation days 14 and 15, paleness on lactation day 15, and piloerection as well as palpable mass through skin, inguinal, between lactation days 14 and 18. These findings were considered to be incidental and spontaneous in origin since no dose-response relationship occurred.
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 in mean body weight change values 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.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
- No test substance-related, adverse changes with regard to food consumption were observed.
- Food consumption in female animals of test group 3 (1000 mg/kg bw/d) was significantly lower between gestation days 7 and 14. This value was still within a normal range typical for this strain of rats and, therefore, the deviations to the control were assessed to be without toxicological relevance.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
No test substance-related changes in water consumption were observed.
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
- No treatment-related changes among haematological parameters were observed.
- At the end of the administration period in males of test group 3 (1000 mg/kg bw/d) red blood cell (RBC) counts were significantly increased. However the mean was within the historical control range (males, RBC 7.91-9.03 Tera/L). Therefore, this change was regarded as incidental and not treatment-related.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
No treatment-related changes among clinical chemistry parameters were observed.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
- Deviations from "zero values" were obtained in several animals. 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. Grip strength of hindlimbs was significantly decreased in female animals of test group 1 (100 mg/kg bw/d). As no dose-response relationship occurred, the change was assessed as being spontaneous in nature and not related to treatment.
- Regarding the overall motor activity as well as single intervals, no test substance-related deviations were noted for male and female animals.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
- 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.
- The female animal, which was neither mated nor pregnant, as well as the male mating partner did not show relevant histopathological findings.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
THYROID HORMONES:
In parental males of 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. The mean estrous cycle duration in the different test groups was 3.9 days in test groups 0 (control) and 2 (300 mg/kg bw/d) and 4.0 days in test groups 1 (100 mg/kg bw/d) and 3 (1000 mg/kg bw/d).
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:
effects observed, non-treatment-related
Description (incidence and severity):
MALE REPRODUCTION DATA
- The male mating index calculated after the mating period for F1 litter was 100% in test groups 0 (control), 1 (100 mg/kg bw/d) and 3 (1000 mg/kg bw/d) and 90% in test group 2 (100 mg/kg bw/d).
- Fertility was proven for most of the F0 parental males within the scheduled mating interval to produce F1 litter. One male animal of test group 2 (300 mg/kg bw/d), which was placed in the same cage with one female animal, did not generate F1 pups. Thus, the male fertility index was 100.0% in test group 0 (control), 1 (100 mg/kg bw/d) and 3 (1000 mg/kg bw/d) and 90% in test group 2 (300 mg/kg bw/d. These values reflected the normal range of biological variation inherent in the strain of rats used for this study.

FEMALE REPRODUCTION DATA
- The female mating index calculated after the mating period for F1 litter was 100% in test groups 0 (control; 0 mg/kg bw/d), 1 (100 mg/kg bw/d) and 3 (1000 mg/kg bw/d) and 90% in test group 2 (300 mg/kg bw/d). The mean duration until sperm was detected (GD 0) was 2.0 days for test group 0, 2.4 days for test group 1, 3.1 days for test group 2 and 3.0 days for test group 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.
- All sperm positive rats delivered pups. Thus, the female fertility index was 100% in all test groups including the control. The mean duration of gestation was similar in all test groups, i.e. 22.5 days (test group 0 and test group 1), 22.2 days (test group 2) and 22.4 days (test group 3). 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.
- The gestation index was 100% in all test groups.
- The rate live birth indices were 100% in all test groups.
- The postimplantation loss was 1.4% in test group 0 (control), 2.8% in test group 1 (100 mg/kg bw/d), 0.9% in test group 2 (300 mg/kg bw/d) and 6.2% 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. A treatment-related increase in postimplantation loss was not observed in any test group.

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
reproductive toxicity
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive performance
Dose descriptor:
NOAEL
Remarks:
systemic toxicity
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: general systemic toxicity

Target system / organ toxicity (P0)

Key result
Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Description (incidence and severity):
All F1 pups of test groups 0 to 3 did not show adverse clinical signs up to scheduled sacrifice on PND 4 or PND 13.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Description (incidence and severity):
- The mean number of delivered F1 pups per dam was equally distributed among test groups 0, 1, 2 and 3 (0, 100, 300 and 1000 mg/kg bw/d). No significant deviations occurred.
- The viability index indicating pup mortality during PND 0-4 was 99.2% in test group 0 (control), 100.0% in test group 1 (100 mg/kg bw/d), 88.9% in test group 2 (300 mg/kg bw/d) and 98.2% in test group 3 (1000 mg/kg bw/d).
The rate of liveborn pups in all test groups was not affected by the test substance, as indicated by live birth indices of 100%.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
- Mean pup body weights/pup body weight changes of all pups in all test groups were comparable to the control group.
- Three males and 1 female runt were seen in 3 litters of test group 0 (control group; 0 mg/kg bw/d), four male and 1 female runt were seen in 2 litters of test groups 1 (100 mg/kg bw/d) and 2 female runts were seen in 2 litters of 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):
- 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 index were not affected in all F1 pups.
- 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:
no effects observed
Description (incidence and severity):
- When compared to control group 0 (set to 100%), no absolute weights were significantly changed.
- When compared to control group 0 (set to 100%), no relative weights were significantly changed.
Gross pathological findings:
no effects observed
Description (incidence and severity):
At pup necropsy, no malformations were found at any dose level.
Histopathological findings:
not examined
Other effects:
no effects observed
Description (incidence and severity):
THYROID HORMONES:
in male and female pups of test groups 11, 12 and 13 (100, 300 and 1000 mg/kg bw/d) at PND13, 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)

Key result
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Any other information on results incl. tables

Stability analysis

The stability of the test substance in0.5% sodium carboxymethyl cellulose in drinking water was demonstrated over a period of 7 days at room temperature. As the test substance preparations were not stored longer than this time period, the stability was guaranteed.

 

Homogeneity control analysis

Considering the low relative standard deviation in the homogeneity analysis, it can be concluded that the substance was distributed homogeneously in 0.5% sodium carboxymethyl cellulose in deionized water.

 

Concentration control analyses

The concentrations of the test substance in 0.5% sodium carboxymethyl cellulose in deionized water were found to be in the range of 90-110% of the nominal concentration. These results demonstrated the correctness of the concentrations ofthe test substancein 0.5% sodium carboxymethyl cellulose in drinking water.

Reproduction data

F0 generation: Parental animals

- Male 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 [%]

100.0

100.0

90.0

100.0

  * p0.05; ** p0.01

 

- 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 [%]

100.0

100.0

100.0

100.0

  * p0.05; ** p0.01

F1 generation (pups)

 

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 [%]

52.8

45.1

42.6

55.6

Live females [%]

47.2

54.9

57.4

44.4

PND 13

 

 

 

 

Live males [%]

50.0

46.2

50.0

56.2

Live females [%]

50.0

53.8

50.0

43.8

 

 

Applicant's summary and conclusion

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