Glycerides, castor-oil mono-, di- and tri-

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

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 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:

28 Jul 2015

Test guideline 2

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

Test material information

Constituent 1

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 oral 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

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 / concentrations

Doses / concentrations 1

Dose / conc.:

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

Remarks:

low-dose level

Doses / concentrations 2

Dose / conc.:

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

Remarks:

mid-dose level

Doses / concentrations 3

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: range finding study
- 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

Observations and examinations performed and frequency:

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).

Sacrifice and pathology:

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

Statistics:

See details about statistics used in this study, in the "Any other information on materials and methods incl.tables" section.

Results and discussion

Results of examinations

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.

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

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:

effects observed, non-treatment-related

Description (incidence and severity):

All findings occurred individually. They were considered to be incidental or spontaneous in origin and without any relation to treatment. The female animal, which was neither mated nor pregnant, as well as the male mating partner did not show relevant gross lesions.

Neuropathological findings:

not examined

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.

Effect levels

Key result

Dose descriptor:

NOAEL

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

Key result

Critical effects observed:

no

Any other information on results incl. tables

Stability analysis

The stability of the test substance in 0.5% 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 thatthe substancewas 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.