Fatty acids, C16-18 and C18-unsatd., Me esters, epoxidized

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

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:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

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:

methyl 8-(3-octyloxiran-2-yl)octanoate; methyl 8-[3-({3-[(3-ethyloxiran-2-yl)methyl]oxiran-2-yl}methyl)oxiran-2-yl]octanoate; methyl 8-{3-[(3-pentyloxiran-2-yl)methyl]oxiran-2-yl}octanoate

EC Number:

605-143-8

Cas Number:

158318-67-3

Molecular formula:

Unspecified (UVCB)

IUPAC Name:

methyl 8-(3-octyloxiran-2-yl)octanoate; methyl 8-[3-({3-[(3-ethyloxiran-2-yl)methyl]oxiran-2-yl}methyl)oxiran-2-yl]octanoate; methyl 8-{3-[(3-pentyloxiran-2-yl)methyl]oxiran-2-yl}octanoate

Test material form:

liquid

Specific details on test material used for the study:

- Test-substance No.: 12/0029-1
- Lot/batch No.: CE80580015
- Homogeneity: Given (visually)
- Expiry date: 27 Feb 2013
- Physical state/appearance: liquid/colorless, clear
- Storage conditions: ambient (room temperature)
- 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.
- Name of test material (as cited in study report): Sovermol 1055

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 GmbH, Sulzfeld, Germany
- Age at study initiation: 42 ± 1 days
- Weight at study initiation: Male: about 154g, females: about 129g
- Housing: Five animals per cage in polysulfonate cages supplied by TECNIPLAST, Hohenpeißenberg, Germany (floor area about 2065 cm2). Dust-free wooden bedding was used. Wooden gnawing blocks (Typ NGM E-022) supplied by Abedd® Lab. and Vet. Service GmbH, Vienna, Austria, were added for environmental enrichment.
- Diet: Ground Kliba maintenance diet mouse/rat “GLP”, meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland, ad libitum
- Water: Ad libitum
- Acclimation period: 9 days

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

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

The test item was applied as a suspension. To prepare this suspension, 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 released with a magnetic stirrer. During administration of the test substance, preparations were kept homogeneous by stirring with a magnetic stirrer. The test substance preparations were produced at least once a week and were stored at room temperature.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- The stability of the test item in corn oil at room temperature for a period of 7 days was demonstrated before the start of the study.
- Homogeneity of the test item was verified in the highest and lowest concentration. Considering the low relative standard deviation in the homogeneity analysis, it was concluded that the test item was distributed homogeneously in corn oil.
- Concentration control was verified in all concentrations at the beginning of the study. The mean values of the test substance in corn oil were always found to be in the range between 90-110% of the nominal concentrations. These results demonstrated the correctness of the concentrations the test substance in corn oil.

Duration of treatment / exposure:

28 days

Frequency of treatment:

once daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Examinations

Observations and examinations performed and frequency:

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
All rats were checked daily for any abnormal clinically signs before the administration as well as within 2 hours and within 5 hours after the administration.

DETAILED CLINICAL OBSERVATIONS
Detailed clinical observations (DCO) were performed in all animals prior to the administration period and thereafter at weekly intervals. The findings were ranked according to the degree of severity, if applicable. The animals were transferred to a standard arena (50 × 37.5 cm with sides of 25 cm high). The following parameters were examined: abnormal behavior when handled, fur, skin, posture, salivation, respiration, activity/arousal level, tremors, convulsions, abnormal movements, impairment of gait, lacrimation, palpebral closure, exophthalmus, feces (appearance/consistency), urine, pupil size.

FOOD CONSUMPTION
Food consumption was determined weekly over a period of 1 day and calculated as mean food consumption in grams per animal and day.

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

BODY WEIGHT
Body weight was determined before the start of the administration period in order to randomize the animals. During the administration period the body weight was determined on day 0 (start of the administration period) and thereafter at weekly intervals. The difference between the body weight on the respective day of weighing and the body weight on day 0 was calculated as body weight change.

FUNCTIONAL OBSERVATIONAL BATTERY
A functional observational battery (FOB) was performed in all animals at the end of the administration period starting at about 10:00 h. At least one hour before the start of the FOB the rats were transferred to single-animal polycarbonate cages. Drinking water was provided ad libitum, but no food was offered during the measurements. The FOB started with passive observations without disturbing the rats, followed by removal from the home cage, open field observations in a standard arena and sensory motor 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 rats 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 rats. Attention was paid to: Posture, Tremors, Convulsions, Abnormal movements, Gait abnormalities. Open field observations: The rats were transferred to a standard arena (50 × 50 cm with sides of 25 cm height) and observed for at least 2 minutes. The following parameters were examined: Behavior on removal from the cage, Fur, Skin, Salivation, Nasal discharge, Lacrimation, Eyes/ pupil size, Posture, Palpebral closure, Respiration, Tremors, Convulsions, Abnormal movements/ stereotypes, Gait abnormalities, Activity/ arousal level, Feces excreted within 2 minutes (number/ appearance/ consistency), Urine excreted within 2 minutes (amount/ color), Rearing within 2 minutes. Sensory motor tests/ reflexes: The rats were then removed from the open field and subjected to following sensory motor or reflex tests: Reaction to an object being moved towards the face (Approach response), Touch sensitivity (Touch response), Vision (Visual placing response), Pupillary reflex, Pinna reflex, Audition (Auditory startle response), Coordination of movements (Righting response), Behavior during handling, Vocalization, Pain perception (Tail pinch), Grip strength of forelimbs, Grip strength of hindlimbs, Landing foot-splay test, Other findings.

MOTOR ACTIVITY ASSESSMENT
Motor activity (MA) was also measured from 14:00 h onwards on the same day as the FOB was performed. The examinations were performed using the TSE Labmaster System supplied by TSE Systems GmbH, Bad Homburg, Germany. For this purpose, the rats 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 number of beam interrupts was counted over 12 intervals for 5 minutes per interval. The sequence in which the rats 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 rats during these measurements and the measurement room was darkened after the transfer of the last rat.

ESTROUS CYCLE DETERMINATION
Vaginal smears for cycle determination were be prepared in the morning and evaluated according to the timetable for at least 3 weeks. The differentiation was conducted according to following stages: 1. Diestrous: leukocytes, few nucleated epithelial cells, 2 Proestrous: single leukocytes, many nucleated and few horny epithelial cells, 3 Estrous only horny epithelial cells, 4 Metestrous leukocytes, some horny epithelial cells and some nucleated epithelial cells.

CLINICAL PATHOLOGY
In the morning blood was taken from the retrobulbar venous plexus from fasted animals. The animals were anaesthetized using isoflurane (Isoba®, Essex GmbH, Munich, Germany). The blood sampling procedure and subsequent analysis of blood and serum samples were carried out in a randomized sequence. For urinalysis the individual animals were transferred to metabolism cages (withdrawal of food and water) and urine was collected overnight. Urine samples were evaluated in a randomized sequence The assays of blood and serum parameters were performed under internal laboratory quality control conditions with reference controls to assure reliable test results.

HAEMATOLOGY
The following parameters were determined in blood with EDTA-K3 as anticoagulant using a particle counter (Advia 120 model; Bayer, Fernwald, Germany): Leukocyte count, Erythrocyte count, Hemoglobin, Hematocrit, Mean corpuscular volume, Mean corpuscular hemoglobin, Mean corpuscular hemoglobin concentration, Platelet count, Differential blood count, Reticulocytes. Furthermore, blood smears were prepared and stained according to WRIGHT without being evaluated, because of non-ambiguous results of the differential blood cell counts measured by the automated instrument. (reference: Hematology: Principles and Procedures, 6th Edition, Brown AB, Lea & Febiger, Philadelphia, 1993, page 101). Clotting tests (Prothrombin time) were carried out using a ball coagulometer (AMAX destiny plus model; Trinity biotech, Lemgo, Germany).

CLINICAL CHEMISTRY
An automatic analyzer (Hitachi 917; Roche, Mannheim, Germany) was used to examine the clinicochemical parameters: Alanine aminotransferase, Aspartate aminotransferase, Alkaline phosphatase, γ-Glutamyltransferase, Sodium, Potassium, Chloride, Inorganic phosphate, Calcium, Urea, Creatinine, Glucose, Total bilirubin, Total protein, Albumin, Globulins, Triglycerides, Cholesterol, Bile acids.

URINALYSIS
The dry chemical reactions on test strips (Combur-10-test M, Roche, Mannheim, Germany) used to determine urine constituents semiquantitatively were evaluated with a reflection photometer (Miditron M; Roche, Mannheim, Germany). Parameters: pH, Protein, Glucose, Ketones, Urobilinogen, Bilirubin, Blood, Specific gravity, Sediment, Color (turbidity), Volume.

SPERM PARAMETERS
Sperm motility, Sperm morphology, Sperm head count (cauda epididymis), Sperm head count (testis).

Sacrifice and pathology:

NECROPSY
The animals were sacrificed by decapitation under isoflurane anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology.

ORGAN WEIGHTS
The following weights were determined in all animals sacrificed on schedule: Anesthetized animals, Adrenal glands, Brain, Epididymides, Heart, Kidneys, Liver, Ovaries, Prostate, Seminal vesicles with coagulating glands, Spleen, Testes, Thymus, Thyroid glands, Uterus with cervix.

ORGAN/TISSUE FIXATION
The following organs or tissues were fixed in 4% buffered formaldehyde solution or in modified Davidson’s solution: All gross lesions, Adrenal glands, Aorta, Bone marrow (femur), Brain, Cecum, Cervix, Coagulating glands, Colon, Duodenum, Epididymis (left), Esophagus, Extraorbital lacrimal glands, Eyes with optic nerve (modified Davidson’s solution), Femur with knee joint, Harderian glands, Heart, Ileum, Jejunum (with Peyer’s patches), Kidneys, Larynx, Liver, Lungs, Lymph nodes (mesenteric and axillary lymph nodes), Mammary gland (male and female), Nose (nasal cavity), Ovaries, Oviducts, Pancreas, Parathyroid glands, Pharynx, Pituitary gland, Prostate, Rectum, Salivary glands (mandibular and sublingual glands), Sciatic nerve, Seminal vesicles, Skeletal muscle, Skin, Spinal cord (cervical, thoracic and lumbar cord), Spleen, Sternum with marrow, Stomach (forestomach and glandular stomach), Testis, left (modified Davidson’s solution), Thymus, Thyroid glands, Trachea, Urinary bladder, Uterus, Vagina. From the livers, one additional slice of the Lobus dexter medialis and the Lobus sinister lateralis was fixed in Carnoy’s solution and embedded in paraplast. The left testis and left epididymis of all animals sacrificed at scheduled dates were fixed in modified Davidson’s solution, whereas the right testis and epididymis were used for sperm parameters. Fixation was followed by histotechnical processing, examination by light microscopy and assessment of findings. All the above mentioned organs/tissues of the control and high dose group were stained with hematoxylin and eosin (H&E) stain, except for the gross lesions (lesions in the low and mid dose groups were also stained with H&E stain) and the liver (livers of the low and mid dose groups were paraplast embed). The immunorelevant organs and tissues were evaluated according to the following parameters: Thymus: Increased/decreased grade of cortico-medullar ratio (related only to area), increase of starry sky cells, changes of cellular density in the cortex, and changes of cellular density in the medulla. Spleen: Changes of the cellularity of PALS, lymphoid follicles, marginal zone, red pulp, altered cellular composition of follicles, altered number of germinal centers. Lymph nodes (mesenteric and axillary lymph nodes): Changes in the cellularity of follicles, interfollicular area, paracortical area, medulla, altered cellular composition of paracortex, altered number of germinal centers, hyperplasia of high endothelial venules. Peyer's patches (of the jejunum): Changes of the cellularity of follicles (including mantle zone and germinal centers) and changes of the cellularity of interfollicular area. Bone marrow: Changes of the cellularity and changes of the myeloid/erythroid ratio. Whenever the histopathologic evaluation of the immunorelevant organs and tissues did not reveal a morphologic alteration of these items and/or whenever no other pathologic finding was noted, these organs were diagnosed as "no abnormalities detected”. Special attention was given for the synchrony of the morphology of the estrous cycle in ovaries, uterus, cervix, and vagina. A correlation between gross lesions and histopathological findings was attempted.

Statistics:

- Body weight, body weight change: A comparison of each group with the control group was performed using DUNNETT's test (two-sided) for the hypothesis of equal means
- Feces, rearing, grip strength forelimbs, grip strength hindlimbs, footsplay test, motor activity and weight parameters: Non-parametric one-way analysis using KRUSKAL-WALLIS test (two-sided). If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON test (two-sided) for the equal medians.
- Blood parameters: For parameters with bidirectional changes: Non-parametric one-way analysis using KRUSKAL-WALLIS test. If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON-test (two-sided) for the hypothesis of equal medians For parameters with unidirectional changes: Pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) for the hypothesis of equal medians.
- Urinalysis parameters (apart from pH, urine volume, specific gravity, color and turbidity): Pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) for the hypothesis of equal medians.
- Urine pH, volume, specific gravity, color and turbidity: Non-parametric one-way analysis using KRUSKAL-WALLIS test. If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON-test (two-sided) for the hypothesis of equal
medians. Urine color and turbidity are not evaluated statistically.
- Sperm analysis parameters: Pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) with Bonferroni-Holm adjustment for the hypothesis of equal medians For the percentage of abnormal sperms (ABNORMAL6_C) values <6% were set to 6% (cut off 6 %).

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

No test substance-related, adverse findings were observed in male and female animals of test groups 1-3 (100, 300 and 1000 mg/kg bw/d).

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 significant differences with regard to the mean body weights and mean body weight change values were noted for male and female animals of test groups 1-3 (100, 300 and 1000 mg/kg bw/d).

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No test substance-related, adverse findings were observed in male and female animals of test groups 1-3 (100, 300 and 1000 mg/kg bw/d).

Food efficiency:

not examined

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

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related changes among hematological parameters were observed. In females of test group 1 (100 mg/kg bw/d) hematokrit values were higher compared to controls and in females of test group 3 (1000 mg/kg bw/d) red blood cell (RBC) counts were lower compared to controls. The hematokrit was not changed dose-dependently. The RBC mean was marginally decreased (6% lower compared to controls), within the historical control range (RBC 6.71-8.41 Tera/L), and no other red blood cell parameter in these rats was altered. Therefore, both red blood cell alterations were 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:

no effects observed

Description (incidence and severity):

No treatment-related changes among urinalysis parameters were observed.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

- Functional observational battery: Deviations from "zero values" were obtained in several rats. However, as most findings were equally distributed between test-substance treated groups and controls, were without a dose-response relationship or occurred in single rats only, these observations were considered to have been incidental. No test substance-related effects were observed for home cage observations, open field observations and sensorimotor tests/reflexes. Feces, rearing, grip strength of fore- and hindlimbs as well as foot splay test did not reveal significant changes in test groups 1-3 (100, 300 and 1000 mg/kg bw/d) when compared to the control animals.
- Motor activity measurement: There were no significant deviations concerning the overall motor activity (summation of all intervals) in male and female animals of all test groups in comparison to the concurrent control group. The single intervals Nos. 4 and 9 were significantly decreased in male animals of test group 3 (1000 mg/kg bw/d). The single interval No. 6 was significantly increased in male animals of test group 1 (100 mg/kg bw/d). The single interval No. 8 was significantly decreased in female animals of test groups 1 and 2 (100, 300 mg/kg bw/d). However, as there were no significant deviations with regard to the overall motor activity (summation of all intervals) in male animals of test groups 1 and 3 (100 and 1000 mg/kg bw/d) as well as in female animals of test groups 1 and 2 (100 and 300 mg/kg bw/d) in comparison to the concurrent control group these findings were assessed as being incidental and not related to treatment.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

The significant absolute and/or relative weight changes noted in the brain of males and in the uterus and liver of females were not considered to be treatment-related since they occurred without any dose-dependent relationship. The significant decrease of the absolute and relative mean weight of the thyroid gland in females of test group 3 (1000 mg/kg bw/d) was not consistent with any histopathological finding. Moreover, the absolute (13.0 mg) and relative (0.007%) mean weight of the thyroid gland was within the historical control range (absolute weight range: 11.8-21.2 mg; relative weight range: 0.007-0.012%). Therefore, the thyroid gland weight decrease was regarded as incidental and not related to treatment. The significant differences in absolute and relative uterus weights of female animals in test groups 1 and 2 (100 and 300 mg/kg bw/d) compared to the control animals were most likely related to the estrous cycle status. In addition, the mean uterus weight of the control group was influenced by the very high value animal No. 23. Thus, the changes were regarded to be incidental and not related to treatment (see 'any other information on results' for details).

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

All findings occurred individually. They were considered to be incidental in nature and not related to treatment.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

All histopathological findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental in nature and not related to treatment.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

- No test substance-related effects on estrous cycle length and the number of cycles were obtained.
- Sperm parameters: Concerning the motility of the sperms and the incidence of abnormal sperms in the cauda epididymidis as well as the sperm head counts in the testis and in the cauda epididymidis no treatment-related effects were observed. Male animal No. 5 of the control group had a low motility (21%) and a high percentage of abnormal sperms in the cauda epididymidis (70%). But even without the values of this rat there was no dose-dependent trend of both parameters when regarding the medians as well as no statistical significant difference between the groups.

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: No toxic effects observed at the highest dose tested.

Target system / organ toxicity

Key result

Critical effects observed:

no

Any other information on results incl. tables

Absolute organ weights

When compared to the control group 0 (set to 100%), the following mean absolute weights were significantly increased or decreased (statistically significant changes printed in bold):

	Male animals			Female animals
Test group (mg/kg bw/d)	1 (100)	2 (300)	3 (1000)	1 (100)	2 (300)	3 (1000)
Brain	106%*	99%	101%
Thyroid gland				117%	103%	74%*
Uterus				55%*	53%*	88%

*p ≤ 0.05

All other mean absolute weight parameters did not show significant differences when compared to the control group 0.

 

Relative organ weights

When compared to the control group 0 (set to 100%), the mean relative weights of the following organs were significantly decreased (statistically significant changes printed in bold):

	Male animals			Female animals
Test group (mg/kg bw/d)	1 (100)	2 (300)	3 (1000)	1 (100)	2 (300)	3 (1000)
Liver				90%	96%	103%
Thyroid gland				112%	104%	73%**
Uterus				53%*	53%**	87%

*p ≤ 0.05; **p ≤ 0.01

All other mean relative weight parameters did not show significant differences when compared to the control group 0.