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Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
09 July 2012 - 03 Jan 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Version / remarks:
03 Oct 2008
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
GLP compliance:
yes (incl. certificate)
Remarks:
22 Okt 2009
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
The rat is a frequently used laboratory animal, and there is comprehensive experience with this animal species.
Moreover, the rat has been proposed as a suitable animal species by the OECD and the EPA for this type of study.
Sex:
male/female
Details on test animals 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 daysENVIRONMENTAL CONDITIONS

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
Details on route of administration:
Substance was corn oil, administration volume: 4 mL/kg body weight
The oral route was selected since this was proven to be suitable for the detection of a toxicological hazard.
Vehicle:
corn oil
Details on oral exposure:
- PREPARATION OF DOSING SOLUTIONS:
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, 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.
- Rate of preparation of dosing (frequency): The test substance preparations were produced at least once a week and were stored at room temperature.

- VEHICLE
- Justification for use and choice of vehicle (if other than water): commonly used vehicle
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- Concentration control was verified in all concentrations at the beginning of the study using a capillary gas chromatograph with internal standard quantification
- Homogeneity of the test item was verified in the highest and lowest concentration.
Duration of treatment / exposure:
28 days
Frequency of treatment:
Daily
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Doses were selected at the request of the sponsor
Positive control:
None

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily (befor, 2h after and within 5h after dosing)
- Cage side observations: any abnormal clinically signs

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: prior to the administration period and thereafter at weekly intervals.
- Observations: 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.

BODY WEIGHT: Yes
- Time schedule for examinations: 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.

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

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At the end of the study (in the morning of day 29)
- Anaesthetic used for blood collection: Yes isoflurane
- Animals fasted: Yes
- Parameters checked: 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: Yes
- Time schedule for collection of blood: At the end of the study (in the morning of day 29)
- Animals fasted: Yes
- Parameters checked: 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: Yes
- Time schedule for collection of urine: day 25
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked: pH, Protein, Glucose, Ketones, Urobilinogen, Bilirubin, Blood, Specific gravity, Sediment, Color (turbidity), Volume.

NEUROBEHAVIOURAL EXAMINATION: Yes / No / Not specified
- Time schedule for examinations: days 26 and 27
- Dose groups that were examined: all
- Battery of functions tested: sensory activity, grip strength, motor activity

IMMUNOLOGY: Yes
- Time schedule for examinations: at necropsy
- How many animals: all
- Dose groups that were examined: all
- Parameters checked:
Thymus: Increased/decreased grade of cortico-medullar ratio (related only to area), Increase of starry sky cells, Changes of cellular density in the cortex, 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), Changes of the cellularity of interfollicular area.
Bone marrow: Changes of the cellularity, Changes of the myeloid/erythroid ratio

Sacrifice and pathology:
GROSS PATHOLOGY: Yes

The following weights were determined in all animals sacrificed on schedule:
1. Anesthetized animals
2. Adrenal glands
3. Brain
4. Epididymides
5. Heart
6. Kidneys
7. Liver
8. Ovaries
9. Prostate
10. Seminal vesicles with coagulating glands
11. Spleen
12. Testes
13. Thymus
14. Thyroid glands
15. Uterus with cervix

The following organs or tissues were fixed in 4% buffered formaldehyde solution or in
modified Davidson’s solution:
1. All gross lesions
2. Adrenal glands
3. Aorta
4. Bone marrow (femur)
5. Brain
6. Cecum
7. Cervix
8. Coagulating glands
9. Colon
10. Duodenum
11. Epididymis, left
12. Esophagus
13. Extraorbital lacrimal glands
14. Eyes with optic nerve (modified Davidson’s solution)
15. Femur with knee joint
16. Harderian glands
17. Heart
18. Ileum
19. Jejunum (with Peyer’s patches)
20. Kidneys
21. Larynx
22. Liver
23. Lungs
24. Lymph nodes (mesenteric and axillary lymph nodes)
25. Mammary gland (male and female)
26. Nose (nasal cavity)
27. Ovaries
28. Oviducts
29. Pancreas
30. Parathyroid glands
31. Pharynx
32. Pituitary gland
33. Prostate
34. Rectum
35. Salivary glands (mandibular and sublingual glands)
36. Sciatic nerve
37. Seminal vesicles
38. Skeletal muscle
39. Skin
40. Spinal cord (cervical, thoracic and lumbar cord)
41. Spleen
42. Sternum with marrow
43. Stomach (forestomach and glandular stomach)
44. Testis, left (modified Davidson’s solution)
45. Thymus
46. Thyroid glands
47. Trachea
48. Urinary bladder
49. Uterus
50. Vagina

HISTOPATHOLOGY: Yes (refer to the table in "any other information")
Other examinations:
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.

Sperm parameters: Sperm motility, Sperm morphology, Sperm head count (cauda epididymis), Sperm head count (testis).
Statistics:
Means and standard deviations of each test group were calculated for several parameters.

Furthermore the below tests were used where appropriate:
- DUNNETT's test (two-sided) for the hypothesis of equal means
- Non-parametric one-way analysis using KRUSKAL WALLIS test (two-sided)
- WILCOXON test (two-sided) for the equal medians

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
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) hematocrit 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 hematocrit 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):
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.
Immunological findings:
no effects observed
Description (incidence and severity):
No effects were observed that could be related to immunological toxicity.
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
(see any other information on results)
- 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. 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.
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:
no effects observed
Description (incidence and severity):
No effects were observed that could be related to neuropathological toxicity.
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):
- Estrous Cycle: No test substance-related effects on estrous cycle length and the number of cycles were obtained.
- Sperm parameters: 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%). However, there was neither a dose-dependent trend of both parameters nor statistical significant difference between the groups.

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects

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.

Applicant's summary and conclusion

Conclusions:
The administration of "Fatty acids, C16-C18 and C18-unsatd., ME esters, epoxidized" by gavage to male and female Wistar rats for 4 weeks did not cause test substance-related adverse signs of systemic toxicity. Therefore, under the conditions of the present study the no observed adverse effect level (NOAEL) was 1000 mg/kg bw/d in male and in female Wistar rats and does not need to be classified for STOT-RE in accordance with the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).
Executive summary:

 The repeated dose toxicity of “Fatty acids, C16-C18 and C18-unsatd., ME esters, epoxidized” was examined in a OECD TG 407 study under GLP conditions. The test substance was administered orally by gavage to groups of 5 male and 5 female Wistar rats at dose levels of 0 mg/kg bw/day, 100 mg/kg bw/day, 300 mg/kg bw/day and 1000 mg/kg bw/day over a period of 4 weeks.

Food consumption and body weight were determined weekly. The animals 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 examinations in an open field were conducted prior to the start of the administration period and weekly thereafter. Beside this, a functional observational battery (FOB) as well as measurement of motor activity (MA) were carried out at the end of the administration period. Biochemical and hematological examinations as well as urinalyses were performed at the end of the administration period. After the administration period all rats were sacrificed and assessed by gross pathology, followed by histopathological examinations.

Clinical examinations did not reveal treatment-related, adverse effects up to a concentration of 1000 mg/kg bw/day. In addition, no test substance-related effects on estrous cycle length and the number of cycles were obtained. Concerning clinical pathology no treatment-related, adverse effects were observed up to a dose of the test substance of 1000 mg/kg bw/day. Furthermore, no test substance-related effects on sperm parameters were obtained. Regarding pathology, no treatment-related organ weight changes, gross lesions or histopathological findings were observed in the treated male and female animals including the reproductive organs. Based on these results, the administration of "Fatty acids, C16-C18 and C18-unsatd., ME esters, epoxidized" the no observed adverse effect level (NOAEL) was set at 1000 mg/kg bw/day in male and in female Wistar rats and the substance does not need to be classified for STOT-RE in accordance with the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).


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