Fatty acids, tall-oil, reaction products with ethanolamine, ethoxylated

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:

Sep. 2012 - May 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

61791-19-3

Cas Number:

61791-19-3

IUPAC Name:

61791-19-3

Test material form:

other: liquid

Details on test material:

Please refer to section "Confidental details on test material"

Test animals

Species:

rat

Strain:

other: Crl:WI(Han)

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
- Age at study initiation: 31 ± 1 day(s) old
- Fasting period before study: no
- Housing: in polysulfonate cages (5 animals per sex per cage)
- Diet (ad libitum): Ground Kliba maintenance diet mouse/rat “GLP”, meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland
- Water (ad libitum): from water bottles
- Acclimation period: 11 days

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

IN-LIFE DATES:
From: 20.09.2012 To: 19.10.2012

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:

For each concentration, respective amount of test item was weighted into a beaker. Then, the respective volume of the carrier was added. Finally, each formulation was stirred with a magnetic stirrer until the test item was completely dissolved. All test item formulations were
prepared daily and discarded after the administration.


VEHICLE
- Justification for use and choice of vehicle: standard vehicle for studies of this type
- Amount of vehicle (if gavage): 4 mL/kg bw

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

The test item was administered daily for at least 4 weeks. Control animals received only the vehicle.

Frequency of treatment:

once daily

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 100, 300 or 1000 mg/kg bw/d
Basis:
actual ingested

No. of animals per sex per dose:

5 animals

Control animals:

yes, concurrent vehicle

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: A check for moribund and dead animals was made twice daily on working days and once daily on Saturday, Sunday and public holidays. If animals were in a moribund state, they were sacrificed and dissected. All rats were checked daily for any abnormal clinical signs prior administration, as well as, within 2 hours and 5 hours post administration.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: 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:

1. abnormal behavior during “handling”
2. fur
3. skin
4. posture
5. salivation
6. respiration
7. activity/arousal level
8. tremors
9. convulsions
10. abnormal movements
11. impairment of gait
12. lacrimation
13. palpebral closure
14. exophthalmos
15. feces (appearance/ consistency)
16. urine
17. pupil size

BODY WEIGHT: Yes
- Time schedule for examinations: 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 body weight on the respective day of weighing and body weight on day 0 was calculated as body weight change.

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

WATER CONSUMPTION: Yes
- Time schedule for examinations: Water consumption was monitored daily by visual inspection of water bottles for any abnormal changes in volume.


HAEMATOLOGY: Yes
- Time schedule for collection of blood: In the morning blood was taken from the retrobulbar venous plexus from fasted animals
- Anaesthetic used for blood collection: Yes , the animals were anaesthetized using isoflurane
- Animals fasted: Yes
- How many animals: 5 animals per test group and sex
- Parameters checked :

Leukocyte count (WBC)
Erythrocyte count (RBC)
Hemoglobin (HGB)
Hematocrit (HCT)
Mean corpuscular volume (MCV)
Mean corpuscular hemoglobin (MCH)
Mean corpuscular hemoglobin concentration (MCHC)
Platelet count (PLT)
Differential blood count
Reticulocytes
Prothrombin time (Hepato Quick’s test)



CLINICAL CHEMISTRY:
- Yes
- Time schedule for collection of blood: In the morning blood was taken from the retrobulbar venous plexus from fasted animals
- Anaesthetic used for blood collection: Yes , the animals were anaesthetized using isoflurane
- Animals fasted: Yes
- How many animals: 5 animals per test group and sex
- Parameters checked :

Alanine aminotransferase (ALT)
Aspartate aminotransferase (AST)
Alkaline phosphatase (ALP)
gamma-Glutamyltransferase (GGT)
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)
Magnesium (MG)
Bile acids (TBA)



URINALYSIS: Yes
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
- Parameters checked:

pH
Protein
Glucose
Ketones
Urobilinogen
Bilirubin
Blood
Specific gravity
Sediment
Color, turbidity
Volume



NEUROBEHAVIOURAL EXAMINATION:
- Yes

A functional observational battery (FOB) was performed in all animals per sex and group at the end of the administration period starting at about 10:00 h. At least one hour prior the start of examination, animals were transferred individually and in a randomized order to polycarbonate cages. 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. 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; any disturbing activities (touching the cage or rack, noise) were avoided during these examinations in order not to influence the behavior of the animals. Attention was paid to:

1. posture
2. tremor
3. convulsions
4. abnormal movements
5. impairment of gait
6. other findings

Open field observations:
The animals were transferred to a standard arena (50 × 50 cm with side height of 25 cm) and observed for at least 2 minutes. The following parameters were examined:

1. behavior when removed from cage
2. fur
3. skin
4. salivation
5. nose discharge
6. lacrimation
7. eyes/pupil size
8. posture
9. palpebral closure
10. respiration
11. tremors
12. convulsions
13. abnormal movements
14. impairment of gait
15. activity/arousal level
16. feces (number of fecal pellets/appearance/consistency) within two minutes
17. urine (appearance/quantity) within two minutes
18. number of rearing within two minutes

Sensorimotor tests/reflexes:
The animals were removed from the open field and subjected to following sensorimotor or reflex tests:

1. approach response
2. touch response
3. vision ("visual placing response")
4. pupillary reflex
5. pinna reflex
6. audition ("startle response")
7. coordination of movements ("righting response")
8. behavior during "handling"
9. vocalization
10. pain perception ("tail pinch")
11. grip strength of forelimbs
12. grip strength of hind limbs
13. landing foot-splay test
14. other findings


Measurement of Motor Activity
The motor activity assessment (MA) was carried out in all animals per sex and group at the end of the administration period. Motor activity was measured on the same day as FOB was performed. 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 for the time of measurement. Eighteen beams were allocated per cage. The numbers of beam interrupts were counted over 12 intervals of 5 minutes. The
sequence at which the animals were placed in the polycarbonate cages was selected at random. Motor activity measurements were carried out starting at 14:00 h. On account of the measuring variant "staggered", the starting time varied according to the time needed to place
the animals in the cages. For each animal, measurement started individually when the 1st beam was interrupted and was finished exactly 1 hour later. The animals did not receive any food or water during the measurements. After the transfer of the last animal the room of measurement was darkened.


OTHER:

Estrous cycle determination:

For estrous cycle determination, vaginal smears were prepared in the morning and evaluated according to the timetable for at least 3 weeks. The differentiation was conducted according to following stages:
Diestrous
Proestrous
Estrous
Metestrous


Sperm parameters:

Immediately after necropsy and organ weight determination right testis and cauda epididymis were taken from all male animals.
Sperm motility examinations were carried out in a randomized sequence.
Parameters:
Sperm motility
Sperm morphology
Sperm head count (cauda epididymis)
Sperm head count (testis)

Sacrifice and pathology:

GROSS PATHOLOGY:
- Yes
The animals were sacrificed by decapitation under isoflurane anesthesia. The exsanguinated animals were dissected and assessed by gross pathology.


Organ weights
The following weights were determined in all male and/or female 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

Organ/tissue fixation
The following organs or tissues were fixed in 4% 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

From each liver, one 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 male animals sacrificed at scheduled dates were fixed in modified Davidson’s solution, whereas the right testis and epididymis were used for sperm parameters.


HISTOPATHOLOGY:
- Yes
Fixation was followed by histotechnical processing (Hematoxylin and eosin (H&E) stain), examination by light microscopy and assessment of findings for following organs from all animals of the control and high dose groups

1. All gross lesions (all test groups, only animals affected)
2. Adrenal glands
3. Bone marrow (femur)
4. Brain
5. Cecum
6. Cervix
7. Coagulating glands
8. Colon
9. Duodenum
10. Epididymis, left
11. Eyes with optic nerve
12. Heart
13. Ileum
14. Jejunum
15. Kidneys
16. Liver (in addidtion: Paraplast embedding for all low dose and mid dose group animals)
17. Lungs
18. Lymph nodes (mesenteric and axillary lymph nodes)
19. Ovaries
20. Peyer’s patches
21. Pituitary gland
22. Prostate
23. Rectum
24. Sciatic nerve
25. Seminal vesicles
26. Skeletal muscle
27. Spinal cord (cervical, thoracic and lumbar cord)
28. Spleen
29. Sternum with marrow
30. Stomach (forestomach and glandular stomach)
31. Testis, left
32. Thymus
33. Thyroid glands
34. Trachea
35. Urinary bladder
36. Uterus
37. Vagina

Other examinations:

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
• 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

Whenever the histopathological evaluation of the immuno-relevant 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:

Statistics of clinical examinations:
- 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, foot-splay test, motor activity:
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

Statistics of clinical pathology:
Means, medians and standard deviations of each test group were calculated for several parameters.
In addition:
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 eachdose 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) with Bonferroni-Holm adjustment for the hypothesis of equal medians
Urinalysis parameters (except 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 thehypothesis of equal medians. Urine color and turbidity are not evaluated statistically.
Spermanalysis parameters: Pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) with Bonferroni-Holm adjustment

Results and discussion

Results of examinations

Details on results:

CLINICAL SIGNS AND MORTALITY

- No premature death or moribund state of animals occurred during the study.
- No adverse findings were observed.
Two of five male animals of the high dose group showed abnormal clinical signs.
Two male animals (nos. 19 and 20) of the high dose group showed respiration sounds on day 26 to day 28. Additionally, one of these males (no. 20) showed piloerection on day 26.
All observed signs occurred only for a short period at the end of administration period. The noted piloerection was most likely secondary to the observed respiration sounds, which in turn were regarded treatment-related but not adverse.
One of five female animals showed clinical signs in the low and mid dose group.
Furthermore, clinical signs were observed in two of five animals of the high dose group.
One female of the high dose group showed severe salivation on day 11. Furthermore, female animal no. 40 of the high dose group exhibited respiration sounds on day 27 to 28.
Sparse fur was noted in female animal no. 27 (frontal region) of the low dose group on day 27 to 29 and in female animal no. 32 (thoracal region). The latter finding was graduated slight (no. 27) and moderate (no. 32), respectively.
All observed signs occurred only for a short period at the end of administration or were a single event. Therefore, the noted salivation and respiration sounds were regarded as treatment-related but not adverse; the fur changes were seen as incidental and spontaneous
in nature.

BODY WEIGHT AND WEIGHT GAIN

- No compound- or dose-related effects on body weight and body weight change were observed.

FOOD CONSUMPTION

- No test substance-related findings were observed.


WATER CONSUMPTION

- No compound- or dose-related effects on water consumption were observed during daily visual inspection.


HAEMATOLOGY

No treatment-related changes among hematological parameters were observed.
At the end of the study, in males of test group 3 (1000 mg/kg bw/d) mean corpuscular volume (MCV) was lower whereas platelet counts were higher compared to controls. Both parameters were within historical control ranges (MCV 49.2-53.7 fL, platelet counts 741-1025
giga/L). Therefore, these alterations were regarded as incidental and not treatment-related.


CLINICAL CHEMISTRY

No treatment-related changes among clinical chemistry parameters were observed.
In males of test groups 2 and 3 (300 and 1000 mg/kg bw/d), urea levels were increased. However, means were within the historical control range (urea 4.69-7.67 mmol/L) In males of test group 2 (300 mg/kg bw/d) calcium levels were lower and in females of the same test group triglyceride levels were lower compared to controls. Both parameters were not dose-dependently changed.
Therefore, all mentioned parameter changes were regarded as incidental and not treatment-related.


URINALYSIS

No treatment-related changes among urinalysis parameters were observed.


NEUROBEHAVIOUR

Home Cage Observation:
No relevant and significant compound- or dose-related effects were observed.
Three males of the control, mid dose (test group 2) and high dose group (group 3), as well as, one male of low dose (test group 1) were sitting or lying. These findings were incidental and regarded spontaneous in nature.
Three female animals of control group and low dose group, as well as, two animals of test group 2 and test group 3 were sitting or lying. These findings were incidental and regarded to be spontaneous in nature.

Open Field Observation
No adverse effects were observed during the 2 minutes observation period.
One male animal of the high dose group showed a reduced exploration of the arena.
Furthermore respiratory sounds were observed in two animals of the high dose group. The latter observation was regarded treatment-related but not adverse. Four male animals of the mid dose and three males of the high dose group urinated during the observation period. The urine of these animals was without pathological findings. One female of the high dose group showed respiratory sounds during the observation period. This finding was regarded as treatment-related but not adverse. Two female animals of the high dose urinated during the observation period. The urine of these animals was without pathological findings.

Sensorimotor tests/ reflex tests
No relevant and significant compound- or dose-related effects were observed.
Male and female animals showed normal variations regarding the approach response.
The ratio of no reaction/approach was transient within and in between each test group.
These findings were incidental and regarded to be spontaneous in nature.

Measurement of Motor Activity (MA):

No relevant and significant compound- or dose-related effects were observed.
There were no significant deviations concerning overall motor activity (summation of all intervals) and individual intervals in male animals.
Female animals of the low dose and high dose group showed a significantly decreased motor activity at interval 11 versus control. However, since this finding was a single event in both test groups and no further significant changes were observed in the respective sum of all
intervals, these results were regarded as incidental and spontaneous in nature.

ORGAN WEIGHTS

Absolute organ weights:
When compared to the control group 0, all mean absolute weight parameters of treated animals showed no statistical significant changes.

Relative organ weights:
When compared to the control group 0 (set to 100%), the mean relative weights of the liver were significantly increased or decreased:
male animals (high dose group: 111 %), female animals (low dose group 91 %).
All other mean relative weight parameters did not show significant differences when compared to the control group 0.
The significant relative weight increase noted in the liver of males of test group 3 (1000 mg/kg bw/d) occurred without a clear histopathological correlate. Furthermore, the mean relative weight in this test group (2.888%) was within the range of the historical control weights (min. 2.505% - max. 3.223%) and thus this change was regarded as incidental. The significant decrease of the mean relative weight in the liver of females of test group 1 (100 mg/kg bw/d) occurred
without any dose-dependent relationship and was therefore considered to be not treatmentrelated.


GROSS PATHOLOGY

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


HISTOPATHOLOGY

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.


OTHER FINDINGS

Estrous cycle:
No relevant and significant compound- or dose-related effects were observed.
Female animals of all test groups showed normal biological variations in cycle length and number of cycles.

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.

Effect levels

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 treatment-related findings were observed in male and female rats orally dosed up to the highest dose of 1000 mg/kg bw/d.

Target system / organ toxicity

Critical effects observed:

not specified