Fatty acids, tall-oil, reaction products with pentaethylenehexamine

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:

November 2012 - June 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 guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

22nd March 1996

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Fatty acids, tall-oil, reaction products with pentaethylenehexamine

EC Number:

288-927-9

EC Name:

Fatty acids, tall-oil, reaction products with pentaethylenehexamine

Cas Number:

85940-40-5

Molecular formula:

Not applicable (UVCB substance)

IUPAC Name:

Fatty acids, tall-oil, reaction products with pentaethylenehexamine

Test material form:

liquid: viscous

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories, B.V. Kreuzelweg 53 5961 NM Horst / Netherlands
- Age at study initiation: 11 weeks
- Weight at study initiation: Males: 260 to 390 g, Females: 175 to 240 g
- Fasting period before study: no
- Housing: individually, following exceptions: During overnight matings, male and female mating partners were housed together. Pregnant animals and their litters were housed together until PND 4 (end of lactation).
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 7d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: August 9th, 2011 To: October 6th, 2011

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Remarks:

suspension

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Test substance concentrations in the dose formulations ranged from 81.0% to 105.2% with reference to the nominal and were within the accepted range of ±20%.

Duration of treatment / exposure:

The duration of treatment covered a 2-week premating and a mating period in both sexes, approximately 1 week post-mating in males,
and the entire gestation period as well as 4 days of lactation and 2 weeks thereafter in females.

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: range finder test 100, 300 and 1000 mg/kg bw

Positive control:

no

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: 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).

FOOD CONSUMPTION
- weekly
- Food consumption was not determined during the mating period (male and female F0 animals).
- Food consumption of the F0 females with evidence of sperm was determined on GD 0, 7, 14 and 20.
- Food consumption of F0 females, which gave birth to a litter, was determined for PND 4

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: in the course of FOB
- Dose groups that were examined: all

HAEMATOLOGY: Yes
- Time schedule for collection of blood: end of administration period
- Anaesthetic used for blood collection: Yes, anaesthetized using isoflurane (Isoba®, Essex GmbH, Munich, Germany)
- Animals fasted: Yes
- How many animals: 5/sex/dose

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: end of administration period
- Animals fasted: Yes
- How many animals: 5/sex/dose

URINALYSIS: Yes
- Time schedule for collection of urine: males: after mating, females: 1 day before end of administration period
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: male: postnatal day 0, female: 10d after gestation
- Dose groups that were examined: all
- Battery of functions tested: sensory activity / grip strength / motor activity

Sacrifice and pathology:

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

HISTOPATHOLOGY: Yes, control and high dose group, gross lesions in all animals
1. All gross lesions
2. Adrenal glands
3. Bone marrow (femur)
4. Brain
5. Cecum
6. Cervix
7. Coagulating glands
8. Colon
9. Duodenum
10. Epididymides
11. Heart
12. Ileum
13. Jejunum
14. Kidneys
15. Liver
16. Lung
17. Lymph nodes (mesenteric and axillary lymph nodes)
18. Ovaries
19. Oviducts
20. Peyer’s patches
21. Prostate
22. Rectum
23. Sciatic nerve
24. Seminal vesicles
25. Spinal cord (cervical, thoracic and lumbar cords)
26. Spleen
27. Stomach (forestomach and glandular stomach)
28. Testes
29. Thymus
30. Thyroid glands
31. Trachea
32. Urinary bladder
33. Uterus
34. Vagina

Other examinations:

Bile acids
Furthermore, an additional blood sample (0.5 mL) was be collected into serum tubes for possible future measurement of thyroid releasing hormone (TSH), and the thyroid hormones triiodothyronine (T3) and thyroxine (T4) (added by amendment if applicable). Serum samples are stored at -80 ± 10°C. Any samples remaining at finalization of the study report, were discarded.

Statistics:

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: WILCOXON-test (one-sided)

Food consumption: DUNNETT-test (two-sided)

fertility indices: FISHER'S EXACT test

Proportions of affected pups per litter with necropsy observations: WILCOXON-test

Weight parameters: KRUSKAL-WALLIS test

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

300 mg/kg bw/d:
- several clinical signs were noted over the whole treatment with earliest onset on the fourth day of treatment: bedding in mouth, breathing noises and labored breathing, ruffled fur, visible weight loss and generally weakened condition

100 mg/kg bw/d:
- isolated breathing noises recorded in two males and one female. Based on the low incidence and transient occurrence, these findings were not considered to be toxicologically relevant.

30 mg/kg bw/d:
- ruffled fur was intermittently noted in one male and in one female. Based on the low incidence and transient occurrence, these findings were not considered to be toxicologically relevant.

Mortality:

mortality observed, treatment-related

Description (incidence):

Three males and one female treated at 300 mg/kg bw/day did not survive until the scheduled necropsy.
- One male and one female were killed for humane reasons after 28 days of treatment. Marked body weight loss, ruffled fur, labored breathing and breathing noises along with a generally weakened condition were observed.
- Two males died spontaneously on day 25 of the pairing period and day 1 of the after pairing period, respectively. Body weight loss, breathing
noises with increasing severity towards the end of pre-pairing and throughout pairing until death were oberved.
- For all decedent animals dark red lungs and/or incompletely collapsed lungs were noted during necropsy.
Although the distribution of the deaths suggested a relationship to treatment the presence of bronchopneumonia, bronchiolitis/peribronchiolitis and/or fluid in the lungs was considered to provide strong evidence that death or moribund condition had, in all of the decedents, resulted from gavage error.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 300 mg/kg bw/day, a slight treatment-related reduction in body weights and body weight gain were noted in males and females.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

At 300 mg/kg bw/day, a slight treatment-related reduction in food consumption was noted in males and females.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

At 300 mg/kg bw/day, 3-fold higher alanine aminotransferase and slightly higher aspartate aminotransferase levels in males and approximately 2-fold higher alanine aminotransferase levels in females were recorded (not reaching statistical significance and still within the range of historical control data). These higher values deemed related to treatment, also in view of the changes in liver weights noted in males.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

In males at 300 mg/kg bw/day, statistically significantly lower absolute liver weights and their ratios to brain were recorded when compared with controls.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no macroscopical findings that were deemed related to treatment with the test item.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Histopathological changes considered to be associated with the administration of the test item were present in the mesenteric lymph nodes of females given 30 mg/kg bw/day and both sexes given 100 or 300 mg/kg bw/day.

Histopathological findings: neoplastic:

no effects observed

Effect levels

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

Target system / organ toxicity

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

Based on these results, the NOAEL (No Observed Adverse Effect Level) for general toxicity was considered to be 100 mg/kg body weight/day based on clinical signs, reduced body weights and
food consumption, and increased serum ALAT- and ASAT activities at 300 mg/kg bw/day.