Fatty acids, C16-18 and C18-unsatd., compds. with triethanolamine

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted according to internationally accepted test guidelines and is considered relevant, adequate and reliable.

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

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Fatty acids, C16-18 and C18-unsatd., compds. with triethanolamine

EC Number:

270-279-3

EC Name:

Fatty acids, C16-18 and C18-unsatd., compds. with triethanolamine

Cas Number:

68424-19-1

Molecular formula:

Not applicable as the substance is a UVCB

IUPAC Name:

Fatty acids, C16-18 (even numbered) and C18-unsatd., compds. with triethanolamine.

Test material form:

semi-solid (amorphous): gel

Remarks:

paste

Details on test material:

- Name of test material (as cited in study report): Fatty acids, C16-18 and C18-unsatd., compds. with triethanolamine.
- Physical state: Viscous, translucent yellowish waxy paste
- Analytical purity: >95%
- Impurities (identity and concentrations):See confidential details
- Composition of test material, percentage of components: See confidential details
- Purity test date: 01/2013
- Lot/batch No.: DDR02/91
- Expiration date of the lot/batch: October 2014
- Storage condition of test material: At +10° to +25°C
- Other: Manufacturer/Supplier: OLEON NV, Assenedestraat 2, 9940 Ertvelde, Belgium

Test animals

Species:

rat

Strain:

other: CD/Crl:CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories Germany GmbH, Sandhofer Weg 7, 97633 Sulzfeld, Germany
- Age at first administration: 63 days
- Weight at first administration: Males: 319.6 – 369.4 g; Females: 212.7 – 246.3 g
- Fasting period before study: Not provided
- Housing: With exception of the mating period, the animals were kept singly in MAKROLON cages (type III plus)with a basal surface of approximately 39 cm x 23 cm and a height of approximately 18 cm. Granulated textured wood (Granulat A2, J. Brandenburg, 49424 Goldenstedt, Germany) was used as bedding material for the cages. The cages were changed and cleaned once a week.
- Diet (e.g. ad libitum): ssniff® R-Z V1324, (ssniff Spezialdiäten GmbH, 59494 Soest, Germany) served as food. This food was offered ad libitum, with exception of the night before the day of blood withdrawal for laboratory examination. Food residue was removed and weighed.
- Water (e.g. ad libitum): Tap water was offered daily ad libitum.
- Acclimation period: 5 adaptation days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C ± 3°C (maximum range)
- Humidity (%): 55% ± 15% (maximum range)
- Air changes (per hr): Not provided
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: February 27, 2013 To: Males: April 4, 2013; Females: April 26, 2013

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was dissolved in the vehicle propylene glycol to concentrations of 50, 150 and 500 mg test item/mL. The test item formulations were freshly prepared and adjusted to the animal's current body weight on each administration day.
The test item was administered orally at a constant dose volume of 2 mL/kg bw/day per animal. The animals of the control group received the vehicle (propylene glycol) at a constant volume of 2 mL/kg bw in the same way.
In addition, the stability, homogeneity and concentration of the test item mixture was monitored


VEHICLE
- Justification for use and choice of vehicle (if other than water): propylene glycol (best solvent for test item)
- Concentration in vehicle: 50 - 150 - 500 mg/mL
- Amount of vehicle (if gavage): 2 mL/kg bw/day (administration volume)
- Lot/batch no. (if required): Batch no.: MKBK1806V, Sigma-Aldrich, USA, storage at room temperature.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

For the analysis of the test item-vehicle mixtures, samples of approx. 5 mL were taken at the following times and stored at -20°C or colder until analysis at LPT.

At study initiation:
Analysis of stability and concentration
Immediately after preparation of the solution as well as after 8 and 24 hours storage of the test item preparations at room temperature (3 samples/dose level group).
Number of samples: 3 x 3 = 9
Homogeneity
At the start of administration, during (middle) administration and before administration to the last animal of the dose level group (3 samples/dose level group).
Number of samples:3 x 3 = 9
At termination of the administration period at a time point when the majority of animals is dosed:
Analysis of concentration
During treatment with the test item always before administration to the last animal of the dose level group (1 samples/dose level group).
Number of samples: 1 x 3 = 3
Sum of all samples: 21

The samples were labelled with the study number, test item, test species, type of sample, aliquot number, concentration, test day, sampling time and date.
The validation of the analytical method is described in LPT Study 29660.

Concentration and stability:
The measured concentrations of the test item in the test item vehicle mixtures were between 100.3% and 104.7% of the nominal concentration. These values indicated correctly prepared test item vehicle mixtures, which were stable at room temperature for at least 24 hours.
Further samples to control the concentration of the test item in the test item vehicle mixtures were taken on test day 30. The concentrations of the test item were between 101.9% and 105.8% of the nominal concentration, indicating correctly prepared test item vehicle mixtures.

Homogeneity
The measured concentrations of the test item in the test item vehicle mixtures were between 100.4% and 104.9%. These values indicated that there was no phase separation between the test item and the vehicle during the procedure of administration of the test item vehicle mixtures to the animals.

Duration of treatment / exposure:

Males: beginning 2 weeks prior to mating lasting up to the day before sacrifice until a minimum dosing period of 28 days was completed.
Females: beginning 2 weeks prior to mating and continuing up to, and including, day 3 post-partum or the day before sacrifice.

Frequency of treatment:

Once daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 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)

Doses / concentrations 4

Dose / conc.:

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

No. of animals per sex per dose:

12

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The dose levels have been selected in agreement with the Sponsor based on the results of a 14-day dose-range-finding study in rats dosed at 100, 300 and 1000 mg (active ingredient)/kg bw by oral gavage (LPT Study No. 29626).
- Rationale for animal assignment (if not random):
The animals were randomly allocated to the test groups.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Throughout the test period, each animal was observed for clinical signs at least once daily. Mortality was recorded twice daily. In addition, animals were checked regularly throughout the working day from 7:00 a.m. to 3:45 p.m. On Saturdays and Sundays animals were checked regularly from 7:00 a.m. to 11 :00 a.m. with a final check performed at approximately 3:30 p.m.
- Individual animals were observed before and after dosing at each time of dosing for any signs of behavioural changes, reaction to treatment or illness. Cageside observations included skin/fur, eyes, mucous membranes, respiratory and circulatory systems, somatomotor activity and behaviour patterns. The onset, intensity and duration of any signs observed were recorded.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Additionally, once before the first exposure (to allow for within-subject comparisons) and once a week thereafter, detailed clinical observations were made in all animals; in test week 4 these observations were performed prior to any laboratory investigations. These observations were made outside the home cage in a standard arena and at the same time, each time. Signs observed included changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g. lacrimation, pilo-erection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g. excessive grooming, repetitive circling) or bizarre behaviour (e.g. self-mutilation, walking backwards) were also recorded.

BODY WEIGHT: Yes. Body weights were recorded individually for each adult animal.
- Time schedule for examinations:
Males and females were weighed on the first day of dosing, weekly thereafter and at termination. During gestation, females were weighed on days 0, 7, 14 and 20 and within 24 hours of parturition (day 0 or 1 post-partum) and day 4 post-partum.

FOOD CONSUMPTION: Yes
Food consumption of each animal was recorded weekly during the pre-mating period, daily during gestation and on day 4 post-partum.
Food intake per rat (g/rat/week) was calculated using the total amount of food given to and left by each rat in each group upon completion of a treatment week (pre-mating and gestation) or treatment period (lactation). From these data the relative food consumption (in g/kg b.w./day) was determined using the following formula:
Relative food consumption (g/kg bw/day) = Total food given (g) - Total food left (g)/Number of animal days# x Body weight (kg)

# The term 'animal days' counts one animal day for each animal alive for a whole
day; it is assumed that on the day of death an animal does not eat.

WATER CONSUMPTION:
- Time schedule for examinations: Water consumption was monitored daily by visual appraisal throughout the study.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At the end of the pre-mating period
- Anaesthetic used for blood collection: Yes, ether anaesthesia
- Animals fasted: Yes , fasted overnight
- How many animals: 5 male and 5 female animals randomly selected from each group.
- Parameters examined:
Differential blood count(relative)
Differential blood count(absolute)
Erythrocytes
Leucocytes
Haematocrit value
Haemoglobin content
Platelets
Reticulocytes
Mean corpuscular volume (MCV)
Mean corpuscular haemoglobin (MCH)
Mean corpuscular haemoglobin concentration (MCHC)


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At the end of the pre-mating period
- Animals fasted: Yes , fasted overnight
- How many animals: 5 male and 5 female animals randomly selected from each group.
- Parameters examined:
Sodium
Potassium
Calcium
Chloride
Albumin
Globulin
Albumin/globulin ratio
Total bilirubin
Total cholesterol
Creatinine
Glucose
Total protein
Blood urea
Alanine amino-transferase (ALAT)
Alkaline phosphatase (aP)
Aspartate aminotransferase (ASAT)
Bile acids

NEUROLOGICAL OBSERVATIONS: Yes
- Time schedule for examinations: The screening was conducted two hours after dosing and before any blood sampling for laboratory examinations.
5 males per group: Shortly before scheduled sacrifice on TD 30 or 31
5 females per group: During lactation, shortly before scheduled sacrifice between test days 39 and 50
- Dose groups that were examined: 5 male and 5 female animals randomly selected from each group
- Battery of functions tested:
Screening of sensory reactivity to stimuli of different types (e.g. auditory, visual and proprioceptive stimuli) (based on Gad ), as well as the assessment of grip strength (Meyer ) and motor activity assessment were conducted in five males and five females randomly selected from each group. The screening was conducted two hours after dosing and before any blood sampling for laboratory examinations:
5 males per group: Shortly before scheduled sacrifice on TD 30 or 31
5 females per group: During lactation, shortly before scheduled sacrifice between test days 39 and 50
Observational screening:
Righting reflex
Body temperature
Salivation
Startle response
Respiration
Mouth breathing
Urination
Convulsions
Pilo-erection
Diarrhoea
Pupil size
Pupil response
Lacrimation
Impaired gait
Stereotypy
Toe pinch
Tail pinch
Wire maneuver
Hind leg splay
Positional passivity
Tremors
Positive geotropism
Limb rotation
Auditory function
Functional tests:
Grip strength
Locomotor activity


OTHER:
COAGULATION
Thromboplastin time
Activated partial thromboplastin time

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
- At the time of sacrifice, or premature death during the study, the adult animals were examined macroscopically for any abnormalities or pathological changes. Special attention was paid to the organs of the reproductive system.
Apparently non-pregnant uteri were placed in a 10% aqueous solution of ammonium sulfide for about 10 minutes to stain possible implantation sites in the endometrium according to SALEWSKI.
The numbers of corpora lutea and implantation sites were recorded in the female adult animals and reported.
- Dead pups and pups sacrificed at day 4 post-partum, or shortly thereafter, were carefully examined externally for gross abnormalities.

ORGAN WEIGHTS: Yes
- The following organs of all adult animals were weighed individually and identified as left or right: Epididymis (2), Testicle (2)
- Determination of the organ weights of the following organs was only performed from 20 adult males and 20 adult females, which were randomly selected: Adrenal gland (2), Heart, Liver, Thymus, Brain, Kidney (2), Spleen. Adrenal glands and kidneys were weighed individually and identified as left or right.
- Animals Nos.
Group 1: 2, 4, 8, 11, 12 15, 18, 20, 23, 24
Group 2: 26, 27, 29, 35, 36 39, 40, 41, 42, 43
Group 3: 49, 52, 53, 54, 55 62, 63, 68, 69, 71
Group 4: 73, 75, 77, 80, 81 87, 90, 93, 95, 96

HISTOPATHOLOGY: Yes
- The following organs or parts of organs of all adult animals were fixed in 7% formalin; testes and epididymides were fixed in Bouin's fixative:
Epididymis (2), Gross lesions, Mammary gland, Ovary (2), Prostate, Seminal vesicle, Testicle (2), Uterus (incl. cervix and oviducts), Vagina
-In addition, the following organs or parts of organs of the selected 20 adult males and 20 adult females (see section above) were fixed in 7% formalin:
Adrenal gland (2)
Bone marrow (os femoris)
Brain (cerebrum, cerebellum, brain stem)
Heart (left and right ventricle, septum)
Intestine, small (duodenum, jejunum, ileum,
incl. Peyer's patches, Swiss roll method)
Intestine, large (colon, rectum)
Kidney and ureter (2)
Liver
Lungs (with mainstem bronchi and
bronchioles), preserved by inflation with
fixative and then immersion
Lymph node (1, cervical), Lymph node (1, mesenteric)
Nerve (sciatic)
Oesophagus
Spinal cord (3 sections)
Spleen
Stomach
Thyroid (incl. parathyroids)
Thymus
Tissue masses or tumours (incl.
regional lymph nodes)
Tongue (incl. base)
Trachea (incl. larynx)
Urinary bladder
-Only the 10 selected animals from the control group and the high dose group (20 animals in total) were considered for histopathological evaluation.

Other examinations:

REPRODUCTIVE PARAMETERS: See Section 7.8.1

DEVELOPMENTAL PARAMETERS: See Section 7.8.2

Statistics:

Toxicology and Pathology data were captured, whenever possible, using the departmental computerized systems (Provantis® Integrated preclinical software, Instem LSS Ltd.). Raw data not fully compatible with the computerized systems were maintained on paper according to appropriate SOPs.
The test item-treated groups (2 - 4) were compared with the control group (1).
The following statistical methods were used:
-STUDENT's t-test: All numerical functional tests (p ≤ 0.05 or p ≤ 0.01)
-Multiple t-test based on DUNNETT, C. W., New tables for multiple Comparisons with a control, Biometrics, 482-491 (Sept 1964): Body weight / Food consumption / Haematology / Clinical chemistry / Absolute and relative organ weights(p ≤ 0.05 or p ≤ 0.01)
- Exact test of R. A. FISHER: Histopathology, if applicable (p ≤ 0.05)

For all numerical values homogeneity of variances was tested by using the BARTLETT chi-square test. If the variances were homogeneous, the DUNNETT test (p ≤ 0.01) was used to compare the experimental groups with the control group.
In case of heterogeneity of variances, the STUDENT's t-test was carried out; limit of significance is p ≤ 0.01.


For the comparison of classification measurements (for example the fertility index) the FISHER's exact test, n < 100 or chi-square test with Yates' correction for continuity, n ≥ 100(p ≤ 0.05 and p ≤ 0.01) were employed.
These statistical procedures were used for all data. Significantly different data were indicated in the tables of the report.
The mean values and standard deviations were calculated to the highest possible degree of accuracy and then rounded to the reported number of decimal places. Hence, deviations to the last decimal place of up to ±1 may occur caused by rounding.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

sporadic and transient salivation and breathing sounds during the pre-mating period for 2 male and 2 female animals of the intermediate and the high dose group, not considered to be adverse.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

basophilic tubules (4 of 5 rats) and interstitial nephritis (3 of 5 rats) in the kidney of the male rats of the high dose group

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
No premature deaths were noted, neither in the control group nor in the treatment groups (100, 300 and 1000 mg test item/kg/bw/day).
One of 10 male animals with moderate salivation on test days 8 and 9 was noted in the intermediate dose group (300 mg test item/kg/bw/day). In the high dose male group (1000 mg test item/kg/bw/day) one of 10 animals with slight salivation was noted on test day 14.
Signs like moderate salivation and/or breathing sounds were noted for two of 10 female animals of the high dose group (1000 mg test item/kg/bw/day) during the pre-mating period on one day each, however they were not considered to be adverse.

BODY WEIGHT AND WEIGHT GAIN
No test item-related differences were noted between the control and the treatment groups.

FOOD CONSUMPTION
No test item-related differences were noted between the control and the treatment groups.

HAEMATOLOGY
No test item-related changes were noted between the control and the treatment groups.

CLINICAL CHEMISTRY
No test item-related changes were noted between the control and the treatment groups.

NEUROBEHAVIOUR
No adverse effects were noted at the observational screening. No test item-related influence was noted at the functional screening.

ORGAN WEIGHTS
No test item-related changes were noted between the control and the treatment groups for the absolute and relative organ weights.

GROSS PATHOLOGY
No test item-related changes were noted between the control and the treatment groups.

HISTOPATHOLOGY: NON-NEOPLASTIC
Test item-related changes like basophilic tubules (p≤0.05; 4 of 5 rats) and interstitial nephritis (3 of 5 rats) were noted in the kidney of the male rats of the high dose group (1000 mg test item/kg bw/day).
No microscopic changes were noted for the reproductive organs of the male and female animals.
No test item-related influence was noted on the stages of spermatogenesis.

Effect levels

1

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

histopathology: non-neoplastic

2

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

other: highest dose tested

Target system / organ toxicity

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Applicant's summary and conclusion

Conclusions:

Effects on the parental generation
NOAEL: 300 mg/kg bw/day in males
NOAEL: 1000 mg/kg bw/day in females

Executive summary:

The aim of the study was to obtain information on possible effects of the test item on general toxicity, reproduction and/or development according to OECD guideline 422. The test item was administered orally to rats at dose levels of 100, 300 or 1000 mg test item/kg bw/day. The application started two weeks before mating on test day one and ended on the day or one day before sacrifice. Dosing lasted for up to 32 days for the male rats and up to 53 days (lactation day 4 or shortly thereafter) for the female rats.

No premature deaths were noted. Sporadic and transient salivation and breathing sounds were noted during the pre-mating period for 2 male and 2 female animals of the intermediate and the high dose group (300 or 1000 mg test item/kg bw/day), however these changes were not considered to be adverse. There were further no test item related changes in neurological observations (including observational screening, grip strength and locomotor activity), body weight and weight gain, food consumption, haematology, clinical biochemistry, macroscopic examination and organ weights (absolute and relative). During microscopic examination, test item-related changes like basophilic tubules and interstitial nephritis were noted in the kidney of the male animals of the high dose group (1000 mg test item/kg bw/day).

NOAEL (no-observed-adverse-effect level) for repeated dose toxicity was 300 mg/kg bw/day in males and 1000 mg/kg bw/day in females.

Effects on reproduction parameters and organs (see section 7.8.1).

Effects on the development of the F1offsprings (pups) (see section 7.8.2).