Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018

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

Constituent 1
Chemical structure
Reference substance name:
Potassium isooctadecanoate
EC Number:
266-369-7
EC Name:
Potassium isooctadecanoate
Cas Number:
66469-15-6
Molecular formula:
C18H36O2.K
IUPAC Name:
potassium 16-methylheptadecanoate
Test material form:
solid: crystalline
Details on test material:
Product description: Potassium isooctadecanoate/potassium isostearate/potassium 16-methylheptadecanoate
Name: Isooctadecanoic acid, potassium salt (1:1)
CAS No.: 66469-15-6
Physical state: off white to yellowish solid at 20 °C
Batch No.: PFS-755-169
Re-certification date of batch: 12 December 2017
Purity: 100 % (mono constituent substance)
pH, 10% in DI water 8.0 - 9.0
Stability: stable under test conditions
Storage condition of test material: Room temperature, protected from light


Specific details on test material used for the study:
Product description: Potassium isooctadecanoate/potassium isostearate
Name: Isooctadecanoic acid, potassium salt
CAS No.: 66469-15-6
Physical state: off white to yellowish solid at 20 °C
Batch No.: PFS-755-169
Re-certification date of batch: 12 December 2017
Purity: 100 % (UVCB)
pH, 10% in DI water 8.0 - 9.0
Stability: stable under test conditions
Storage condition of test material: Room temperature, protected from light

Test animals

Species:
rat
Strain:
Crj: CD(SD)
Details on species / strain selection:
Rat / CD / Crl:CD(SD)

Charles River Laboratories, Research Models and Services, Germany GmbH
Sandhofer Weg 7
97633 Sulzfeld
Germany
Sex:
male/female
Details on test animals or test system and environmental conditions:
For this study CD rats bred by Charles River Laboratories Germany GmbH were used. The healthy nulliparous adult animals were randomised and assigned to the treatment groups and cages. The body weight range did not exceed 20% of the mean weight for each sex at the time of selection. The animals were held for 5 days for adaptation. Health checks were performed on the day of delivery and at first administration.

Body weight (at 1st administration, TD15)
Males: 383.7 g - 468.9 g
Females: 257.5 g - 302.0 g

Age (at 1st administration)
Males and Females: 80 days

Selection of species: The rat is a commonly used rodent species for such studies.

Housing and feeding

Diet: A certified commercial diet (ssniff® R/Z V1324) served as food. This food was offered ad libitum. Food residue was removed and weighed.
Samples of the food are analysed for contaminants based on EPA/USA by LUFA-ITL at least twice a year. Certificates of analysis of the composition and for contaminants are provided by the manufacturer and are included in the raw data. No contaminants above the limitations were noted.

Drinking water: Tap water was offered ad libitum. Samples of the drinking water are taken periodically by the Wasserwerk Wankendorf and periodic analyses are performed by LUFA-ITL according to the 'Deutsche Trinkwasserverordnung, Bundesgesetzblatt 2001' [German Regulations on drinking water, public notice of the law, 2001]. In addition, drinking water samples taken at LPT are analysed by LUFA-ITL once a year for means of bacteriological investigations according to the "Deutsche Trinkwasserverordnung 2001, Anlage 1".

Housing: With the exception of the mating period, the males and females (F0-Generation) were kept singly in MAKROLON cages (type III plus) with a basal surface of approx. 39 cm x 23 cm and a height of approx. 18 cm. The room temperature was 22°C ± 3°C (maximum range) and the relative humidity was 55% ± 15% (maximum range).

Granulated textured wood released for animal bedding (Granulat A2) was used as bedding material in the cages. The cages were changed and cleaned once a week. The rooms were alternately lit (about 150 lux at approximately 1.5 m room height) and darkened in a 12 hours dark/12 hours light cycle. The ventilation rate of the animal room was between fifteen to twenty air changes per hour.



Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
Administration volume: 10 mL/kg b.w./day
Vehicle:
water
Remarks:
Tap water
Details on oral exposure:
Administration

Route of administration: Oral, by gavage.
Frequency of administration: Once daily.
Administration volume: 10 mL/kg b.w./day
Vehicle: Tap water

Selection of route of administration: According to international guidelines.

The test item was dissolved in the vehicle to provide dose concentrations of 10, 30 or 100 mg/mL. The test item formulations were freshly prepared every day and were adjusted to the animal's actual body weight daily. The control animals received the vehicle at the same administration volume daily in the same way.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
For the analysis of the test item-vehicle formulations, samples of approximately 5 mL were taken at the following times and stored at ≤ -20°C until analysis.

At start of the treatment period (first dosing day):
Analysis of stability and concentration

Immediately after preparation of the test item-vehicle formulation as well as after 8 and 24 hours storage at room temperature
(3 samples / dose level group; groups 2 - 4).
Number of samples: 3 x 3 = 9

Towards the end of the treatment period (when the majority of animals was dosed):
Analysis of concentration

During treatment always before administration to the last animal/dose level group
(1 sample / dose level group; groups 2 - 4).
Number of samples: 3 x 1 = 3

Sum of all samples: 12

The samples were labelled with the study number, species, type of sample, concentration, test day, sampling time and date.

Analysis of the test item in the test item formulations

The analytical method was validated by LPT, whereby the following parameters were determined:
- Linearity
- Accuracy
- Precision
- Sensitivity
- Specificity
- Stability

The investigation of the above mentioned validation parameters confirmed that the method employed was suitable for the determination and quantification of the test item Potassium isostearate.

Validation of the method
The analytical method was successfully validated by with HPLC-UV detection.
Analysis of the test item-formulation

The results of the test item-formulation analyses for the investigated parameters are listed in the table below:

Parameter Sampling / Dealing Percent of nominal concentration [%]

Concentration immediately after preparation on test day 15 101.5 % - 103.3 %
Stability left at room temperature after preparation for 8h or 24 h on test day 15 102.7 % - 105.2 %
Concentration before administration to the last animal per group on test day 58 99.7 % - 104.5%

These results indicated correctly prepared test item vehicle mixtures, which were stable for at least 24 hours.
Duration of treatment / exposure:
The study animals were treated during the following periods:

Males: 2 weeks prior to mating (from test day 15 until test day 29), during the mating period (from test day 30# until test day 33# at maximum) and during the post-mating period until test day 47 (one day before sacrifice).

Females: 2 weeks prior to mating (from test day 15 until test day 29), during the mating period (from test day 30# until test day 33# at maximum) and during the lactation period until test day 64 to 68 (corresponding to lactation days 13 to 15). The last dosing was always one day before sacrifice.

Frequency of treatment:
Once daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
negative control
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
(low dose)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Remarks:
(intermediate dose)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
(high dose)
No. of animals per sex per dose:
10 male and 10 female rats
Control animals:
yes, concurrent vehicle
Details on study design:
The dose levels were selected by the sponsor based on available toxicological data and a 14-day dose range finding study. In the 14-day dose range finding study, Potassium isostearate was administered orally to male and female rats at dose levels of 100, 300 or 1000 mg/kg b.w./day for 2 weeks.
Changes in behaviour or the external appearance were noted in all test item treated groups (100, 300 or 1000 mg/kg b.w./day) in the form of salivation and nose discharge. Furthermore, a reduced motility and gasping were noted for 2 or 1 animal at the high dose level (1000 mg/kg b.w./day). No test item-related changes were noted on body weight and body weight gain, whereas slight reductions in food consumption where noted during the first and the second test week (at maximaum 20 % below the value of the control group; p ≤ 0.01). Based on the data obtained in this dose range finding study, dose levels of 100, 300 and 1000 mg Isooctadecanoic acid, potassium salt /kg b.w./day were selected for the main study in agreement with the Sponsor.
Positive control:
no

Examinations

Observations and examinations performed and frequency:
Clinical signs

Daily observations: Throughout the test period, each animal (parental animals and pups) was observed for clinical signs at least once daily. Behavioural changes, signs of difficult or prolonged parturition, and all signs of toxicity were recorded. Mortality was recorded twice daily. Animals which died prematurely were necropsied as soon as possible after exitus. Individual animals were observed before and after dosing at each time of dosing for any signs of behavioural changes, reaction to treatment or illness. Any signs of illness or reaction to treatment were recorded for each individual animal.
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. 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. Dated and signed records of appearance, change and disappearance of clinical signs were maintained on clinical history sheets for individual animals.

Detailed clinical observations

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 of the parental generation. These detailed clinical observations were performed at least 2 hours after dosing. These observations were made outside the home cage in a standard arena and at the same time, each time preferably by observers unaware of the treatment. Signs observed included changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g. lacrimation, piloerection, pupil size, and unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypy (e.g. excessive grooming, repetitive circling), bizarre behaviour (e.g. self-mutilation, walking backwards) were also recorded. Dated and signed records of appearance, change, and disappearance of clinical signs were maintained on clinical history sheets for individual animals.

Neurological screening

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 as described on the following pages in five males and five females randomly selected from each study group. The screening was conducted at least two hours after dosing and before any blood sampling for laboratory examinations:

5 male F0 animals per group (randomly selected) on test days 43 and 45.

5 female F0 animals per group (randomly selected) on test days 63 and 64 (between lactation days 10 to 12).

Observational screening

Righting reflex

The animal was grasped by its tail and flipped in the air approximately 60 cm above the cart surface so that it turned head over heels. The normal animal should land squarely on its feet; that means zero (0) points were scored. If it landed on its side, 1 point was scored; if it landed on its back, 2 points were scored. This test was repeated five times and the total scores were recorded.

Body temperature

An electronic probe thermometer with a blunt probe was used to take the rectal temperature, being allowed to equilibrate for 30 seconds before the reading was recorded.

Salivation

Discharge of clear fluid from the mouth is most frequently seen as beads of moisture on lips in rats. The normal state is to see none, in which case a zero (0) was recorded in the blank space of the scoring sheet. If present, a plus sign (+) was recorded in the blank.

Startle response

With the animal on the cart, the metal cage was struck with the blunt probe. The normal animal should exhibit a marked but short-lasting response, whereby a zero (0) was recorded in the blank space of the scoring sheet. If there was no response, a plus sign (+) was recorded.

Respiration

While at rest on the cart, the animal's respiration cycle was observed and evaluated in terms of a scale from 1 (reduced) to 5 (increased), with 3 being normal.

Mouth breathing

Rats are normally obligatory nose-breathers. Each animal was observed whether or not it was breathing through its mouth. If the rat was breathing through its nose, a zero (0) was recorded; mouth breathing was documented by a plus sign (+).

Urination

When an animal was removed from its cage, the pan beneath the animal's cage was examined while returning the animal to its cage. The signs of urination were evaluated on a scale of 0 (lacking) to 5 (polyurea) with 3 being normal.

Convulsions

If clonic or tonic convulsions were observed, their intensity was graded on a scale of 1 (minor) to 5 (marked) and the type was recorded. In the normal animal no convulsions should be observed, in which case a score of zero (0) was recorded.

Pilo-erection

The fur of the animal's back was observed whether it was raised or elevated. In the normal animal no pilo-erection should be observed and a score of zero (0) was recorded. If pilo-erection was present, a plus sign (+) was recorded.

Diarrhoea

In examining the pan beneath an animal's cage, it was noted if there were any signs of loose or liquid stools. The normal state is for there to be none (0); in case of diarrhoea the intensity was recorded on a scale of 1 (slight) to 5 (much increased).

Pupil size

It was determined if the pupils were constricted or dilated and the observations were evaluated in terms of a scale from 1 (constricted) to 5 (dilated), with 3 being normal.

Pupil response

The beam of light from the pen light was played across the eyes of the animal and the changes in pupil size were noted. In the normal animal, the pupil is constricted when the beam is on it and then dilates back to normal when the light is removed, whereby a score of zero (0) was recorded. If there was no pupil response, a minus sign (-) was recorded in the blank space.

Lacrimation

The animal was observed for the secretion and discharge of tears. The tears of rats contain a reddish pigment. No discharge is normal, whereby a score of zero (0) was recorded in the blank space of the scoring sheet. If a discharge was present, a plus sign (+) was recorded.

Impaired gait

The occurrence of abnormal gait was evaluated. The most frequent impairments are waddling (W), hunched gait (H), or ataxia (A, the inability of all the muscles to act in unison). The extent of any impairment was recorded on a scale of 1 (slight) to 5 (marked). A normal gait was documented by a score of zero (0).


Stereotypy

Each animal was evaluated for stereotypic behaviour (isolated motor acts or partial sequences of more complex behavioural patterns occurring out of context and with an abnormally high frequency). These were graded on a scale of 1 (slight) to 5 (marked). Normal behaviour was documented by a score of zero (0).

Toe pinch

The blunt probe was used to bring pressure to bear on one of the digits of the hind limb. This should evoke a response from the normal animal. The response or lack thereof was graded on a scale from 1 (absent) to 5 (exaggerated) with 3 being the normal response.

Tail pinch
The toe pinch procedure was utilized with the animal's tail instead of its hind limb and was graded on the same scale from 1 (absent) to 5 (exaggerated), with 3 being the normal response.

Wire manoeuvre

The animal was placed on the metal rod suspended parallel to the cart approximately 60 cm above the cart's surface. The animal's ability to move along the rod was evaluated. If impaired, a score from 1 (slightly impaired) to 5 (unable to stay on wire) was recorded. Normal movement was documented by a score of zero (0).

Hind leg splay

The hind paws were marked with ink using an ink pad. The rat was then held 30 cm above a sheet of blotting paper on the cart. The animal was dropped and the distance between the prints of the two hind paws was measured (in cm).

Positional passivity

The animal was observed after being placed in an awkward position, such as on the edge of the top of the wire-bottomed cage on the cart surface. If the animal immediately moved into a normal position, a score of zero (0) was recorded. If not, a score was recorded on a scale of 1 (slightly impaired) to 5 (cataleptic).

Tremors

Periods of continued fine movements, usually starting in the limbs and perhaps limited to them. The normal case is to have none, in which case a score of zero (0) was recorded. If tremors were present, they were graded on a scale of 1 (slight and infrequent) to 5 (continuous and marked). Positive geotropism The animal was placed on the inclined (approximately 30°) top surface of the wire cage with its head facing downward. It should turn 180° and face "uphill", in which case a score of zero (0) was recorded in the blank space of the scoring sheet. If this did not occur, a negative sign was recorded in the blank.

Limb rotation

One of the animal's hind limbs was taken and moved through its normal plane of rotation. In the normal state, it should rotate readily but there should be some resistance. The variations from normal were from no resistance (1) to markedly increased resistance or rigidity (5), with 3 being normal.
Auditory function Each animal was placed in a container and observed for Preyer's reflex (twitching of the pinna) in response to a high frequency sound stimulus. The stimulus was repeated, if necessary, up to 3 times. A normal response was recorded with a plus sign (+); if there was no response a zero (0) was recorded.

Functional tests

Grip strength

Prior to testing, the gauge (Chatillon, Modell DPP - 1.0 kg) was calibrated with a set of known weights and the apparatus adjusted for the size of the animal (about 1 cm clearance on both sides of the animal). After the strain gauge was zeroed and set in the record mode, the animal was placed into the trough with the forepaws inside the triangular grasping ring. Using one hand, the animal was grasped about 2.5 cm of the way up toward the base of the tail and steadily pulled (approx. 2.5 cm/sec) away from the ring until the grip was broken. It was continued to pull the animal along the trough until the hind limbs grasp the T-bar. The trial was completed when grip of the hind limbs was broken. Three successive readings were taken for each animal with an intertrial interval long enough to record the data and zero both meters for the next trial.

Locomotor activity

The motility was measured using the TSE InfraMot system . The infrared sensor was placed on the cage and any movements were measured for a duration of 12 min by sensing the body heat image, i.e. the infrared radiation, and its spatial displacement over time.
Any movements within the cage, even brief movement events of only a few milliseconds duration, were detected and included in the activity data.

Mortality

Further checks were made early in the morning and again in the afternoon of each working day to look for dead or moribund animals.

Body weight

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 1 post-partum) and on day 4 and 13 post-partum. Body weights were recorded individually for each adult animal.

Food and drinking water consumption

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.



Sacrifice and pathology:
Gross necropsy

Vaginal smears were examined on the day of necropsy to determine the stage of the oestrus cycle and allow correlation with the histopathology of the female reproductive organs. The animals were euthanized by carbon dioxide (CO2) inhalation, exsanguinated by cutting the aorta abdominalis, weighed, dissected and inspected macroscopically (gross necropsy) at the following times:
males - on test day 49 (after a dosing period of at least 4 weeks)
pups - on PND 13
dams - on lactation day 14, 15 or 16.

Dissection of adult animals

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.
During necropsy the number of implantation sites was recorded in the female animals.
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.
All superficial tissues were examined visually and by palpation and the cranial roof removed to allow observation of the brain, pituitary gland and cranial nerves. After ventral midline incision and skin reflection all subcutaneous tissues were examined. The condition to the thoracic viscera was noted with due attention to the thymus, lymph nodes and heart.
The abdominal viscera were examined before and after removal; the urinary bladder was examined externally and by palpation. The gastro-intestinal tract was examined as a whole and the stomach and the caecum were incised and examined. The lungs were removed and all pleural surfaces were examined under suitable illumination. The liver and the kidneys were examined. Any abnormalities in the appearance and size of the gonads, adrenals, uterus, intra-abdominal lymph nodes and accessory reproductive organs were recorded.

Organs to be weighed

The weight of the following organs of all adult male and female animals was determined before fixation (where applicable):
Adrenal gland (2), Brain, Epididymis (2), heart, kidney (2), liver, ovary (2), prostate, seminal vesicles with coagulating glands, thyroid, spleen testicle (2), thymus, uterus including cervix.

Thyroid weight was determined after fixation; paired organs were weighed individually and identified as left or right.

Organs for histopathology
Fixed organs from the 5 randomly selected male and female animals per group.
Fixative: Davidson'solution
Eye with optic nerve (2) - Fixative: modified Davidson'solution
Epididymis (left) - Fixative: 7 % buffered formalin
Adrenal gland (2)
Bone
Bone marrow (os femoris)
Brain (cerebrum, cerebellum, brain stem (pons))
Gross lesions observed
Heart (3 levels: right and left 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)
Mammary gland
Muscle (skeletal)
Nerve (sciatic)
Oesophagus
Pituitary
Ovary and oviduct
Prostate and seminal vesicles with coagulating glands
Spinal cord (3 sections)
Spleen
Stomach
Testicle (left) - Fixative: 7 % buffered formalin
Thyroid (including parathyroids)
Tissue masses or tumors including regional lymph nodes)
Thymus
Tongue (including base)
Trachea (including larynx)
Urinary bladder
Uterus (including cervix)
Vagina

Any other organs displaying macroscopic changes were also preserved.

Full histopathology was performed on the preserved organs of the selected parental animals of groups 1 and 4, and the thyroid of the selected pups. The organs listed above were examined histologically after preparation of paraffin sections and haematoxylin-eosin staining. Parathyroids cannot always be identified macroscopically. They were examined microscopically if in the plane of section and in all cases where they are noted were grossly enlarged. In addition, frozen sections of the heart, liver and one kidney were prepared, stained with Oil Red O and examined histologically. Detailed histopathologic examination was performed on one testicle and one epididymis (with special emphasis on the qualitative stages of spermatogenesis and histopathology or interstitial testicular structure) of the selected males of groups 1 and 4 following H-E and PAS staining.

Sperm viability and morphology (spermiogram)

The right epididymis and the right testis of the 5 selected male animals from each group were used for sperm count and the examination of sperm viability and sperm morphology. The examinations were performed according to the methods described by L. Chahoud and R. Franz (1993) as well as by S. Plassmann and H. Urwyler (2001).


Other examinations:
Laboratory examinations

Blood samples were taken from the retrobulbar venous plexus under isoflurane anaesthesia from animals fasted overnightat the following times:
At the end of the premating period on test day 29 (male and female animals): 5 male and 5 female F0 animals randomly selected from each group.

The blood samples collected were divided into tubes as follows:
EDTA anticoagulant (whole blood) for haematological investigations
Citrate anticoagulant (plasma) for coagulation tests
Li-Heparin anticoagulant (plasma) for clinical chemistry tests

Haematology

The parameters listed below were determined:

Haemoglobin content (HGB), mmol/L blood
Erythrocytes (RBC): 10^6/µL blood
Leucocytes (WBC): 10^3/µL blood
Differential blood count (relative and absolute): % and 10^3/µL blood
Reticulocytes (Reti): ‰ of the erythrocytes
Platelets (PCT or PLT): 10^3/µL blood
Haematocrit value: %
Mean corpuscular volume (MCV): fL
Mean corpuscular haemoglobin (MCH): fmoL
Mean corpuscular haemoglobin concentration (MCHC): mmol/L

Following the haematological examinations, blood smears were prepared from all samples, dried and stained for possible histopathological examinations in case of pathological findings.

Coagulation
The parameters listed below were determined:

Thromboplastin time (TPT)
Activated partialthromboplastin time (aPTT)

Clinical biochemistry

Albumin: g/L plasma
Globulin by substraction: g/L plasma
Albumin/Globulin ratio by calculation: non-dimensional
Bile acids: µmol/L plasma
Bilirubin (total): µmol/L plasma
Cholesterol (total): mmol/L plasma
Creatinine: µmol/L plasma
Glucose: mmol/L plasma
Protein (total): g/L plasma
Blood urea (BUN): mmol/L plasma
Calcium, Chloride. Potassium, Sodium: mmol/L plasma
Sodium/Potassium ratio by calculation: non-dimensional
BUN/Creatinine ratio by calculation: non-dimensional
Lactate dehydrogenase (LDH): U/L plasma
Alanine aminotransferase (ALAT): U/L plasma
Alkaline phosphatase (aP): U/L plasma
Aspartate aminotransferase (ASAT/GOT): U/L plasma
T4 ELISA: Total Thyroxine (T4)
TSH ELISA: TSH (Rat)

Urinalysis
Urine samples were collected from animals fasted overnight at the following times and the parameters listed below are determined:
Main study animals - 5 male animals randomly selected from each group.
At the end of the premating period on test day 29. The urine was collected for 16 hours in a URIMAX funnel cage. The collection of urine was terminated immediately prior to starting the blood withdrawals for the haematological and clinical chemical examinations (for the main study male animals on test day 29, for the toxicity study male animals at the end of the recovery periode before sacrifice on test day 66).

The following parameters were measured using the methods given below:

Volume, mL
pH
Specific gravity, g/mL (Sample compared with water )
Protein, bg/L
Glucose, bmmol/L
Bilirubin, (neg, +/++/+++ scale))
Urobilinogen, µmol/L
Ketones, (neg or +/++/+++ scale)
Haemoglobin (Hb) (approx. values), Ery/µL
Nitrite, - v()positive or negative)

Microscopic examination of urine samples were carried out by centrifuging samples and spreading the resulting deposit on a microscope slide. The deposit was examined for the presence of the following parameters:
- Epithelial cells (E)
- Leucocytes (L)
- Erythrocytes (R)
- Organisms(B)
- Further constituents (i.e. sperm, casts) (C)
- Crystalluria (A)

The colour and the turbidity of the urine were examined visually.



Statistics:
Parametrical data

The statistical evaluation of the parametrical values was done by Provantis (Provantis® integrated preclinical software, version 9.4.0.1, Instem LSS Ltd) using the following settings:
Homogeneity of variances and normality of distribution were tested using the BARTLETT’s and SHAPIRO-WILKS test. In case of heterogeneity and/or non-normality of distribution, stepwise transformation of the values into logarithmic or rank values was performed prior to ANOVA. If the ANOVA yielded a significant effect (p ≤ 0.05), intergroup comparisons with the control group were made by the DUNNETT’s test (p ≤ 0.01 and p ≤ 0.05).

Non-parametrical data

The statistical evaluation of non-parametrical values was done by comparison of the group values using the FISHER or the Chi2 test with the following software:
- Using Provantis: Statistical evaluation of histopathology findings
- Using StatXact 4.0.1: Statistical evaluation of the fertility index, the gestation index, the birth and live birth index, the viability indices and the post-implantation loss

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Males and females:At 300 or 1000 mg test item/kg b.w./day, a clear post-dosing salivation was noted in a dose related manner for the male and female animals. The saliva was reddish discoloured for one observation at the intermediate dose level and a few observations at the high dose level. The observation of salivatio disappeared again after several minutes. This transient form of salivation maybe related to the taste or irritant properties of the test item rather than an indication of adverse toxicity.
Males: At 1000 mg test item/kg b.w./day nose discharge was noted for several male animals (reddish discoloured for a few occasions).
Mortality:
mortality observed, treatment-related
Description (incidence):
Males: No test item-related premature death was noted for the animals treated with 100, 300 or 1000 mg test item/kg b.w./day.
Females: No test item-related premature death was noted for at the low and the intermediate dose level (100 or 300 mg test item/kg b.w./day).
At 1000 mg test item/kg b.w./day 2 of 10 pregnant females died on their gestation days 17 or 18. The deaths were considered as test item-related. Test item-related changes for both prematurely deceased animals were noted during the macroscopic examination at necropsy (dark-red discoloured lungs, reddened thymus, haemorrhagic nose/snout, enlarged adrenal glands) and during the histopathological examination (marked congestion in the lungs, adrenal cortical hypertrophy, thymus atrophy).
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Males: No test item-related influence on body weight and body weight gain was noted for the male animals.
Females: At 1000 mg test item/kg b.w./day slight reductions in body weight were noted at the end of the gestation period and during the lactation period (5.4 % below the value of the control group on gestation day 20 and 6.7 % or 4.9 % below the values of the control group on lactation days 4 and 13). None of these changes were statistically significant. Body weight gain of the female animals of the high dose group was reduced during the gestation period (39.8% in comparison to 52.1% in the control group).
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Males and females: No test-item related changes were noted.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No test item-related influence was noted on the haematological parameters of the male and female animals in any of the treatment groups (100, 300 or 1000 mg test item/kg b.w./day) for the haemoglobin content, the number of erythrocytes, the number of leucocytes, the number of reticulocytes and platelets, the haematocrit value, the mean corpuscular volume (MCV), the mean corpuscular haemoglobin (MCH), the mean corpuscular haemoglobin concentration (MCHC) and the parameters of coagulation (TPT, aPTT). No test item-related changes were noted in the relative and absolute differential blood counts. However, statistically significantly decreased values in comparison to the control group were noted for the haemoglobin concentration, the number of reticulocytes and the haematocrit value at the intermediate and or the high dose level (see table below). As no dose response relationship was noted and no statistically significant changes were noted for the female animals, the statistically significant changes that were noted for the male animals were considered as spontaneous and not as test item related.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Males and females: No test item related influence was noted for the examined plasma levels of the biochemical parameters in any of the treatment groups (100, 300 or 1000 mg test item/kg b.w./day), i.e. levels of albumin, globulin, the albumin/globulin ratio, bile acids, bilirubin (total), cholesterol (total), creatinine, glucose, protein (total), urea in blood, calcium, chloride, potassium, sodium, sodium/potassium ratio, urea/creatinine, the activity of the alanine aminotransferase (ALAT), of the alkaline phosphatase (aP), of the aspartate aminotransferase (ASAT) and the lactate dehydrogenase (LDH).
However, slightly decreased protein concentrations were noted for the male animals of the intermediate and the high dose group (4.6 % or 6.3 % below the value of the control group, statistically significant at p ≤ 0.05 at the high dose group). For the female animals slightly decreased protein levels were noted for all treatment groups (between 5.5 % and 6.4 % below the value of the control group, statistically not significant. This led to decreased globulin concentrations and increased albumin/globulin ratios for the male and the female animals (statistically significant or not, see table below).
The decreased protein concentration was due to slightly increased mean values of the protein concentrations of the male and the female animals of the control group (60.8 g/L for the males and 65.2 g/L for the females). For both, the male and the female animals the mean protein concentrations of the high dose group (57.0 g/L for the males and 61.6 g/L for the females) were at the level of the mean protein concentration of LPT background data (57.8 g/L for the males and 61.2 g/L for the females). Therefore, the observations were considered as spontaneous and not as test item-related.


Urinalysis findings:
no effects observed
Description (incidence and severity):
Males and females: No test-item related changes were noted.
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
Males and females: Changes in behaviour or the external appearance were noted in all test item treated groups (100, 300 or 1000 mg/kg b.w./day) in the form of salivation and nose discharge. Furthermore, a reduced motility and gasping were noted for 2 or 1 animal at the high dose level (1000 mg/kg b.w./day).
Immunological findings:
no effects observed
Description (incidence and severity):
Males and females: No test-item related changes were noted.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Males and females: At 1000 mg test item/kg b.w./day increased absolute and relative liver weights were noted for the male and female rats.
The absolute liver weight for the male and female animals was 28.0 % or 15.2 % above the value of the control group and the relative liver weight was 28.1 % or 18.2 % above the value of the control group (statistically significant at p ≤0.05 / 0.01 or not statistically significant).
Gross pathological findings:
no effects observed
Description (incidence and severity):
Males: Necropsy revealed no test item-related changes during the macroscopic examination of the internal organs and tissues of the male rats of the test item treated groups (100, 300 or 1000 mg test item/kg b.w./day).

Females: Necropsy revealed no test item-related changes during the macroscopic examination of the internal organs and tissues of the surviving female rats of the treatment groups (100, 300 or 1000 mg test item/kg b.w./day).
Neuropathological findings:
no effects observed
Description (incidence and severity):
Males and females: No changes were noted during the neurological screening.
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
Males and females: No test-item related changes were noted for the 5 selected males and the 5 selected female animals from the surviving 8 animals of the high dose group.

Microscopic examination of the reproductive organs:
No test item-related microscopic changes were observed in the reproductive organs for group 4 males and females that were examined microscopically.
The histopathological examination performed on one testicle and one epididymis of the selected males of groups 1 and 4 (with special emphasis on the qualitative stages of spermatogenesis (proliferative, meiotic and spermiogenic phases) and histopathology of the interstitial testicular structure), did not reveal any test izem-related effects. The mammary glands of the observed female animals showed prominent mammary development. No test item-related microscopic changes could be seen in the reproductive organs of the female animals of group 4.
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
Males and females: No test-item related changes were noted.
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
T4 Hormone Determination

Male and female animals: No toxicologically relevant changes were noted for the T4 levels of all treatment groups (100, 300 or 1000 mg test item/kg b.w./day).
The statistically significantly decreased T4 levels that were noted at the intermediate and the high dose level were not considered to be of toxicological relevance (see assessment below).

TSH Hormone Determination

Male animals: No test item-related changes were noted between the TSH levels of the male animals of the control group and the male animals of the treatment groups (100, 300 or 1000 mg test item/kg b.w./day).

Asssessment of the maternal T4 / TSH levels

As in the present study no influence of the decreased T4 level was noted on the TSH level and no changes were noted in the thyroid weight and the histopathology of the thyroids, the decreased T4 levels that were noted for the maternal animals were not considered as of toxicological relevance for the maternal animals and were not considered for the determination of the NOAEL. This is in accordance with a published 90 day rat toxicity study.[1] In this study dose levels with significant changes for the T4 and the TSH level, but without changes of the thyroid (weight and histopathology) or other indicators of toxicity (body weight, adverse clinical signs) were not considered for the NOAEL.
The mode of toxicological action on the thyroid gland by several substances is quite similar, independently of the mechanism that led to the reduction of the T4 level. As a response to a decreased circulating T4 level an increased release of TSH from the pituitary gland is observed. The increased TSH level results in thyroid follicular cell hyperplasia and hypertrophy.[2] It is generally known that histopathological examination of the thyroid is usually more sensitive than thyroid weight and hormone levels.[3] The validation report of OECD 407 states that “thyroid histopathology was consistently the most reliable and most sensitive endpoint for the detection of thyroid modulation. Thyroid weigth was reliable, but was somewhat less sensitive when compared to thyroid histopathology. Circulating thyroid hormone levels (T3, T4, and TSH) were not always reliable and sensitive, but the standard operating procedures for blood sampling and for thyroid hormone analyses were not standardized to reduce stress induced variability and to reduce analytical variability, respectively. Circulating T4 levels were the most promising of the three thyroid hormonal values”.[3]

[1] Siglin JC et al.: A 90-day drinking water toxicity study in rats of the environmental contaminant ammonium perchlorate, Toxicol Sci (2000) 57(1): 61-74.
[2] G. Coelho-Palermo Cunha and B. van Ravenzwaay: Evaluation of mechanisms inducing thyroid toxicity and the ability of the enhanced OECD Test Guideline 407 to detect this changes, Arch Toxicol (2005) 79: 390 - 405.
[3] Manon Beekhuijzen et al.: Update of OECD DART guidelines with endocrine disruptor relevant endpoints: Practical considerations, Reproductive Toxicology (2016) 64: 64 - 71.


Details on results:
Mortality

Males: None of the male animals died prematurely.
Females: Two of 10 female animals of the high dose group (1000 mg test item/kg b.w./day) died prematurely at the end of the gestation period. Both prematurely deceased animals were pregnant as determined by the presence of fetuses in the uterus. The death of both animals was considered as test item-related.

Clinical signs

Male and female animals - daily observations:

A red nose discharge was noted at the control group for one male animal once on test day 32 for a short period after dosing with the vehicle. The observation was considered as incidental as the animals of the control group received no test item. In rats a red nose discharge is probably caused by the secretion of a red pigment from the eyes due to stress or illness. This pigment drains from the eye into the nose, leading to reddish coloured nose discharge and / or in some cases to a reddish discoloured saliva.At the low dose level (100 mg test item/kg b.w./day) a red nose discharge and salivation were noted for 1 or 4 male animals on one test day each. For the female animals of the low dose group the only observation was salivation of one animal on 2 test days.
As the observations of a red nose discharge and salivation at the low dose level were only noted on 1 or 2 test days and the fact that a singular observation of a red nose discharge was also noted at the control group, the observations noted at the low dose group were considered as spontaneous and not as test item-related. At the intermediate and the high dose level (300 or 1000 mg test item/kg b.w./day) a dose related increased incidence of salivation (clear or sometimes red) was noted that mostly disappeared between 5 and 20 min after dosing. The effect observed maybe a reaction to the taste or irritation of the test article, rather than an indication of adverse toxicity. With the exception of the above mentioned salivation (that was not considered as a sign of adverse toxicity), no changes in behaviour, the external appearance or the faeces that were considered as test item-related were noted at the intermediate dose level. The observation of a clear nose discharge (noted for the female animals no. 57 and no. 58 on altogether 3 test days) was considered as spontaneous and not as test item-related due to the low incidence. An adverse toxic effect of the test item on the behaviour and the external appearance of the male and female animals (only the 8 surviving female animals were considered) was considered for the observations that were noted at the high dose level (1000 mg test item/kg b.w./day). This assessment was due to a high incidence of nose discharge in combination with further observations that appeared at the high dose level (reduced motility, breathing sounds, gasping, piloerection).

Start and duration of symptoms

Male and female animals (including the 2 prematurely deceased animals):

Salivation (clear or red): With one exception (no. 71 on gestation day 18), all observations of salivation started immediately to 5 min after dosing. In most cases salivation disappeared between 20 and 60 min after dosing. Only in 4 cases salivation disappeared earlier between 5 and 20 min after dosing.

Nose discharge (clear or red): For all observations, nose discharge was noted immediately to 5 min after dosing and disappeared between 20 and 60 min after dosing.

Reduced motility: Reduced motility was noted immediately to 5 min after dosing and disappeared again between 5 and 20 min or 20 to 60 min after dosing.

Gasping: Gasping was noted for female animal no. 72 on lactation day 1. It started immediately to 5 min after dosing and disappeared between 20 and 60 min after dosing.

Decreased respiratory rate: A decreased respiratory rate was noted for female animal no. 79 on gestation day 18 immediately to 5 min after dosing. The animal was found dead approx. one hour later.

Detailed clinical observations

The detailed clinical observations were performed once weekly, at least 2 h after dosing.
Males and females: No external observations and no changes in body posture, movement and coordination and in behaviour were noted for the animals of the low and the intermediate dose group (100 or 300 mg test item/kg b.w./day).

Neurological screening

The observational and functional neurological screenings were performed at least 2 hours after dosing on test day 43 for the male rats and on test days 63 (between lactation days 8 and 12) for the female rats.

Observational screening
Males and females: No test item-related observations of abnormal behaviour, changes in the external appearance, the appearance of the faces or test item-related differences in body temperature or the hind-leg splay in comparison to the control group were noted for the male and female animals of all treatment groups (100, 300 or 1000 mg test item/kg b.w./day).

Functional tests
Grip strength
Males and females: No test item-related influence on the fore- and hindlimb grip strength was noted for the male and female animals of all treatment groups (100, 300 or 1000 mg test item/kg b.w./day).

Body weight and body weight gain

Males:
Pre-mating-, mating- and post-mating period: No test item-related changes in body weight and body weight gain were noted for the male rats between the control group and the treatment groups.

Females:
Pre-mating-, gestation- and lactation period: No test item-related differences in body weight and body weight gain were noted between the female rats of the control group and the female rats of the low and the intermediate dose group (100 or 300 mg test item/kg b.w./day) during the pre-mating, the gestation and the lactation period. At 1000 mg test item/kg b.w./day a slightly decreased body weight was noted at the end of the gestation period (5.4 % below the value of the control group, statistically not significant). This difference in body weight was nearly diminished at the start of the lactation period (2.6 % below the value of the control group), but the difference increased again during the further course of the lactation period (minus 6.7 % or minus 4.9 % below the value of the control group on lactation days 4 and 13). Though the differences were only small and not statistically significant, the reduced body weight of the high dosed females in comparison to the control group was considered as test item-related.

Food consumption

Males: Pre-mating period
Food consumption of the male animals was only measured for the 2 weeks between the start of dosing and the day before the begin of the pairing period (between test days 15 to 28). No food intake of male animals was recorded during the mating period as both sexes were housed together and the following 2 weeks before necropsy. During these 2 weeks no test item-related difference in food consumption was noted between the rats of the control group and the rats of the treatment groups (100, 300 or 1000 mg test item/kg b.w./day). The slight reduction in food intake that was noted for the rats of the high dose group (at maximum 6.2 % below the value of the control group during the first week after the start of treatment, p ≤ 0.05) can be considered as an adaptation to the start of dosing with a high amount of 1000 mg/kg b.w/day. Hence, this was not considered to be of toxicological relevance.

Females: Pre-mating, gestation and lactation period
No test item-related differences in food consumption were noted between the female rats of the control group and the female rats of the treatment groups (100, 300 or 1000 mg test item/kg b.w./day) during the pre-mating, the gestation and the lactation period. However, slight and statistically not significant reductions in food consumption were noted during the first week after the start of treatment (8.9 % below the value of the control group, statistically not significant). As for the male animals the slight reduction was considered as an adaptation to the start of dosing (during the second test week after the start of dosing the food intake of the high dosed females reached again the level of the control group) and was not considered to be of toxicological relevance.
Further reductions at the high dose level were noted during the lactation period (10.9 % below the value of the control group during the first lactation week and 6.0 % during the second week of lactation, statistically not significant for both weeks). These slight reductions were considered to be spontaneous and not test item-related, as they showed no statistically significance.

Haematology

Males and females: No test item-related influence was noted on the haematological parameters of the male and female animals in any of the treatment groups (100, 300 or 1000 mg test item/kg b.w./day) for the haemoglobin content, the number of erythrocytes, the number of leucocytes, the number of reticulocytes and platelets, the haematocrit value, the mean corpuscular volume (MCV), the mean corpuscular haemoglobin (MCH), the mean corpuscular haemoglobin concentration (MCHC) and the parameters of coagulation (TPT, aPTT). No test item-related changes were noted in the relative and absolute differential blood counts.
However, statistically significantly decreased values in comparison to the control group were noted for the haemoglobin concentration, the number of reticulocytes and the haematocrit value at the intermediate and or the high dose level. As no dose response relationship was noted and no statistically significant changes were noted for the female animals, the statistically significant changes that were noted for the male animals were considered as spontaneous and not as test item related.

Clinical biochemistry

Males and females: No test item related influence was noted for the examined plasma levels of the biochemical parameters in any of the treatment groups (100, 300 or 1000 mg test item/kg b.w./day), i.e. levels of albumin, globulin, the albumin/globulin ratio, bile acids, bilirubin (total), cholesterol (total), creatinine, glucose, protein (total), urea in blood, calcium, chloride, potassium, sodium, sodium/potassium ratio, urea/creatinine, the activity of the alanine aminotransferase (ALAT), of the alkaline phosphatase (aP), of the aspartate aminotransferase (ASAT) and the lactate dehydrogenase (LDH). However, slightly decreased protein concentrations were noted for the male animals of the intermediate and the high dose group (4.6 % or 6.3 % below the value of the control group, statistically significant at p ≤ 0.05 at the high dose group). The decreased protein concentration was due to slightly increased mean values of the protein concentrations of the male and the female animals of the control group (60.8 g/L for the males and 65.2 g/L for the females). For both, the male and the female animals the mean protein concentrations of the high dose group (57.0 g/L for the males and 61.6 g/L for the females) were at the level of the mean protein concentration of LPT background data (57.8 g/L for the males and 61.2 g/L for the females). Therefore, the observations were considered as spontaneous and not as test item-related.
The determination of the bilirubin concentration revealed slightly reduced values for the male animals at the high dose level (14.3 % below the value of the control group, statistically not significant). For the female animals slightly reduced bilirubin concentrations were noted at all dose levels in comparison to the control group (between 12.8 % and 23.2 % below the value of the control group, statistically significant for the female animals at the high dose level at p ≤0.05).
However, all individual values of bilirubin were in the range of LPT background data. The mean value of the high dose level of the male animals (2.40 µmol/L) and the mean values of the intermediate and the high dose level of the female animals (2.74 µmol/L or 2.52 µmol/L) were at the level of the mean value of LPT background data (2.38 µmol/L for the males and 2.71 µmol/L for the females). Hence, the observed decreases in the bilirubin concentration were considered as spontaneous and not as test item-related.

Urinalysis

Males: No test item-related changes were noted for the urinary parameters of the male rats treated with 100, 300 or 1000 mg test item/kg b.w./day.

Body weight at autopsy

Males: No test item-related differences were noted on the body weight at autopsy between the control group and the treatment groups (100, 300 or 1000 mg test item/kg b.w./day).
Females: No differences were noted on the body weight at autopsy between the control group and the treatment groups (100, 300 or 1000 mg test item/kg b.w./day).

Macroscopic post mortem findings

Males: Necropsy revealed no test item-related changes during the macroscopic examination of the internal organs and tissues of the male rats of the test item treated groups (100, 300 or 1000 mg test item/kg b.w./day).
Females: Necropsy revealed no test item-related changes during the macroscopic examination of the internal organs and tissues of the surviving female rats of the treatment groups (100, 300 or 1000 mg test item/kg b.w./day).

Sperm parameter
Sperm count: No test item-related differences were noted for the number of spermatids in the right testis between the rats of the control group and the rats of the treatment groups (100, 300 or 1000 mg test item/kg b.w./day).

Sperm motility
The examination of the spermatids from the cauda of the right epidimydes revealed no test item-related differences between the control group and the treatment groups (100, 300 or 1000 mg test item/kg b.w./day).

Organ weights (relative and absolute)

Male and female animals: No test item-related influence was noted for the relative and the absolute organ weights of the male and female animals of the low and the intermediate group (100 or 300 mg test item/kg b.w./day). At the high dose level (1000 mg test item/kg b.w./day) an increased organ weight was noted for the absolute and the relative liver weight of the male and the female rats (statistically significant at p ≤ 0.05 / 0.01 or not statistically significant). These increased liver weights, noted at the high dose level, were considered as test item-related for the male and the female rats









Effect levels

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat. (total fraction)
Sex:
male/female
Basis for effect level:
clinical signs
organ weights and organ / body weight ratios
other: general toxicity
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat. (total fraction)
Sex:
female
Basis for effect level:
other: reproductive parameters of the parental females

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Table 1: Body Weight - Summary - Male Rats





































































Sex: male Day(s) relative to start date                  
  TD15TD22TD29 pairingTD36TD43TD48
Gr. 1: control

Mean


SD


N



413.07


18.07


10



433.82


22.88


10



430.41


32.70


10



454.28


31.97


10



465.95


30.22


10



457.06


31.96


10


Gr. 2: 100 mg/kg bw

Mean


SD


N


%Diff



415.22


18.07


10


0.5



440.57


19.13


10


1.6 



434.71


18.94


10


1.0



460.69


25.34


10


1.4



476.98


28.97


10


2.4



468.74


27.50


10


2.6


Gr. 3: 300 mg/kg bw

Mean


SD


N


%Diff



416.93


22.58


10


0.9



439.46


28.46


10


1.3



438.82


25.10


10


2.0



458.92


27.11


10


1.0 



470.97


33.63


10


1.1



459.92


31.60


10


0.6 


Gr. 4: 1000 mg/kg bw

Mean


SD


N


%Diff



408.99


18.77


10


-1.0



429.92


24.68


10


-0.9



424.05


30.34


10


-1.5 



449.47


26.18


10


-1.1



469.15


30.07


10


0.7



458.35


29.75


10


0.3


 

 



 



 



 



 



 



 



Body Weight (g)


 


Table 2: Body Weight - Summary - Female Rats












































Sex: femaleDay(s) relative to start date         
  TD15TD22TD29 pairing
Gr. 1: control

Mean


SD


N

278.27


13.87


10



281.14


15.64


10



275.56


20.97


10


Gr. 2: 100 mg/kg bw

Mean


SD


N


%Diff



278.99


11.46


10


0.3



279.56


12.99


10


-0.6



271.02


14.74


10


-1.6


Gr. 3: 300 mg/kg bw

Mean


SD


N


%Diff



277.36


7.78


10


-0.3



280.52


9.55


10


-0.2



271.40


10.39


10


-1.5


Gr. 4: 1000 mg/kg bw

Mean


SD


N


%Diff



281.39


13.61


10


1.1



278.89


16.98


10


-0.8



274.21


24.43


10


-0.5



Body Weight (g)


 


Table 3: Body Weight - Summary - Females - Gestation Period

















































Sex: femaleDay(s) relative to start date            
  GD0GD7GD14GD20
Gr. 1: control

Mean


SD


N

275.47


11.97


10 



307.56


18.88


10



339.98


19.09


10



418.73


22.74


10


Gr. 2: 100 mg/kg bw

Mean


SD


N


%Diff



283.55


13.53


10


2.9



310.65


17.34


10


1.0



349.36


21.14


10


2.8



421.19


25.99


10


0.6


Gr. 3: 300 mg/kg bw

Mean


SD


N


%Diff



282.03


11.36


10


2.4 



307.51


15.14


10


0.0



337.62


13.93


10


-0.7



408.57


15.28


10


-2.4


Gr. 4: 1000 mg/kg bw

Mean


SD


N


%Diff



287.06


22.15


10


4.2



300.28


16.75


10


-2.4



335.12


17.71


10


-1.4



396.10


27.96


8


-5.4



 


Table 4: Body Weight - Summary - Females - Lactation Period












































Sex: femaleDay(s) relative to start date         
  LD1LD4LD13
Gr. 1: control

Mean


SD


N



322.17


17.61


10



337.81


15.71


10



358.39


19.05


10


Gr. 2: 100 mg/kg bw

Mean


SD


N


%Diff



328.81


23.92


10


2.1



342.20


23.36


10


1.3



365.51


28.27


10


2.0


Gr. 3: 300 mg/kg bw

Mean


SD


N


%Diff



317.71


19.35


10


-1.4



325.38


13.14


10


-3.7



355.34


16.26


10


-0.9


Gr. 4: 1000 mg/kg bw

Mean


SD


N


%Diff



313.66


24.95


8


-2.6



315.06


25.60


8


-6.7



340.75


17.23


8


-4.9



 


 


Table 5: Haematological Parameters - Summary 1 - Males (Day: 29 Relative to Start Date)



























































Sex: maleHaematological Parameters                  
 

 



 


HGB


(mmol/L)


[a]



 


RBC


(x10E6/µL)


[a]



 


WBC


(x10E3/µL)


[a]


  

Reti


(%)


[a1]


 


  

PLT


(x10E3/µL)


[a]


 

HCT


(%)


[a]


Gr. 1: control

Mean


SD


N



10.12


0.34




 8.796


0.190


5



 10.076


2.955


5



 1.92


0.35


5



 997.6


161.1


5



 49.40


0.92


5


Gr. 2: 100 mg/kg bw

Mean


SD


N


%Diff



 9.90


0.19


5


-2.2



 8.592


0.185


5


-2.3



 10.010


1.255


5


-0.7



 2.20


0.23


5


14.6



 791.0


64.4


5


-20.7



 48.02


0.85


5


-2.8


Gr. 3: 300 mg/kg bw

Mean


SD


N


%Diff



9.60**


0.12


5


-5.1 



 8.194*


0.427


5


-6.8



 11.658


2.372


5


15.7



 2.52


0.37


5


31.3



 910.0


124.7


5


-8.8



 46.78**


0.76


5


-5.3


Gr. 4: 1000 mg/kg bw

Mean


SD


N


%Diff



9.74


0.26


5


-3.8 



 8.194*


0.427


5


-6.8



10.214


1.982


5


1.4 

1.72


0.73


5


-10.4



893.2


149.6


5


-10.5



47.58*


1.29


5


-3.7



[a] - Anova & Dunnett: * = p ≤ 0.05; ** = p ≤ 0.01


[a1] - Anova & Dunnett(Log)


 


Table 6: Haematological Parameters - Summary 1 - Females (Day: 29 Relative to Start Date)





































































Sex: femaleHaematological Parameters                  
  

HGB


(mmol/L)


[a]



RBC


(x10E6/µL)


[a]


  

WBC


(x10E3/µL)


[a]


  

Reti


(%)


[a1]


  

PLT


(x10E3/µL)


[a]


 

HCT


(%)


[a]


 
Gr. 1: control 

Mean


SD


N



9.62


0.28


5



8.570


0.351


5



7.254


2.673


5



1.98


0.31


5



800.2


101.6


5



46.50


1.66


5


Gr. 2: 100 mg/kg bw

Mean


SD


N


%Diff



9.34


0.21


5


-2.9



8.110


0.487


5


-5.4



7.382


1.252


5


1.8



2.20


0.50


5


11.1



962.0


146.8


5


20.2



45.26


1.15


5


-2.7


Gr. 3: 300 mg/kg bw

Mean


SD


N


%Diff



9.38


0.36


5


-2.5



8.148


0.246


5


-4.9



7.888


1.858


5


8.7



2.28


0.41


5


15.2



853.0


90.2


5


6.6



45.22


1.30


5


-2.8


Gr. 4: 1000 mg/kg bw

Mean


SD


N


%Diff



9.58


0.54


5


-0.4



8.468


0.471


5


-1.2



8.696


1.663


5


19.9



1.86


0.80


5


-6.1



861.6


163.5


5


7.7



46.12


2.68


5


-0.8


 

 



 



 



 



 



 



 



[a] - Anova & Dunnett: * = p ≤ 0.05; ** = p ≤ 0.01


[a1] - Anova & Dunnett(Log)


 


Table 7: Haematological Parameters - Summary 2 - Males (Day: 29 Relative to Start Date)






















































Sex: maleHaematological Parameters
  

TPT


(Seconds)


[a]



aPTT


(Seconds)


[a1]


 

MCV


(fL)


[a1]



MCH


(fmol)


[a1]



MCHC


(mmol/L)


[a1]


 
Gr. 1: control

Mean


SD


N



11.12


2.28


5



15.48


0.69


5



56.20


2.27


5



1.15


0.060


5



20.486


0.370


5


Gr. 2: 100 mg/kg bw

Mean


SD


N


%Diff



10.14


0.34


5


-8.8



15.20


0.85


5


-1.8 



55.90


1.09


5


-0.5



1.152


0.024


5


0.2



20.598


0.171


5


0.5


Gr. 3: 300 mg/kg bw

Mean


SD


N


%Diff



9.46


0.58


5


-14.9



15.56


0.96


5


0.5



57.14


1.84


5


1.7



1.172


0.043


5


1.9



20.504


0.250


5


0.1


Gr. 4: 1000 mg/kg bw 

Mean


SD


N


%Diff



9.88


0.35


5


-11.2



15.00


0.92


5


-3.1



58.12


2.52


5


3.5



1.190


0.045


5


3.5



20.480


0.224


5


0.0



 


Table 8: Haematological Parameters - Summary 2 - Females (Day: 29 Relative to Start Date)






















































Sex: femaleHaematological Parameters
  

TPT


(Seconds)


[a]



aPTT


(Seconds)


[a1]


 

MCV


(fL)


[a1]



MCH


(fmol)


[a1]



MCHC


(mmol/L)


[a1]


 
Gr. 1: control

Mean


SD


N



8.96


0.31


5



14.74


0.29


5



54.28


1.22


5



1.120


0.032


5



20.678


0.190


5


Gr. 2: 100 mg/kg bw

Mean


SD


N


%Diff



8.72


0.45


5


-2.7



14.22


0.70


5


-3.5



55.94


2.32


5


3.1



1.154


0.064


5


3.0



20.608


0.338


5


-0.3


Gr. 3: 300 mg/kg bw

Mean


SD


N


%Diff



9.06


0.48


5


1.1



13.96


0.63


5


-5.3



55.50


0.89


5


2.2



1.156


0.015


5


3.2



20.800


0.286


5


0.6


Gr. 4: 1000 mg/kg bw 

Mean


SD


N


%Diff



9.00


0.42


5


0.4



14.20


0.57


5


-3.7



54.44


1.17


5


0.3



1.128


0.028


5


0.7



20.750


0.112


5


0.3



 


Table 9-1: T4 - TSH - Level Analysis - Summary - Males  Rat







































Sex: male     
    

T4


concentrat.


(nmol/L)


[a]



TSH


concentrat.


(ng/mL)


[a1]


 
Gr. 1: control

Mean


SD


N



52.3392


6.5992


10



2.9689


2.0892


10


Gr. 2: 100 mg/kg bw

Mean


SD


N


%Diff



52.6401


6.7382


10


0.6 



2.4916


2.0318


10


-16.1 


Gr. 3: 300 mg/kg bw

Mean


SD


N


%Diff



45.1468*


4.7078


10


-13.7



3.2874


2.3184


10


10.7


Gr. 4: 1000 mg/kg bw

Mean


SD


N


%Diff



35.6372**


5.4689


10


-31.9



2.9258


2.4144


10


-1.5



 


Table 9-2: T4 Level Analysis - Summary - Females Rat


 

































Sex: female 
 

T4 concentrat. (nmol/L)


[a]


Gr. 1: control

Mean


SD


N



46.0962


6.2515


10


Gr. 2: 100 mg/kg bw

Mean


SD


N


%Diff



42.6422


2.4854


10


-7.5


Gr. 3: 300 mg/kg bw

Mean


SD


N


%Diff



35.2265**


6.0244


10


-23.6


Gr. 4: 1000 mg/kg bw

Mean


SD


N


%Diff



28.9446**


6.4852


8


-37.2



 


 


[a] - Anova & Dunnett: * = p ≤ 0.05; ** = p ≤ 0.01


[a1] - Anova & Dunnett


T4: Lower Level of Quantification (LLOQ) = 25 nmol/L


TSH: Lower Level of Quantification (LLOQ) = 2.5 ng/mL


TSH: Lower Level of Detection (LLOD) = 0.1 ng/mL


 


Table 10: Biochemical Parameters - Summary 1 - Males Rat (Day: 29 Relative to Start Date)



























































Sex: maleBiochemical Parameters                  
  

Albumin


(g/L)


[a]



Globulin


(g/L)


[a]



Alb./Glob.


Ratio


[a]


 

Bile


Acids


(µmol/L)


[a1]



Bilirubin


(total)


(µmol/L)


[a]


 

Choleste-


rol (total)


(mmol/L)


[a]


 
Gr. 1: control

Mean


SD


N



29.78


0.40


5



31.02


1.86


5



0.962


0.048


5



8.12


4.43


5



2.80


0.14


5



1.174


0.129


5


Gr. 2: 100 mg/kg bw

Mean


SD


N


%Diff



30.10


0.63


5


1.1



31.10


1.07


5


0.3



0.969


0.037


5


0.7



7.96


3.16


5


-2.0



2.86


0.36


5


2.1



1.832**


0.133


5


56.0


Gr. 3: 300 mg/kg bw

Mean


SD


N


%Diff



28.92


0.31


5


-2.9



29.08


2.45


5


-6.3



1.001


0.092


5


4.0



6.08


0.72


5


-25.1



2.80


0.43


5


0.0



1.238


0.316


5


5.5


Gr. 4: 1000 mg/kg bw

Mean


SD


N


%Diff



29.46


0.90


5


-1.1



27.54*


2.00


5


-11.2



1.073*


0.057


5


11.5


 



12.64


8 .00


5


55.7



2.40


0.34


5


-14.3



1.240


0.168


5


5.6



[a] - Anova & Dunnett: * = p ≤ 0.05; ** = p ≤ 0.01


[a1] - Anova & Dunnett(Rank)


 


Table 11: Biochemical Parameters - Summary 2 - Males Rat (Day: 29 Relative to Start Date)











































































Sex: male Biochemical Parameters                     
 

 



 


Crea-


tinine


(µmol/L)


[a]



 


BUN/Creat.


Ratio


[a]



 


Glucose


(mmol/L)


[a]



 


Protein


(total)


(g/L)


[a]



 


Urea


(in blood)


(mmol/L)


[a]


 



  


Calcium


(mmol/L)


[a1]



 


Chloride


(mmol/L)


[a]



Gr. 1: control



 Mean


SD


N



38.8


1.8




118.964


6.603


5



7.022


0.877


5



60.8


2.2


5



4.622


0.431


5



2.594


0.073


5



102.2


0.8


5



Gr. 2: 100 mg/kg bw



Mean


SD


N


%Diff



38.6


2.3


5


-0.5



104.903


11.232


5


-11.8



6.822


0.297


5


-2.8



61.2


1.3


5


0.7



4.058


0.573


5


-12.2



2.648


0.073


5


2.1



101.4


1.5


5


-0.8



Gr. 3: 300 mg/kg bw



Mean


SD


N


%Diff



37.2


2.5


5


-4.1



109.925


12.089


5


-7.6



7.174


1.196


5


2.2



58.0


2.2


5


-4.6



4.074


0.324


5


-11.9



2.586


0.073


5


-0.3



102.0


0.7


5


-0.2



Gr. 4: 1000 mg/kg bw



Mean


SD


N


%Diff


 

41.8


2.3


5


7.7



108.882


11.008


5


-8.5



6.188


0.526


5


-11.9 



57.0*


2.7


5


-6.3



4.536


0.305


5


-1.9



2.590


0.084


5


-0.2



101.6


1.5


5


-0.6



 



 



 



 



 



 



 



 



 



[a] - Anova & Dunnett: * = p ≤ 0.05; ** = p ≤ 0.01


[a1] - Anova & Dunnett(Rank)


 


Table 12: Biochemical Parameters - Summary 3 - Males Rat (Day: 29 Relative to Start Date)











































































  Sex: male  Biochemical Parameters                           
 

 



Potas-


sium


(mmol/L)


[a]


Sodium

(mmol/L)


[a]


 

Sod./Potass.


Ratio


[a]


 

ALAT


(U/L)


[a]



aP


(U/L)


[a1]


 

ASAT


(U/L)


[a2]


 

LDH


(U/L)


[a]


 
Gr. 1: control

Mean


SD


N



3.362


0.232


5



140.4


0.5


5



41.915


3.244


5



38.6


4.2


5



144.8


29.3


5



95.8


8.8


5



86.0


22.8


5



Gr. 2: 100 mg/kg bw



Mean


SD


N


%Diff



3.698


0.313


5


9.9



139.0


1.4


5


-1.0



37.804


3.195


5


-9.8



35.2


6.6


5


-8.8



132.6


15.9


5


-8.4



87.6


4.3


5


-8.7



93.8


34.6


5


9.1



Gr. 3: 300 mg/kg bw



Mean


SD


N


%Diff



3.746


0.242


5


11.4



139.2


0.8


5


-0.9



37.285


2.437


5


-11.0



38.4


3.6


5


-0.5



148.0


27.9


5


2.2



97.6


26.3


5


1.9



122.4


41.3


5


42.3


  

Gr. 4: 1000 mg/kg bw



Mean


SD


N


%Diff



 3.674


0.302


5


9.2



139.0


0.7


5


-1.0



38.039


3.129


5


-9.2



41.0


2.7


5


6.2



223.2


97.4


5


54.1



85.0


12.6


5


-11.3



111.0


62.5


5


29.1


 

 



 



 



 



 



 



 



 



[a] - Anova & Dunnett


[a1] - Anova & Dunnett(Log)


[a2] - Anova & Dunnett(Rank)

Applicant's summary and conclusion

Conclusions:
The following no-observed adverse-effect (NOAEL) levels were established:
Paternal/Maternal toxicity: NOAEL= 300 mg/kg bw/day, p.o.
Reproductive/Development toxicity: NOAEL=1000 mg/kg bw/day, p.o.
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 Potassium isostearate was administered orally to rats at dose levels of 100, 300 or 1000 mg test item/kg b.w./day.


General toxicity F0 - Generation


Two pregnant female animals of the high dose group (1000 mg test item/kg b.w./day) died prematurely during the gestation period.


Furthermore, changes in behaviour were noted at the high dose level (1000 mg test item/kg b.w./day) in the form of nose discharge for the male and female animals. Several female animals additionally showed a reduced motility. Breathing sounds, piloerection and / or gasping were further noted for one or 2 female animals of the high dose group (1000 mg test item/kg b.w./day). The body weight of the high dosed (1000 mg test item/kg b.w./day) females was slightly reduced at the end of the gestation period and during the lactation period. No influence was noted on the body weight of the male animals and on the food consumption of the male and female animals. Necropsy revealed an increased absolute and relative liver weight for the male and female animals of the high dose group (1000 mg test item/kg b.w./day). The statistically significantly reduced T4 levels that were noted at the intermediate and the high dose level for the male and the female animals were not considered as of toxicological relevance, as no changes were noted for the TSH level, the thyroid weights and during the histopathological examination of the thyroid glands. A test item-related post dosing salivation was noted in a dose related manner at the intermediate and the high dose level dose level (100, 300 or 1000 mg test item/kg b.w./day). However, this maybe a reaction to the taste or irritation of the test article, rather than an indication of adverse toxicity.


No changes were noted during the neurological screening, the examination of the haematological and the biochemical parameters, the urinalysis, the examination of the sperm parameter and during the macroscopic examination at necropsy.


NOAEL for systemic toxicity = 300 mg test item/kg b.w./day, p.o.


NOAEL for adverse effects on the reproductive parameters of the parental females = above 1000 mg test item/kg b.w./day, p.o

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