Fatty acids, tall-oil, reaction products with 2-[(2-aminoethyl)amino]ethanol

Endpoint summary

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

June 2015 - November 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

other: Crl:CD(SD)

Details on species / strain selection:

The rat was selected because of its proven suitability in toxicology studies and to comply with regulatory requirements for testing in a rodent animal species.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Breeder: Charles River Laboratories, Research Models and Services, Germany GmbH, Sulzfeld, Germany
- Body weight (at 1st administration): males: 134.4 - 160.5 g; females: 126.2 - 161.0 g
- Age (at 1st administration): males: 35 days; females: 39 days
- Fasting period before study:
- Housing: The animals were kept singly in MAKROLON cages (type III plus) with a basal surface of approx. 39 cm × 23 cm and a height of approx. 18 cm.
- Diet: A certified commercial diet (ssniff® R/M-H V1534, ssniff Spezialdiäten GmbH, 59494 Soest, Germany) served as food. This food was offered ad libitum. Food residue was removed and weighed.
- Water: Tap water was offered ad libitum.
- Adaptation period: males: 7 days, females: 8 days

DETAILS OF FOOD AND WATER QUALITY:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C ± 3°C (maximum range)
- Humidity (%): 55% ± 15% (maximum range)
- Photoperiod (hrs dark / hrs light): 12/12

Start of the exposure period:
Male main study and recovery animals: June 24, 2015
Female main study and recovery animals: June 25, 2015

Termination of the in-life period:
Male main study animals: September 22, 2015
Female main study animals: September 23, 2015
Male recovery animals: October 20, 2015
Female recovery animals: October 21, 2015

Route of administration:

oral: gavage

Details on route of administration:

Frequency of administration: once daily for 90 consecutive days

Vehicle:

corn oil

Details on oral exposure:

Administration volume: 2 mL/kg b.w./day
The test item was administered orally at a constant volume per kilogram body weight once daily for 90 days.
The dose of the test item was adjusted to each animal's body weight daily up to and including TW 6, and once weekly thereafter.
The control animals received the vehicle orally at a constant volume in the same way once daily.
The administration formulations were freshly prepared daily.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

For each test item mixed with the vehicle, tests by appropriate analytical methods were conducted to determine the concentration and homogeneity of the test item in the formulations.
For the analysis of the test item-vehicle mixtures, samples of at approximately 10 mL were taken at the following times and stored at -20°C or colder until analysis.

The analysis of the test item-vehicle mixtures sampled on TD 1 and 90 was performed using an HPLC-UV detection method re-validated by LPT.
The results of the analysis of WS400112 concentrations in the administration formulations indicate that the test item formulations were correctly prepared by LPT. The actual concentrations of WS400112 determined in the test item formulations on TD 1 and TD 90 for groups 2 to 4 ranged from 92.8% to 100.0%. These values were well within the admissible limits of 90% to 110%.

Duration of treatment / exposure:

90 test days
28-day recovery period for selected animals

Frequency of treatment:

once daily

Dose / conc.:

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

Remarks:

vehicle control

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

Main study animals: 20 animals (10 males and 10 females)
Recovery animals: 10 animals (5 males and 5 females)

Control animals:

yes, concurrent vehicle

Details on study design:

The dose levels for this study were selected based on the results of a 14-day dose range-finding study. In that study, male and female rats were treated once daily with 100, 300 or 1000 mg WS400112/kg b.w. by oral administration.

Observations and examinations performed and frequency:

1 Clinical signs
The animals were observed individually before and after dosing at each time of dosing for any signs of behavioural changes, reaction to treatment or illness.
In particular, the nature of the test item implied that administration may lead to signs of sedation like ataxia and reduced motility, which were to be described in terms of duration and intensity, if present.
In addition, the animals were checked regularly throughout the working day from 07:30 a.m. to 04:30 p.m. On Saturdays and Sundays, the animals were checked regularly from 08:00 a.m. to 12:00 a.m. with a final check performed at approximately 04:00 p.m.
Any signs of illness or reaction to treatment were recorded individually for each 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.
Dated and signed records of appearance, change, and disappearance of clinical signs were maintained on clinical history sheets for individual animals.
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 (TW) 13, these observations were performed prior to any laboratory investigations. The observations were made in a standard arena outside the home cage, each time at the same time of day. Signs noted 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.

2 Neurological screening
At the end of the treatment period (in TW 13) and at the end of the recovery period (in TW 17, before blood sampling for laboratory examinations), 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 was conducted for all animals.

2.1 Observational screening
Righting reflex
The animal was grasped by its tail and flipped in the air (approx. 60 cm) above the cart surface so that it turned head over heels. The normal animal should have landed squarely on its feet in which case 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, the thermometer was 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. If present, a plus sign was recorded in blank.
Startle response
With the animal on the cart, the metal cage was struck with the blunt probe. The normal animal should have exhibited a marked but short-lasting response, in which case a zero (0) was recorded. If there was no response, a plus sign was entered.
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 it was breathing through its mouth or not (if it did, a plus sign (+) was placed in the appropriate box, if it did not, a zero (0) was recorded).
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 to occur, they were graded on intensity from 1 (minor) to 5 (marked) and the type and intensity were recorded. In the normal animal, no convulsions were observed, in which case a score of zero (0) was recorded.
Piloerection
The fur of the animal's back was observed to determine whether it was raised or elevated. In the normal case (no piloerection) a score of zero (0) was recorded. If piloerection was present, a plus sign (+) was entered in the blank.
Diarrhoea
Upon examining the pan beneath an animal's cage, it was noted if there were any signs of loose or liquid stools. The normal state was 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. It was noted if there was no response (in which case a minus sign (-) was recorded in the blank space, otherwise a zero (0) was entered).
Lacrimation
The animal was observed for the secretion and discharge of tears. In rats the tears contain a reddish pigment. No discharge is normal and in this case a score of zero (0) was recorded. If the discharge was present, a plus sign (+) was entered.
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 abnormal high frequency). These were graded on a scale of 0 (= absent ^ normal) to 5 (marked) if such signs were present.
Toe pinch
The blunt probe was used to bring pressure to bear on one of the digits of the hind limb. This should have evoked a response from the normal animal, graded on a scale from 1 (absent) to 5 (exaggerated), with 3 being the normal response.
Tail pinch
The procedure detailed above was utilized with the animal's tail instead of its hind limb and was graded on the same scale.
Wire manoeuvre
The animal was placed on the metal rod suspended parallel to the cart approx. 60 cm above it. Its ability to move along the rod was evaluated. If impaired, a score of 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
Using an ink pad, the hind paws were marked with ink. 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.
Positional passivity
When placed in an awkward position (such as on the edge of the top of the wire bottomed cage) on the cart surface, it was examined whether the animal immediately moved into a more normal position. If it did, a score of zero (0) was recorded. If it did 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 zero (0) was recorded. If 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 (at an angle of approx. 30°) top surface of the wire cage with its head facing downward. It should have turned turn 180° and faced "uphill", in which case a score of zero (0) was recorded. 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 have rotated readily but there should be some resistance. The variations from normal are 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.

2.2 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 was 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. The animal was continually pulled along the trough until the hind limbs grasped 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 inter-trial interval long enough to record the data and zero both meters for the next trial.
Locomotor activity
The motility of the animals was measured using a TSE InfraMot system9. The infrared sensor was mounted on top of the home cage. Movements were detected 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.
The movement counters were scanned with a measuring interval of 1 minute for a duration of 12 minutes. The data given in the result tables represent the sum of movements counted during the 12-minute period of motion detection.

3 Mortality
Checks were made early in the morning and again in the afternoon of each working day to look for dead or moribund animals. On Saturdays and Sundays a similar procedure was followed with a final check at approximately 04:00 p.m. These provisions allowed a post mortem examination to be carried out during the working period of a day and to record premortal symptoms in detail. As soon as possible after exitus of animal no. 87, a post mortem examination was performed. No premature sacrifice was necessary.

4 Body weight
The weight of each rat was recorded at allocation (TD -7 for the males and TD -8 for the females), on TD 1 (day of first administration), and once a week thereafter always on the same day of the week throughout the experimental period (TD 8, 15, 22, 29, 36, 43, 50, 57, 64, 71, 78, 85 and 90 during the treatment period, and TD 97, 104, 111, and 118 during the recovery period).

5 Food and drinking water consumption
The quantity of food left by individual animals was recorded on a weekly basis throughout the experimental period. The food intake per animal (g/animal/week) was calculated using the total amount of food given to and left by each rat in each group on completion of a treatment week. The drinking water consumption was monitored daily by visual appraisal throughout the study.

6 Laboratory examinations
Blood samples were taken from the retrobulbar venous plexus under isoflurane anaesthesia from animals fasted overnight. 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
No anticoagulant (serum) ............................................ for bile acid determination

6.1 Haematology
The haematological parameters listed below were determined for all animals on TD 91 (end of the treatment period), and for all recovery animals on TD 119 (end of the recovery period).
Haemoglobin content (HGB)
Erythrocytes (RBC)
Leucocytes (WBC)
Reticulocytes (Reti)
Platelets (PLT)
Haematocrit value (HCT)
Differential blood count (relative)
Differential blood count (absolute)
Mean corpuscular volume (MCV)
Mean corpuscular hemoglobin (MCH)
Mean corpuscular hemoglobin concentration (MCHC)

6.2 Coagulation
The coagulation parameters listed below were determined for all animals on TD 91 (end of the treatment period), and for all recovery animals on TD 119 (end of the recovery period).
Thromboplastin time (TPT)
Activated partial thromboplastin time (aPTT)

6.3 Clinical chemistry
The biochemical parameters listed below were determined for all animals on TD 91 (end of the treatment period), and for all recovery animals on TD 119 (end of the recovery period).
Albumin
Globulin
Albumin/globulin ratio
Bile acids
Bilirubin (total)
Cholesterol (total)
Creatinine
Glucose
Protein (total)
Triglycerides
Urea (in blood)
Calcium
Chloride
Potassium
Sodium
Alanine amino-transferase (ALAT)
Alkaline phosphatase (aP)
Aspartate aminotransferase (ASAT)
Lactate dehydrogenase (LDH)

6.4 Urinalysis
Urine samples were collected from all animals fasted overnight on TD 91 (TW 13, end of treatment period), and from all recovery animals on TD 119 (end of recovery period).
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 chemistry examinations at study termination.
Parameters:
volume
pH
specific gravity
Protein
Glucose
Bilirubin
Urobilinogen
Ketones
Haemoglobin
Nitrite
A microscopic examination of urine samples was 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
- Leucocytes
- Erythrocytes
- Organisms
- Further constituents (i.e. sperm, casts)
- Crystalluria

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

7 Ophthalmological examination
All animals were examined prior to the start of administration (TW -1), at main study termination (TW 13), and at the end of the recovery period (TW 17, before blood sampling for laboratory examinations). The eyes were examined with a HEINE ophthalmoscope. Prior to examination, mydriasis was produced after instillation of MYDRUM®10 eye drops into the conjunctival sacs. The following ocular structures were examined: Adnexa oculi, conjunctiva, cornea, anterior chamber, iris (pupil dilated), lens, vitreous body, fundus.

Sacrifice and pathology:

Necropsy
On TD 91 (approx. 24 hours after the last administration), all main study animals were dissected following a randomisation scheme. Necropsy of all animals allocated to the recovery period was performed on TD 119.
The animals were euthanized by carbon dioxide (CO2), exsanguinated by cutting the aorta abdominalis, weighed, dissected, and inspected macroscopically under the direction of a pathologist.
All superficial tissues were examined visually and by palpation and the cranial roof was 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 of 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 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, adrenal glands, uterus, intra-abdominal lymph nodes and accessory reproductive organs were recorded.

The weights of the following organs of all animals were determined before fixation: Adrenal gland (2), Kidney (2), Spleen, Uterus (incl. cervix), Brain, Liver, Testicle (2), Prostate and seminal vesicles, Epididymis (2), Ovary (2), Thymus, Heart, Pancreas.

Histopathology
The organs listed above of all main study and recovery animals of groups 1 and 4 (control and high dose, including the prematurely deceased female animal no. 87) were examined histologically after preparation of paraffin sections and haematoxylin-eosin (H&E) staining.
In addition, frozen sections of the heart, liver and one kidney were prepared and stained with Oil Red O and examined microscopically.
It was planned to examine the corresponding organs of the animals of group 2 (low dose) and group 3 (intermediate dose) only in case test item-related changes were present in the organs of group 4 (high dose). However, as no test item-related changes were noted at the high dose, no additional examinations were performed.
Parathyroids were examined only if they could be identified macroscopically and were in the plane of section microscopically, or in case they were noted as grossly enlarged.

Bone marrow
During dissection fresh bone marrow was obtained from the os femoris (3 air-dried smears/animal) of all main study and recovery animals and stained according to PAPPENHEIM. The myeloid : erythroid ratio was determined by cell differentiation (counting of 200 nuclei-containing cells) for groups 1 and 4 (control and high dose).

Statistics:

Data for Toxicology and Pathology were captured, as far as possible, using the departmental computerized systems (Provantis® Integrated preclinical software, version 8.2.0, Instem LSS Ltd., Stone, Staffordshire ST15 0SD, United Kingdom). Raw data not fully compatible with the computerized systems were maintained on paper according to appropriate SOPs.
The test item-treated groups 2 to 4 were compared with the control group 1.

The following statistical methods were used for the data captured with the Provantis system:

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 and Coagulation / Clinical chemistry / Relative and absolute organ weights / Urinalysis (p ≤ 0.05 and p ≤ 0.01)

Exact test of R. A. FISHER: Histology (p ≤ 0.05)

The following statistical methods were used for the data not captured with the Provantis system:

STUDENT's t-test Numerical functional tests: Body temperature / Hind leg splay / Grip
strength / Spontaneous motility (p ≤ 0.05 and p ≤ 0.01)
chi² test: Bone marrow (p ≤ 0.01)

These statistical procedures were used for all data.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Detailed clinical observations in form of an assessment of external appearance, body posture, movement and coordination capabilities, and behaviour were performed for all animals pre- and post-dose on TD 1, and once weekly thereafter for the respective animals scheduled for the treatment period (TW 1 to 13) and the recovery period (TW 14 to 17).
All parameters of the detailed clinical observations of all animals scheduled for the control or treatment groups were in the normal range at pre-dose examination on TD 1.
All male and female control animals revealed the respective normal value for the parameter examined throughout the treatment and recovery period.

Treatment period:
None of the animals treated with 5 or 15 mg WS400112/kg b.w./day revealed any changes in external appearance, body posture, movement and coordination capabilities in TW 1 to 13. All male and female animals treated with the high dose of 45 mg WS400112/kg b.w./day revealed salivation for a period of approx. 30 minutes following administration in TW 4 to 9.

Recovery period (restricted to groups 1 and 4):
None of the animals previously treated with the high dose of 45 mg WS400112/kg b.w./day revealed any changes in external appearance, body posture, movement and coordination capabilities in TW 14 to 17.

Mortality:

mortality observed, treatment-related

Description (incidence):

Treatment period
All animals repeatedly treated orally with 5 or 15 mg WS400112/kg b.w./day and all male animals treated with 45 mg WS400112/kg b.w./day survived until their scheduled sacrifice at the end of the treatment period. No premature deaths were
noted.
One of 15 females animals (no. 87) treated with 45 mg WS400112/kg b.w./day was found dead in the morning of TD 78. A very low body weight at autopsy was noted for this animal. In advance to its death, the animal had revealed a body weight loss of 21% on TD 71 compared to TD 64. Further, pilo-erection and a reduced drinking water intake (estimated by visual observation) were observed on TD 77. The macroscopic inspection at necropsy revealed emphysematous lungs and that spleen and thymus were reduced in size. These findings were confirmed by histopathology. The macroscopically visible reduced organ sizes correlate with low relative and absolute organ weights noted for the respective organs. Additionally, a minimal focal myofiber atrophy/ degeneration in the skeletal muscle of the leg, and a moderate haemorrhage in the bone marrow of the os femoris were noted at histopathological examination.
The death is considered to be test item-related.
Also in the initial 14-day dose-range-finding study one of 10 animals treated with 100 mg/kg b.w./day and 2 of 10 animals treated with 300 mg/kg b.w./day died.

Recovery period (restricted to groups 1 and 4):
None of the animals treated previously with 45 mg WS400112/kg b.w./day died during the recovery period.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Treatment period and recovery period (restricted to groups 1 and 4):
No test item-related influence was observed on the body weight and the body weight gain of the male and female animals repeatedly treated orally with 5, 15 or 45 mg WS400112/kg b.w./day compared to the control animals throughout the treatment and recovery period.
No test item-related differences were noted for the body weight at autopsy between the test item-treated animals and the control animals.
However, the body weight of the female animals, previously treated with 45 mg WS400112/kg b.w./day, was approx. 10% higher than that of the control animals at the end of the recovery period (TD 118, statistically significant at p ≤ 0.05). This slight difference in body weight is not a delayed effect of the previous high dose treatment but due by coincidence to the comparatively high (group 4) or low (control group) weight gain of those animals scheduled for the recovery at the beginning of the study in comparison to the main study animals of the respective group during the treatment period.
The female animal no. 87 (scheduled for the treatment period) which was found dead on TD 78 as well as the male animal no. 83 (scheduled for the recovery period) were considered to be out of range regarding their respective body weights at the time of termination of the in-life period.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Treatment period and recovery period (restricted to groups 1 and 4):
No test item-related influence was observed on the food consumption of the male and female animals treated orally with 5, 15 or 45 mg WS400112/kg b.w./day compared to the control animals throughout the treatment period and the recovery period.
The food consumption of the female animals treated with 5 mg WS400112/kg b.w./day appeared to be slightly increased between TW 7 and 13 by up to 14% compared to the control group (statistically significant at p ≤ 0.05 in TW 11). However, this is considered to be a coincidental effect that is due to the relatively high food intake of only 3 animals. In addition no dose-dependency was detected.

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

no effects observed

Description (incidence and severity):

The visual appraisal of the drinking water consumption did not reveal any test item related differences between the test item-treated animals and the control animals throughout the treatment and the recovery period.
A reduced drinking water intake (estimated by visual observation) was observed for the prematurely deceased animal no. 87 on TD 77, the day before it was found dead. This finding is considered to be due to the moribund condition of this animal on that day and not to be an effect of the test item treatment.

Ophthalmological findings:

no effects observed

Description (incidence and severity):

Treatment period and recovery period (restricted to groups 1 and 4):
The ophthalmological examination did not reveal any test item-related changes of the eyes and the optic region in any animal after repeated oral treatment with 5, 15 or 45 mg WS400112/kg b.w./day, neither at the end of treatment (TD 91), nor at the end of the 4-week recovery (TD 119).
No test item-related pathological changes were noted on the adnexa oculi, conjunctiva, cornea, anterior chamber, iris (pupil dilated), lens, vitreous body and fundus.

Haematological findings:

no effects observed

Description (incidence and severity):

Treatment period and recovery period (restricted to groups 1 and 4):
No test item-related influence was observed on the haematological parameters for the male and female animals treated orally with 5, 15 or 45 mg WS400112/kg b.w./day compared to the control animals at the end of the treatment period (TD 91) and at the end of the recovery period (TD 119).
No test item-related effects were observed for the haemoglobin content (HGB), the numbers of erythrocytes (RBC), leucocytes (WBC) and platelets (PLT), the relative reticulocyte count (Reti), the haematocrit value (HCT), the relative and absolute differential blood count, the thromboplastin time (TPT), the activated partial thromboplastin time (aPTT), the mean corpuscular volume (MCV), the mean corpuscular haemoglobin (MCH), and the mean corpuscular haemoglobin concentration (MCHC) on TD 91 and on TD 119.
The following statistically significant differences in haematological parameters compared to the control animals noted on TD 91 or on TD 119 are not considered to be test item-related but to be coincidental:
Increased reticulocyte count in male rats of the treatment group 45 mg WS400112/kg b.w./day,
Decreased MCH in male rats of the treatment group 45 mg WS400112/kg b.w./day,
Decreased MCHC in female rats of the treatment group 45 mg WS400112/kg b.w./day

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Treatment period and recovery period (restricted to groups 1 and 4):
No test item-related influence was noted on the biochemical parameters of the male and female animals treated with 5, 15 or 45 mg WS400112/kg b.w./day at the end of the treatment period (TD 91) and at the end of the recovery period (TD 119).
No test item-related effects were noted on the plasma levels of albumin and globulin and on the albumin/globulin ratio, on the serum level of bile acids, as well as the plasma levels of total bilirubin, total cholesterol, creatinine, glucose, total protein, triglycerides, urea, calcium, chloride, potassium, and sodium on TD 91 and on TD 119. No test item-related influence was noted on the plasma enzyme activities of alanine amino-transferase (ALAT), alkaline phosphatase (aP), aspartate aminotransferase (ASAT), and lactate dehydrogenase (LDH). All data are considered to be within the limits of normal biological variability.
The statistically significant differences in biochemical parameters compared to the control animals noted on TD 91 are not considered to be test item-related but to be coincidental. The values obtained for albumin, cholesterol, protein and urea were within the range of historical control values for rats in LPT studies generated in the years 2012-2015. The mostly marginal changes in the respective parameter were always noted in only one sex and were considered to be within the normal range of biological variation.

Urinalysis findings:

no effects observed

Description (incidence and severity):

Treatment period and recovery period (restricted to groups 1 and 4):
Daily oral treatment with 5, 15 or 45 mg WS400112/kg b.w./day did not lead to any test item-related changes of the urinary parameters of the male and female animals compared to the control group at the end of the treatment period (TD 91) and at the end of the recovery period (TD 119).
No test item-related changes were noted for the specific gravity and the pH value of the urine and the urine volume. The analyte concentrations of nitrite, protein, glucose, ketones, urobilinogen, bilirubin and haemoglobin were not influenced in male and female animals. No test item-related changes were observed in the urine colour and the microscopically analysed urine sediments.
However, the pH value of the urine of the female high-dose animals, previously treated with 45 mg WS400112/kg b.w./day, was increased by approx. 20% compared to the control animals at the end of the recovery period (TD 119, statistically significant at p ≤ 0.05). This slight difference in the pH value of the urine is not considered to be a delayed effect of the previous high dose treatment but to be a coincidental finding. Historical pH control values of the urine of female rats in LPT studies range from 5.9 to 8.3.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Behaviour, external appearance, and faeces
Treatment period:
None of the male and female rats repeatedly treated orally with 5, 15, and 45 mg WS400112/kg b.w./day revealed any changes in behaviour or external appearance. In particular, no signs of sedation like ataxia and reduced motility were observed.

Recovery period (restricted to groups 1 and 4):
No abnormalities in behaviour and external appearance were observed for the male and female animals previously treated with 45 mg WS400112/kg b.w./day during the recovery period.

Neurological screening
The neurological screening was performed on all main study and recovery animals (group 1: n = 15 per sex, groups 2 and 3: n = 10 per group and sex, group 4: n = 15 for the males and n = 14 for the females) at the end of treatment (in TW 13), and on all recovery animals (groups 1 and 4: n = 5 per group and sex) at the end of the recovery period (in TW 17).

Treatment period and recovery period (restricted to groups 1 and 4):
No test item-related influence was noted on any of the parameters examined during the functional observation tests, on the fore- and hind limb grip strength, or on the spontaneous motility for any of the male and female animals after repeated oral treatment with 5, 15 or 45 mg WS400112/kg b.w./day in TW 13 and in TW 17.
One of 15 male animals (no. 77) treated with 45 mg WS400112/kg b.w./day revealed scores that were slightly different from the normal score for the following parameters examined in TW 13: respiration, mouth breathing, pilo-erection, toe pinch, tail pinch, wire maneuver, positional passivity, and resistance to limb rotation. However, the scores being slightly different from the normal score exhibited by this animal may occur for individual animals and are not considered to be of toxicological relevance. All other male and female high-dose animals examined revealed normal scores for all parameters tested.
A statistically significant change in comparison to the control animals was noted for the hind leg splay in TW 13. This minor chang is considered to be a coincidental effect and not to be related to the treatment with the test item.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Treatment period and recovery period (restricted to groups 1 and 4):
No test item-related influence was noted on the relative or absolute organ weights of the male and female animals treated with 5, 15 or 45 mg WS400112/kg b.w./day at the end of treatment (TD 91) and at the end of the recovery period (TD 119).
Statistically significant differences in relative and absolute organ weights compared to the control animals on TD 91 or TD 119 that are not considered to be test itemrelated are listed in the table on the following page. All organ weight values measured are within the range of historical control values for rats in LPT studies.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Treatment period and recovery period (restricted to groups 1 and 4):
The macroscopic inspection at necropsy did not reveal any test item-related changes in the organs and tissues of the animals repeatedly treated with 5, 15 or 45 mg WS400112/kg b.w./day after terminal sacrifice at the end of the treatment period (TD 91). No abnormal findings were noted at recovery sacrifice at the end of the recovery period (TD 119).
A few minor macroscopic findings were noted in various organs of individual animals which are not considered to be test item-related but to be of spontaneous nature. In detail, these findings were:
Enlarged spleen, enlarged mandibular lymph node, enlarged cervical lymph node, testes and epididymides reduced in size (males treated with 45 mg WS400112/kg b.w./day), a spleen with separated yellow foci with a diameter of approx. 2 to 3 mm (one female treated with 15 mg WS400112/kg b.w./day).
The findings mentioned above are considered to be normal spontaneous background changes due to their generally isolated occurrence.

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

Treatment period and recovery period (restricted to groups 1 and 4):
The histomorphological examination of a variety of organs and tissues was restricted to the control group 1 and the high dose group 4 treated with 45 mg WS400112/kg b.w./day (including the prematurely deceased female animal no. 87).
No morphological differences were noted at the microscopic level between the control group and the high-dose group 4. Type, incidence and severity of any lesions observed in the high-dose group 4 were comparable to those observed in control animals.
The cause of death of animal no. 87 could not be determined on the microscopic level.
The coincidental findings in various organs in a small number of control and test item-treated animals are considered to be spontaneous organ changes and are thus not test item-related.

Other effects:

no effects observed

Description (incidence and severity):

Bone marrow
Treatment and recovery period (restricted to groups 1 and 4):
The myeloid : erythroid ratio of the male and female animals treated with 45 mg WS400112/kg b.w./day was not influenced in comparison to the control group at the end of the treatment period (TD 91) and at the end of the recovery period (TD 119).

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

mortality

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

other: the reason for the death of one high dose animal is not known.

Organ:

not specified

Treatment related:

yes

Dose response relationship:

no

Relevant for humans:

not specified

In this sub-chronic oral gavage study no adverse systemic effects were observed up to and including the highest dose of 45 mg/kg body weight/day. At the highest dose level one female died after 78 days of daily dosing.

A very low body weight at autopsy was noted for this animal. In advance to its death, the animal had revealed a body weight loss of 21% from treatment day 64 through TD 71. Further, pilo-erection and a reduced water intake (estimated by visual observation) were observed on TD 77. Macroscopic inspection at necropsy revealed emphysematous lungs and that spleen and thymus were reduced in size. These findings were confirmed by histopathology. The macroscopically visible reduced organ sizes correlate with low relative and absolute organ weights noted for the respective organs. The death is considered to be test item-related.

No effects on stomach or the gastrointestinal system were reported. However, in the OECD 421 study performed with higher dose levels of 20, 60, and 200 mg/kg/d three animals at 60 mg/kg and seven animals at 200 mg/kg died between TD 11 and TD 41. In necropsy gastric and/or intestinal lesions were observed in nearly all deceased animals. Therefore, it is rather likely that also the animal which died in the sub-chronic study suffered from gastrointestinal effects leading to significant body weight loss before death.

Conclusions:

A study according to OECD Guideline 408 was conducted to obtain information on the toxicity of WS400112 given to rats by daily oral administration via gavage at dose levels of 5, 15 or 45 mg/kg b.w./day for 90 days, and to assess the reversibility of any affects following a recovery period of 28 days.
In the 90-day repeated-dose study one of 15 female animals repeatedly treated orally with the high dose of 45 mg WS400112/kg b.w./day was found dead in the morning of TD 78. The death is considered to be test item-related. All male and female animals treated with 45 mg/kg b.w./day revealed slight salivation between test days 17 to 81 and 16 to 80, respectively. No test item-related influence was observed on the body weight and the body weight gain, the food and drinking water consumption, haematological, biochemical and urinary parameters, the eyes and optic region, the relative and absolute organ weights, and on the myeloid : erythroid ratio at any of the tested dose levels. Observational screening and functional tests did not reveal any test item-related neurological effects. No test item-related macroscopic changes in organs or tissues were noted at necropsy. The histopathological examination of the high-dosed animals did not reveal any test item-related morphological lesions. No abnormalities related to the previous treatment were noted for the previously high-dosed animals during the 4-week recovery period.
Under the present test conditions of this study, the No-Observed-Adverse-EffectLevel (NOAEL) was 15 mg WS400112/kg b.w./day by oral administration.

Executive summary:

A study according to OECD Guideline 408 was conducted to obtain information on the toxicity of WS400112 given to rats by daily oral administration via gavage at dose levels of 5, 15 or 45 mg/kg b.w./day for 90 days, and to assess the reversibility of any affects following a recovery period of 28 days. The dose levels for this study were selected based on the results of a 14-day dose range-finding study with doses of 100, 300, and 1,000 mg/kg b.w./day. At 1,000 mg/kg b.w./day all rats of both sexes died prematurely in test week (TW) 1. One of 10 animals treated with 100 mg/kg b.w./day and 2 of 10 animals treated with 300 mg/kg b.w./day died in a 14-day dose-range finding study. In the 90-day repeated-dose study one of 15 female animals repeatedly treated orally with the high dose of 45 mg WS400112/kg b.w./day was found dead in the morning of TD 78. The death is considered to be test item-related. No test item-related changes in behaviour or external appearance were observed by daily cageside observations and detailed clinical observations once per week for the male and female animals treated with 5 or 15 mg/kg b.w./day. All male and female animals treated with 45 mg/kg b.w./day revealed slight salivation between test days 17 to 81 and 16 to 80, respectively. No test item-related influence was observed on the body weight and the body weight gain, the food and drinking water consumption, haematological, biochemical and urinary parameters, the eyes and optic region, the relative and absolute organ weights, and on the myeloid : erythroid ratio at any of the tested dose levels. Observational screening and functional tests did not reveal any test item-related neurological effects. No test item-related macroscopic changes in organs or tissues were noted at necropsy. The histopathological examination of the high-dosed animals did not reveal any test item-related morphological lesions. No abnormalities related to the previous treatment were noted for the previously high-dosed animals during the 4-week recovery period. Under the present test conditions of this study, the No-Observed-Adverse-EffectLevel (NOAEL) was 15 mg WS400112/kg b.w./day by oral administration.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

15 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

reliable without restriction

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Mode of Action Analysis / Human Relevance Framework

The substance 400112 has surface active properties. It exhibited strong cytotoxicity in cell cultures used in in vitro genotoxicity tests; genotoxic activity was not observed. It was corrosive in eye and irritant to skin.

 

In the OECD 421 study for screening of reproduction toxicity doses of 20, 60, and 200 mg/kg/d were applied repeatedly by oral gavage to rats. Three animals at 60 mg/kg and seven animals at 200 mg/kg died between treatment day (TD) 11 and TD 41. In necropsy gastric and/or intestinal lesions were observed in nearly all deceased animals. Therefore, in the absence of any systemic toxic effects it is rather likely that the surface active / corrosive properties of the substance caused the gastrointestinal effects which finally resulted in death of animals. In all surviving animals no effects in stomach or intestines were observed. There were no effects on reproductive parameters in parental animals up to and including the highest dose of 200 mg/kg/d. Also in offspring and during its growth for the first three days after parturition no effects were observed up to the highest dose level.

 

In the sub-chronic oral gavage study in rats 45 mg/kg/d was applied as highest dose level. No test item-related influence was observed on body weight and body weight gain, food and water consumption, haematological, biochemical and urinary parameters, the eyes and optic region, relative and absolute organ weights, and on myeloid : erythroid ratio at any of the tested dose levels. Observational screening and functional tests did not reveal any test item-related neurological effects. No test item-related macroscopic changes in organs or tissues were noted at necropsy. The histopathological examination of the high-dose animals did not reveal any test item-related morphological lesions. The only severe effect observed was the death of one animal of the high dose group (45 mg/kg/d) after 78 days of repeated dosing. Effects on the intestines were not reported after necropsy. The No-Observed-Adverse-Effect Level (NOAEL) was derived at 15 mg 400112/kg b.w./day by oral gavage administration (due to the death of one animal in the high dose group).

 

In conclusion, the substance 400112 has surface active / corrosive properties but did not exhibit any systemic toxic effects, no toxicity to reproduction and no genotoxic properties. Based on the effects observed in the gastrointestinal tract of animals that died in the OECD 421 study and in the absence of any systemic effects in the sub-chronic study it is likely that the mode of action leading to death of animals in the OECD 421 and in the sub-chronic oral gavage studies is based on the surface active / corrosive properties of the substance on the gastrointestinal tract. This effect is considered a local effect after oral gavage exposure and not a systemic effect.

Justification for classification or non-classification

Based on death of animals in sub-acute and sub-chronic oral toxicity studies between doses of 45 mg and 100 mg/kg/d the substance 400112 has to be classified for specific target organ toxicity after repeated exposure in category 2. Target organs are stomach and gastrointestinal tract.