Registration Dossier

Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
7 December 2018 - 2 July 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020
Report date:
2020

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
adopted June 25, 2018
Qualifier:
according to guideline
Guideline:
EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
Council Regulation (EC) No. 440/2008, adopted May 30, 2008, published in the Official Journal of the European Union L142/1, dated May 31, 2008.
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Butanedioic acid, 2(or3)-sulfo-, 4-[2-[(1-oxo(C12-C18(even numbered) and C18 unsaturated)alkyl)amino]ethyl]esters, disodium salts
Molecular formula:
Molecular formula cannot be given as the substance is a mixture
IUPAC Name:
Butanedioic acid, 2(or3)-sulfo-, 4-[2-[(1-oxo(C12-C18(even numbered) and C18 unsaturated)alkyl)amino]ethyl]esters, disodium salts
Test material form:
liquid
Specific details on test material used for the study:
Test material contains 41.2% solid content, therefore a correction factor of 2.43 was applied.
See confidential information

Test animals

Species:
rat
Strain:
other: CD® / Crl:CD(SD)
Details on species / strain selection:
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
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sandhofer Weg 7, 97633 Sulzfeld, Germany
- Age at 1st dosing: Males: 57 days; Females: 59 days
- Weight at 1st dosing: Males: 292.6 to 344.7 g; Females: 208.3 to 264.8 g
- Fasting period before study: not reported
- 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. Granulated textured wood (Granulat A2, J. Brandenburg, 49424 Goldenstedt, Germany) was used as bedding material for the cages. The cages were changed and cleaned once a week. Periodic analysis of the bedding material for contaminants based on EPA/USA is conducted at least once a year by LUFA-ITL. The animals received one piece of wood (certified for animal use) to gnaw on once weekly at change of the cages. Octagon-shaped red-tinted huts (polycarbonate) were placed in the cages to offer the animals a resting and hiding place.
- Diet (e.g. ad libitum): A certified commercial pelleted diet (ssniff® R/M-H V1534, ssniff Spezialdiäten GmbH, 59494 Soest, Germany served as food. The food was offered ad libitum. Food residue was removed and weighed.
- Water (e.g. ad libitum): Drinking water was offered ad libitum.
- Acclimation period: Males: 13 days; Females: 15 days

DETAILS OF FOOD AND WATER QUALITY:
Periodic analysis of the food for contaminants based on EPA/USA is conducted at least twice a year by LUFA-ITL. Certificates of analysis of the composition and for contaminants were provided by the manufacturer and included in the raw data.
Drinking water is examined according to the 'Deutsche Trinkwasserverordnung 2001' [German Regulations on Drinking Water 2001] by the Hamburger Wasserwerke, 20539 Hamburg, Germany, at least four times a year. 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' [German Regulations on Drinking Water 2001, Addendum 1].

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C±3°C (maximum range)
- Humidity (%): 55% ± 10% (maximum range)
- Air changes (per hr): not reported
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES:
From: Start of exposure period: Male animals: 11 December 2018; Female animals: 13 December 2018
To: Dissection of main study animals: Male animals: 11 March 2019; Female animals: 13 March 2019
Dissection of recovery animals: Male animals: 08 April 2019; Female animals: 10 April 2019

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
Selection of administration route according to international guidelines.
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: The administration formulations were freshly prepared daily.
The test item was diluted in the vehicle to the appropriate concentrations.
Administration volume 5 mL/kg bw/day

VEHICLE
- Justification for use and choice of vehicle (if other than water): Tap water (the test item was well soluble)
- Concentration in vehicle: The test item was orally administered at a constant volume per kilogram body weight once daily for 90 days. The test item doses were adapted to each animal's current body weight daily up to and including test week 6, and once weekly thereafter.
- Amount of vehicle (if gavage): Administration volume 5 mL/kg bw/day
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The stability, homogeneity and concentration of the administration formulations were analysed. Samples of approximately 10 mL administration formulation were taken at the times given below and stored at -20°C±2°C until analysis.
-At first administration (males on test day 1)
*Analysis of stability and concentration
Immediately after preparation of the formulations as well as after 8 and 24 hours storage at room temperature.
(3 samples per test item-treated group).
*Number of aliquots: 3× 3 = 9
*Analysis of homogeneity
At start of administration, during administration (in the middle), and before administration to the last animal per dose group.
(3 samples per test item-treated group)
*Number of aliquots: 3 × 3 = 9

-At last administration (females on test day 90)
*Analysis of concentration
During treatment always before administration to the last animal per group.
(one sample per test item-treated group).
*Number of aliquots: 3
*Total number of samples: 21

The samples were labelled with study number, species, type of sample, concentration, test day, sampling time, and date.
The analysis of the test item-vehicle mixtures from test days 1 and 91 was performed by LPT using an HPLC-UV detection method that was validated at LPT The following parameters were determined according to ICH Q2 (2005):
- Linearity
- Accuracy
- Precision
- Sensitivity
- Specificity
- Stability
The results indicate that all test item formulations were correctly prepared by LPT, were very homogeneous, and were stable for at least 24 hours.
Duration of treatment / exposure:
90 days
Frequency of treatment:
once daily
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
active ingredient; concentration: 20 mg/mL
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Remarks:
active ingredient; concentration: 60 mg/mL
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
active ingredient; concentration: 200 mg/mL
No. of animals per sex per dose:
main study: 40
recovery period: 10
In addition, 10 spare animals (5 males and 5 females) were available for possible replacement during the adaptation period.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels for the study had been selected to be 100, 300 and 1000 mg act. ingr./kg bw/day by the Sponsor based on the available toxicological data of an OECD 422 study (LPT report no. 28640). The dose of 1000 mg/kg bw was considered to be a maximum tolerated dose.
- Rationale for animal assignment (if not random): The animals were weighed and allocated to the test groups by means of a computerised randomisation program. Animals of the extremes of the weight distribution and/or any animal having shown signs of illness were to be excluded and replaced by spare animals. However, no replacements were necessary. The body weight variation of the animals used did not exceed ±20% of the mean weight per sex at start of the study.
None of the animals was replaced after the first dose had been administered.
- Fasting period before blood sampling for clinical biochemistry: overnight
- Rationale for selecting satellite groups: Post-exposure recovery groups were included for the control and high dose group (5m + 5f per group)
- Post-exposure recovery period in satellite groups: 28 days.
- Section schedule rationale: computer randomisation model.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule:
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 addition, the animals were checked regularly throughout the working day from 7.30 a.m. to 4.30 p.m. by cageside observations. On Saturdays and Sundays, the animals were checked regularly from 8.00 a.m. to 12.00 noon with a final check performed at approximately 4.00 p.m.
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 of individual animals were maintained on clinical history sheets.
Special attention was paid to ascertain if there were any signs of irritation after oral dosing, such as increased salivation, redness of the oral cavity, etc.
Further 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 4.00 p.m. These provisions allowed for post mortem examinations to be carried out during the working period of the respective day, and to record premortal symptoms in detail. However, no deaths occurred and no premature sacrifice was necessary.
- Cage side observations checked in table [No.?] were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once before the first exposure (to allow for within-subject comparisons) and once a week thereafter until test week 13, detailed clinical observations were made in all animals. In test week 13 the observations were performed prior to any laboratory investigations. The observations were made outside the home cage in a standard arena and at the same time, each time. 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.
The detailed clinical observations were discontinued for the animals scheduled for the recovery period after test week 13 as none of the animals had ever revealed any abnormalities during the treatment period.

BODY WEIGHT: Yes
- Time schedule for examinations: The weight of each rat was recorded at the time of group allocation, daily from the day of start of treatment up to and including test week 6 (for dose adjustment), and once weekly thereafter throughout the experimental period. Weekly values are reported for the entire study period.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
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 determined using the total amount of food given to and left by each animal in each group on completion of a treatment week.
The relative food consumption (in g/kg bw/day) was calculated as follows:
Relative food consumption (g/kg bw/day) = (Total food given (g) - Total food left (g)) / (Number of animal days# × Body weight (kg))
#The term 'animal days' counts one animal day for each animal alive for a whole day; it is assumed that on the day of death an animal does not eat.

WATER CONSUMPTION: No
- Time schedule for examinations: The drinking water consumption was recorded once weekly by weighing the water bottles after being filled and the water residues upon removal of the bottles at the end of the test week. The residue was discarded. Weekly means are reported.
Visual appraisal did not reveal any noteworthy water loss due to spilling. No data were recorded.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Examinations were performed pre-dose (males: test day -1, females: test day -3), in test week 13 (end of the treatment period, males: test day 91, females: test day 89) and in test week 17 (end of recovery, males: test day 119, females: test day 117).
The eyes were examined with a HEINE ophthalmoscope. After examination of the pupillary reflex, mydriasis was produced by instillation of STULLN® eye drops (Ankerpharm GmbH, 07407 Rudolstadt, Germany) onto the cornea.
The following ocular structures were examined:
• Adnexa oculi (i.e. lids, lacrimal apparatus), conjunctiva
• Cornea, anterior chamber
• Lens, vitreous body, fundus (retina, optic disc)
- Dose groups that were examined: Treatment and recovery period (recovery restricted to control/high dose, groups 1/4)

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood samples were taken from the retrobulbar venous plexus at the following times:
At the end of the treatment period (test day 91, before necropsy): all main study animals
At the end of the recovery period (test day 119, before necropsy): all recovery animals
- Anaesthetic used for blood collection: Yes, under isoflurane anaesthesia
- Animals fasted: Yes, fasted overnight
- How many animals:
At the end of the treatment period (test day 91, before necropsy): all main study animals
At the end of the recovery period (test day 119, before necropsy): all recovery animals
- Parameters examined: Haemoglobin content, Erythrocytes, Leucocytes, Reticulocytes, Platelets, Haematocrit value, Differential blood count relative), Differential blood count (absolute), Mean corpuscular volume, Mean corpuscular haemaglobin, Mean corpuscular haemoglobin concentration, Prothrombin time, Activated partial thromboplastin.
Following the haematological examinations using the ADVIA system, blood smears were prepared from all samples, dried and stained for possible histopathological examinations in case of pathological findings. So far, no histopathological examinations were performed.


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood samples were taken from the retrobulbar venous plexus at the following times:
At the end of the treatment period (test day 91, before necropsy): all main study animals
At the end of the recovery period (test day 119, before necropsy): all recovery animals
- Animals fasted: Yes, fasted overnight
- How many animals:
At the end of the treatment period (test day 91, before necropsy): all main study animals
At the end of the recovery period (test day 119, before necropsy): all recovery animals
- Parameters examined: Albumin, Bile acids, Bilirubin (total), Cholesterol (total), High density lipoprotein (HDL cholesterol), Low density lipoprotein ‘LDL cholesterol), Creatinine, Glucose, Protein (total), Urea (in blood), Calcium, Chloride, Potassium, Sodium, Alanine anmino transferase (ALAT), Alkaline phosphatase (aP),Aspartate aminotransferase (ASAT), Lactate dehydrogenase (LDH)

Thyroid hormones: In order to obtain approximately 2×150 µL serum each for determination of the thyroid hormones triiodothyronine (T3), thyroxine (T4), and thyroid-stimulating hormone (TSH), a sufficient volume of blood was taken from the retrobulbar venous plexus under isoflurane anaesthesia from animals fasted overnight. Blood sampling was performed at the same time of day (at approx. 7:00 a.m. to 10:00 a.m.).
The serum samples were divided into aliquots and stored at -20°C±2°C at LPT until analysis.
The T3, T4 and TSH ELISA analyses were performed by LPT using commercial kits by IBL International GmbH (Hamburg, Germany) and a Tecan Sunrise microplate reader (Tecan Deutschland GmbH, Crailsheim, Germany). The evaluation was performed by LPT.

URINALYSIS: Yes
- Time schedule for collection of urine: The collection of urine was terminated immediately prior to starting the blood withdrawals for laboratory examinations at study termination.
- Metabolism cages used for collection of urine: Yes. The urine was collected in URIMAX funnel cages from individual animals for 16 hours. - Animals fasted: Yes, fasted overnight
- Parameters examined: Volume, pH, Specific gravity, Protein, Glucose, Bilirubin, Urobilinogen, Ketones, Haemoglobin (Hb, approx.. values), 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 frequency of the above parameters in the centrifugal deposit was recorded as follows:
0 None found in any field examined
+ Few in some fields examined
++ Few in all fields examined
+++ Many in all fields examined
The colour and the turbidity of the urine were examined visually.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: The neurological screening was performed at the following times: in test week 13 (at the end of the treatment period): all study animals
In test week 17 (at the end of the recovery period): all recovery animals
Screening of sensory reactivity to stimuli of different types (e.g. auditory, visual and proprioceptive stimuli; based on Gad), as well as an assessment of grip strength (Meyer) and motor activity assessment was conducted for all animals. The neurological screening in test week 13 was performed approximately 1 to 2 hours after dosing.
- Dose groups that were examined: all animals
- Battery of functions tested:
Observational screening: righting reflex, body temperature, salivation, startle response, respiration, mouth breathing, urination, convulsions, piloerection, diarrhoea, pupil size, pupil response, lacrimation, impaired gait, stereotypy, toe pinch, tail pinch, wire manoeuvre, hind leg splay, positional passivity, tremors, positive geotropism, limb rotation, auditory function.
Functional tests: grip strength, locomotor activity

IMMUNOLOGY: No

OTHER:
AUDITORY EXAMINATIONS: Examinations were performed pre-dose (males: test day -1, females: test day -3), in test week 13 (end of the treatment period, males: test day 91, females: test day 89) and in test week 17 (end of recovery, males: test day 119, females: test day 117).
The auditory acuity was checked with a simple noise test.


Sacrifice and pathology:
GROSS PATHOLOGY: Yes. On test day 91 (approx. 24 hours after the last dosing), all main study animals were dissected following a randomisation scheme. All animals allocated to the recovery period were sacrificed and dissected on test day 119.
At necropsy, the oestrus cycle of all main study and recovery females was determined by taking vaginal smears. These observations provided information regarding the stage of oestrus cycle at the time of humane killing and assisted in histopathological evaluation of oestrogen sensitive tissues.
The animals were sacrificed by carbon dioxide (CO2) inhalation, 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. 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, the lymph nodes and the 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 organs listed below were weighed before fixation: Adrenal gland (2), Brain, Epididymis (2), Heart, Kidney (2), Liver, Ovary (2), Spleen, Pituitary, Testicle (2), Thymus, Thyroid (2, including parathyroids), Uterus including cervix, Prostate and seminal vesicles with coagulating glands (as a whole)
The eyes were fixed in Davidson's solution and the testes in modified Davidson’s solution for optimum fixation. The organs or parts of organs of all animals of all test groups as listed below were fixed in 7% neutral buffered formalin: Adrenal gland (2), Aorta abdominalis, Bone (os femoris with joint), Bone marrow (os femoris), Brain (cerebrum, cerebellum, brain stem), Epididymis (2), Eye with optic nerve, Gross lesions (if any observed), Heart (left and right ventricle, septum), Intestine, small (duodenum, jejunum, ileum, Swiss roll method), Intestine, large (colon, rectum), Kidney and ureter (2), Liver (2 lobes), 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, leg), Nerve (sciatic), Oesophagus, Ovary with oviducts (2), Pancreas, Pituitary, Prostate and seminal vesicles with coagulating glands, Salivary glands (mandibular, parotid, sublingual), Skin (left flank), Spinal cord (3 sections: cervical, mid-thoracic, lumbar), Spleen, Stomach, Testicles (2), Thyroid (2, incl. parathyroids), Tissue masses or tumours (including regional lymph nodes), Trachea (including larynx), Urinary bladder, Uterus (including cervix), Vagina.

HISTOPATHOLOGY: Yes. The organs listed above (fixed in 7% neutral buffered formalin) of all main study and recovery animals of groups 1 (control) and 4 (high dose) were examined histopathologically after preparation of haematoxylin-eosin stained paraffin sections. Frozen sections of the heart, liver, and one kidney were prepared, stained with Oil Red O, and examined histopathologically.
A detailed histopathological examination was performed on one testicle and one epididymis of the males of groups 1 and 4 following H-E and PAS staining in order to identify treatment related effects such as retained spermatids, missing germ cell layers or types, multinucleate giant cells or sloughing of spermatogenic cells into the lumen. Special attention was given to possible stage-specific changes.
Parathyroids were examined microscopically if identified macroscopically and if in the plane of section and in cases they are noted as grossly enlarged.
The histopathological evaluation of group 4 animals (high dose) identified the stomach as a target organ. Therefore, the stomachs of all group 2 and 3 animals (low and intermediate dose) were H-E stained and examined histopathologically, too.
The histotechnique was performed by LPT. The slides of groups 1 and 4 (labelled with study number, species, animal number, block number) were dispatched to Test Site for histopathology on 28 May 2019. The slides with the stomach sections for the additional examinations of groups 2 and 3 were dispatched on 06 August 2019.
The transport of slides to the Test Site was arranged by LPT, whereas the return transport to the Test Facility was arranged by the Test Site.
Histopathology was performed by Dr. R.J.M.M. Thoolen, Global Pathology Support B.V., Benoordenhoutseweg 23, 2596 BA The Hague, The Netherlands, under the Test Site reference 544 according to all relevant Test Site SOPs.
The histopathological examination included the organs and tissues as defined in the Study Plan and any subsequent Study Plan amendments.
All observations upon final assessment were reported per animal and the findings considered relevant for the treatment in an incidence and occurrence table. All microscopic findings were recorded, reported and archived with the Provantis system.
The report of the histopathology phase of the study comprises a description of objective, materials and methods, results (including results of macroscopic and microscopic changes) and conclusions. The unaudited Draft Phase Report was presented electronically to the Study Director for comments/review. Comments, if any, were addressed and the Draft Final Phase Report of the histopathology phase of the study was presented to the TSQAU for auditing.
The audited Draft Final Phase Report of the histopathology phase of the study was sent electronically to the Study Director who provided documented approval on the contents of the Phase Report by fax or e-mail to the Principal Investigator. An original of the final, signed Phase Report was sent (on paper and electronically) to the Study Director; a copy is archived by the Test Site.
The Phase Report on histopathology is included ('Pathologist's Report').
The blood smears prepared from all animals during the haematological examination were retained for possible examination of pathological changes but were not examined and evaluated so far as there were no noteworthy necropsy findings and no request by the Sponsor.
Other examinations:
The oestrus cycle stages (proestrous, oestrous, metoestrous, dioestrous) of all main study and recovery females was determined by analysis of vaginal smears taken at necropsy at terminal sacrifice (test day 91) or recovery sacrifice (test day 119).
Statistics:
All toxicology and pathology data were captured, as far as possible, using the departmental computerised systems (Provantis® Integrated preclinical software, version 10.2, Instem LSS Ltd., Stone, Staffordshire ST15 0SD, United Kingdom). Raw data not fully compatible with the computerised systems were maintained on paper according to appropriate SOPs.
The test item-treated groups 2, 3 and 4 were compared with the control group 1.
The statistical methods below 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 / Thyroid hormones / Relative and absolute organ weights / Water consumption / Urinalysis (p ≤ 0.05 and p ≤ 0.01)
- Exact test of R. A. FISHER (if applicable): Histopathology (p ≤ 0.05)
Homogeneity of variances and normality of distribution were tested using BARTLETT's test and the SHAPIRO-WILK's 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 DUNNETT's test.
The following statistical methods were used for the data NOT captured with the Provantis system:
STUDENT's t-test: All numerical functional tests: Body temperature / Hind leg splay / Grip strength / Spontaneous motility (p ≤ 0.05 and p ≤ 0.01. The following limits were used:
- p = 0.05 / 0.01 ^ t = 2.05 / 2.76 (for 28 degrees of freedom)
- p = 0.05 / 0.01 ^ t = 2.07 / 2.81 (for 23 degrees of freedom)
- p = 0.05 / 0.01 ^ t = 2.31 / 3.36 (for 8 degrees of freedom).
The statistical procedures were used for all data. Statistically significantly different data are indicated in the tables of the report.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
Treatment period: None of the male and female rats treated orally with 100, 300, or 1000 mg act. ingr./kg bw/day revealed any test item-related changes in behaviour, external appearance, consistency of faeces, body posture, and movement and coordination capabilities.
Recovery period: None of the animals previously treated with 1000 mg act. ingr./kg bw/day revealed any abnormalities related to the previous treatment during the 4-week recovery period.
Mortality:
no mortality observed
Description (incidence):
Treatment period No deaths were noted at any dose level. All animals survived until their scheduled sacrifice.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Treatment period: The mean body weight of the male and female animals treated with 1000 mg act. ingr./kg bw/day was slightly reduced by up to 7.4% in males and up to 10.7% in females in comparison to the control group. The body weight gain and the body weight at autopsy of both sexes were reduced accordingly. This effect is considered to be test item-related.
Recovery period: The body weight of the male and female animals recovered towards the normal range, no noteworthy difference compared to the control group was noted at the end of the recovery period.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
No test item-related toxicologically relevant influence was noted on absolute and relative food consumption.For the male high-dosed animals, a slight but statistically significant (at p ≤ 0.05 or p ≤ 0.01) increase by up to 14.4% was noted for the relative food consumption compared to the control animals starting in test week 2 during the treatment period and lasting until test week 16 during the recovery period, however no significant changes were seen in the absolute food consumption.These changes are within the normal range of biological variation. In test week 17 (end of recovery), when the previously high-dosed male animals revealed no evident difference in the body weight compared to the control animals, there was also no noteworthy difference in the food consumption between the two groups.
For the female animals treated with 1000 mg act. ingr./kg bw/day, a slight but statistically significant (at p ≤ 0.01) decrease in absolute food consumption of up to 14.6% was noted in test weeks 1 and 10 to 12. In test week 1, the relative food consumption was also slightly decreased (statistically significant at p ≤ 0.01), however in test weeks 10 to 12 no significant changes were noted for the relative food consumption. Accordingly, high-dosed female animals showed slightly lower body weights of up to 10.7% (statistically significant at p ≤ 0.01) in test weeks 10 to 12. All changes are within the normal range of biological variation.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
The female animals treated with the intermediate dose of 300 mg act. ingr./kg bw/day revealed a slight increase of the drinking water consumption by up to 25.9% (in test week 13) compared to the control group being statistically significant (at p ≤ 0.05 or p ≤ 0.01) in test weeks 1 to 8, 11, and 13.
At the high dose of 1000 mg act. ingr./kg bw/day, both the male and the female animals revealed an increase of the drinking water consumption by up to 64.0% (in test week 4) for the male animals and by up to 60.6% (in test week 3) for the female animals in comparison to the control group as of test week 1. The effect was statistically significant (at p ≤ 0.05 or p ≤ 0.01) in test weeks 1 to 13 for the male and female animals and appeared to be slightly more pronounced in the female animals than in the male animals. . The increased drinking water consumption is considered a test item-related effect.
Recovery period: No noteworthy difference was noted for the drinking water consumption of the male animals previously treated with the high dose in comparison to the control group during the recovery period.
The drinking water consumption of the female animals previously treated with the high dose was still slightly increased by 17.5% (statistically significant at p ≤ 0.05) compared to the control group in test week 14. Afterwards, there was no noteworthy difference between the previously high-dosed animals and the control group.

Ophthalmological findings:
no effects observed
Description (incidence and severity):
Treatment period: No test item-related changes were noted.
Recovery period: No test item-related changes were noted.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Treatment period: No test item-related changes were noted in haematology and coagulation.
Recovery period: No test item-related changes were noted.
Some statistically significant differences in haematological parameters were noted in comparison to the control group that are not considered to be test item-related but to be coincidental.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Treatment period: No test item-related changes were noted for clinical biochemistry and serum levels of triiodothyronine) (T3), thyroxine (T4), and thyroid-stimulating hormone (TSH).
Recovery period: No test item-related changes were noted.
Some statistically significant differences in biochemical parameters were noted in comparison to the control group that are not considered to be test item-related but to be coincidental.
The serum levels of T4 appeared to be decreased by 24.4% or 25.5% in the male animals treated with 300 or 1000 mg act. ingr./kg bw/day (statistically significant at p ≤ 0.01 for both groups) in comparison to the control animals at the end of the treatment period, respectively (test day 91, only the main study animals). The female animals revealed a decrease by 22.4% (not statistically significant at p ≤ 0.05 or p ≤ 0.01) at 300 mg act. ingr./kg bw/day but an increase by 8.7% at 1000 mg act. ingr./kg bw/day in comparison to the control (also not statistically significant).
The mode of toxicological action on the thyroid gland by various substances is quite similar, independently of the mechanism that leads to the reduction of the T4 level. Increased release of TSH from the pituitary gland is observed as a response to a decreased circulating T4 level. However, no influence was noted on the thyroid weight, and no histopathological findings were observed for the thyroids in animals treated with the test item. Therefore, a toxicological relevance of the decreased T4 serum levels cannot be confirmed for the test item due to the absence of any of the described changes expected to follow a toxicity-related decrease in T4 serum levels. Hence, these changes are considered coincidental alterations within the range of normal biological variability, as only 1 animal in the intermediate and high dose group each showed T4 serum levels outside the historical range.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
Treatment period: No test item-related changes were noted for urinalysis.
Recovery period: No test item-related changes were noted.
A large amount ('+++') of erythrocytes in the centrifugal deposits and an increased haemoglobin content (250 erythrocytes/µL) were observed in the urine of 4 of 10 male animals (nos. 71, 72, 75, and 77) treated with 1000 mg act. ingr./kg bw/day at the end of the treatment period and one male control animal. For all animals, all findings observed (increased number of erythrocytes, increased haemoglobin content, urine colour) are not regarded to be test item-related effects. For animals no. 71, 72 and 75 as well as the single control animal, we consider these changes most likely to be due to small injuries at the animals' claws caused by the metal grid of the funnel cages that might not have been detected at necropsy. For animal no. 77, there are two possible causes for the findings. However, due to animal no. 77 being the only high dose animal to show macroscopic and microscopic changes in the related organs, we consider these to be spontaneous and not test item-related.
Some other statistically significant differences were noted for the urinary parameters in comparison to the control group that are not considered to be test item-related but to be coincidental.
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
No test item-related and/or statistically significant influence was noted on any of the parameters examined during the functional observation tests, on the fore- and hindlimb grip strength, or on the spontaneous motility for any of the male and female animals after repeated oral treatment with 100, 300, or 1000 mg act. ingr./kg bw/day in test week 13 and in test week 17. In female animals treated with either 100, 300 or 1000 mg act. ingr./kg bw/day, a decrease in spontaneous motility was noted compared to control animals. This difference was even more pronounced at the end of the recovery period for females previously treated with 1000 mg act. ingr./kg bw/day. However, during the daily observation of the animals no corresponding clinical signs were noted, and the neurological screening revealed no differences in behaviour between the treated and control animals. Therefore, these apparent effects observed in a single test are considered to be spontaneous and not related to any toxic effect of the test item.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Treatment period: No test item-related changes were noted in organ weights.
Recovery period: No test item-related changes were noted.
Any differences noted for the relative or absolute organ weights of the test item-treated animals at the end of the treatment period or at the end of the recovery period in comparison to the control animals are not considered to be test item-related and to be within the normal range of biological variation. The increased relative brain weight noted in females treated with 300 or 1000 mg act. ingr./kg bw/day is considered a secondary effect to the reduced body weight at necropsy, as the absolute brain weight does not differ between the control group animals and the animals treated with the test item.
Statistically significant differences in organ weights compared to the control animals that are not considered to be test item-related are listed.
Any differences noted for the relative or absolute organ weights of the test item-treated animals at the end of the treatment period or at the end of the recovery period in comparison to the control animals are not considered to be test item-related and to be within the normal range of biological variation. The increased relative brain weight noted in females treated with 300 or 1000 mg act. ingr./kg bw/day is considered a secondary effect to the reduced body weight at necropsy, as the absolute brain weight does not differ between the control group animals and the animals treated with the test item.
Some statistically significant differences in organ weights compared to the control animals were not considered to be test item-related.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Treatment period: One of 10 female animals treated with 1000 mg act. ingr./kg bw/day revealed test item-related changes in form of a thickened mucosa in the cardia region of the stomach with multiple elevated foci with a diameter of 1 to 3 mm that were partly crater-like. Histopathological correlates were noted at microscopic examination.
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Treatment period: Histopathology was restricted to control/high dose group, and stomachs of low and intermediate dose groups. Test item-related local changes were noted in the stomach (non-glandular) of almost all animals treated with 1000 mg act. ingr./kg bw/day in form of squamous cell hyperplasia and hyperkeratosis (10 of 10 males, 9 of 10 females) and submucosal mixed inflammatory cell infiltrate (4 of 10 animals per each sex). No local changes were noted in the stomachs at 100 and 300 mg act. ingr./kg bw/day
Recovery period: The histomorphological examination did not reveal any findings related to the previous test item-treatment, in particular no abnormalities were noted in the stomach.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
AUDITORY EXAMINATIONS:
-Treatment and recovery period (recovery restricted to control/high dose, groups 1/4): There was no indication of any impairment to auditory acuity.

OESTRUS CYCLE:
-The oestrus cycle stages (proestrous, oestrous, metoestrous, dioestrous) of all main study and recovery females was determined by analysis of vaginal smears taken at necropsy at terminal sacrifice (test day 91) or recovery sacrifice (test day 119). All groups showed a normal variation regarding the frequency of the different oestrus cycle stages.
Details on results:
CLINICAL SIGNS AND MORTALITY
-Treatment and recovery period (recovery restricted to control/high dose, groups 1/4):
No test item-related changes in behaviour, external appearance, or consistency of faeces were observed for the male and female rats following repeated oral treatment with 100, 300, or 1000 mg act. ingr./kg bw/day for 90 days.
No signs of irritation were observed following the oral dosing, in particular no salivation or redness of the oral cavity.
None of the animals died prematurely, all animals survived until their scheduled sacrifice at terminal sacrifice or recovery sacrifice.
Detailed clinical observations in form of an assessment of external appearance, body posture, movement and coordination capabilities, as well as behaviour were performed for all animals pre- and post-dose on test day 1 (= TW 1), and always on the first day of the week in test weeks 2 to 13. The observations were made within approx. 2 hours after dosing.
All parameters of the detailed clinical observations of all animals scheduled for the control group or the treatment groups were in the normal range at pre-dose examination on test day 1.
-Treatment period:
None of the animals treated with 100, 300, or 1000 mg act. ingr./kg bw/day revealed any changes in external appearance, body posture, movement and coordination capabilities in test weeks 1 to 13. All male and female control animals revealed normal values for each parameter set examined throughout the treatment period.
The detailed clinical observations were discontinued for the animals scheduled for the recovery period after test week 13 as none of the animals had ever revealed any abnormalities during the treatment period.

BODY WEIGHT AND WEIGHT GAIN
-Treatment period:
No test-item related influence was noted on the body weight, the body weight gain and the body weight at autopsy for the male and female animals treated orally with 100 or 300 mg act. ingr./kg bw/day for 90 days in comparison to the control group during the treatment period.
The mean body weight of the female animals treated with 1000 mg act. ingr./kg bw/day was slightly but statistically significantly (at p ≤ 0.05 or p ≤ 0.01, day 43-50 and 64-90) decreased by up to 10.7% in comparison to the control group as of test day 43. Body weight gain appeared to be slightly lower in females dosed at 1000 mg/kg bw.
The body weight of the male high-dosed animals was also slightly reduced by up to 7.4% (on test day 78) in comparison to the control group, but none of the decreases attained statistical significance (at p ≤ 0.05 or p ≤ 0.01). Furthermore, body weight gain appeared to be slightly lower as well.
The body weight at autopsy of male and female animals treated with 1000 mg act. ingr./kg bw/day was reduced accordingly.
This effect is considered to be test item-related.
The low-dosed male animals revealed a marginal decrease of the body weight (by up to 5.8% in comparison to the control group on test day 85), whereas at the intermediate dose the body weight was always nearly identical to that of the control group.
Given these observations, the marginal decreases of the body weight noted for the male and female animals at the intermediate and/or low dose is not considered to be test item-related but to be within the range of normal biological variation.
- Recovery period (restricted to control and high dose, groups 1 and 4):
A recovery of the reduced body weight of the female animals was noted during the course of the recovery period as the difference to the control group (statistically significant on test days 97 (-8.7%; p ≤ 0.01) and 104 (-7.3%; p ≤ 0.05)) declined to be only -4.5% at study termination (not statistically significant).
The slight difference in body weight noted between the male high-dosed animals and the control animals at the end of the treatment period also declined during the recovery period such that the body weight of the previously high-dosed animals and the control animals was nearly identical at study termination.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
-Treatment and recovery period (recovery restricted to control/high dose, groups 1/4):
No test-item related influence was noted on the absolute and relative food consumption for the animals treated orally with 100, 300, or 1000 mg act. ingr./kg bw/day for 90 days in comparison to the control group throughout the treatment and recovery period.
For the male high-dosed animals, a slight but statistically significant (at p ≤ 0.05 or p ≤ 0.01) increase by up to 14.4% was noted for the relative food consumption compared to the control animals starting in test week 2 during the treatment period and lasting until test week 16 during the recovery period, however no significant changes were seen in the absolute food consumption. The changes seen for the relative food consumption are within the normal range of biological variation.
In test week 17 (end of recovery), when the previously high-dosed male animals revealed no evident difference in the body weight compared to the control animals, there was also no noteworthy difference in the food consumption between the two groups.
For the female animals treated with 1000 mg act. ingr./kg bw/day, a slight but statistically significant (at p ≤ 0.01) decrease in absolute food consumption of up to 14.6% was noted in test weeks 1 and 10 to 12. In test week 1, the relative food consumption was also slightly decreased (statistically significant at p ≤ 0.01), however in test weeks 10 to 12 no significant changes were noted for the relative food consumption. Accordingly, high-dosed female animals showed slightly lower body weights of up to 10.7% (statistically significant at p ≤ 0.01) in test weeks 10 to 12. In test week 1, no significant differences in bodyweight between female high-dosed and control animals were noted, however the decrease in absolute and relative food consumption is considered spontaneous and not test-item related due to its transient nature. Furthermore, all changes are within the normal range of biological variation.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study)
-Treatment period:
No test-item related influence was noted on the drinking water consumption for the male and female animals treated orally with 100 mg act. ingr./kg bw/day and for the male animals treated with 300 mg act. ingr./kg bw/day for 90 days in comparison to the control group throughout the treatment and recovery period.
The female animals treated with the intermediate dose of 300 mg act. ingr./kg bw/day revealed a slight increase of the drinking water consumption by up to 25.9% (in test week 13) compared to the control group being statistically significant (at p ≤ 0.05 or p ≤ 0.01) in test weeks 1 to 8, 11, and 13.
At the high dose of 1000 mg act. ingr./kg bw/day, both the male and the female animals revealed an increase of the drinking water consumption by up to 64.0% (in test week 4) for the male animals and by up to 60.6% (in test week 3) for the female animals in comparison to the control group as of test week 1. The effect was statistically significant (at p ≤ 0.05 or p ≤ 0.01) in test weeks 1 to 13 for the male and female animals and appeared to be slightly more pronounced in the female animals than in the male animals. Visual appraisal did not reveal any noteworthy water loss due to spilling.
The increase in the drinking water consumption noted for the females at the intermediate dose level and for the males and females at the high dose level is considered to be test item-related and regarded as an adverse finding. No increase in urinary volume was observed in animals of the intermediate and high dose groups, however, this parameter was determined only once at the end of the treatment period. Therefore, changes in urinary volumes as expected following an increase in drinking water consumption might have occurred during the study without being detected.
-Recovery period (restricted to control and high dose - groups 1 and 4):
No noteworthy difference was noted for the drinking water consumption of the male animals previously treated with the high dose in comparison to the control group during the recovery period.
The drinking water consumption of the female animals previously treated with the high dose was still slightly increased by 17.5% (statistically significant at p ≤ 0.05) compared to the control group in test week 14. Afterwards, there was no noteworthy difference between the previously high-dosed animals and the control group.

OPHTHALMOSCOPIC EXAMINATION
-Treatment and recovery period (recovery restricted to control/high dose, groups 1/4):
Ophthalmological examination did not reveal any changes of the eyes and the optic region in the animals treated orally with 100, 300, or 1000 mg act. ingr./kg bw/day for 90 days at the end of the treatment period.
No changes of the eyes and the optic region were noted for the animals previously treated with 1000 mg act. ingr./kg bw/day for 90 days at the end of the recovery period.

HAEMATOLOGY
-Treatment and recovery period (recovery restricted to control/high dose, groups 1/4):
No test item-related influence was observed on the haematological parameters for the male and female animals treated orally with 100, 300, or 1000 mg act. ingr./kg bw/day compared to the control animals at the end of the treatment period (test day 91, only the main study animals) and at the end of the recovery period (test day 119, groups 1 and 4 only).
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 prothrombin time (PT), the activated partial thromboplastin time (aPTT), the mean corpuscular volume (MCV), the mean corpuscular haemoglobin (MCH), and the mean corpuscular haemoglobin concentration (MCHC) at the end of the treatment period and at the end of the recovery period. All data were within the limits of normal biological variability.

CLINICAL CHEMISTRY
-Treatment and recovery period (recovery restricted to control/high dose, groups 1/4):
No test item-related influence was observed on the biochemical parameters for the male and female animals treated orally with 100, 300, or 1000 mg act. ingr./kg bw/day compared to the control animals at the end of the treatment period (test day 91, only the main study animals) and at the end of the recovery period (test day 119, groups 1 and 4 only).
No test item-related effects were noted on the plasma levels of albumin, bile acids, bilirubin, total cholesterol, HDL cholesterol, LDL cholesterol, creatinine, glucose, total protein, urea (in blood), calcium, chloride, potassium, and sodium at the end of the treatment period and at the end of the recovery period. 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 within the range of normal biological variability.
No test item-related influence was observed on the thyroid hormone levels for the male and female animals following daily oral treatment with 100, 300, or 1000 mg act. ingr./kg bw/day compared to the control animals at the end of the treatment period (test day 91, only the main study animals) and at the end of the recovery period (test day 119, groups 1 and 4 only).
No test item-related effects were noted on the serum levels of T3 (thyroid hormones triiodothyronine), T4 (thyroxine), TSH (thyroid-stimulating hormone). All data are considered to be within the range of normal biological variability.
The serum levels of T4 appeared to be decreased by 24.4% or 25.5% in the male animals treated with 300 or 1000 mg act. ingr./kg bw/day (statistically significant at p ≤ 0.01 for both groups) in comparison to the control animals at the end of the treatment period, respectively (test day 91, only the main study animals). The female animals revealed a decrease by 22.4% (not statistically significant at p ≤ 0.05 or p ≤ 0.01) at 300 mg act. ingr./kg bw/day but an increase by 8.7% at 1000 mg act. ingr./kg bw/day in comparison to the control (also not statistically significant).
The mode of toxicological action on the thyroid gland by various substances is quite similar, independently of the mechanism that leads to the reduction of the T4 level. Increased release of TSH from the pituitary gland is observed as a response to a decreased circulating T4 level. However, no influence was noted on the thyroid weight, and no histopathological findings were observed for the thyroids in animals treated with the test item. Therefore, a toxicological relevance of the decreased T4 serum levels cannot be confirmed for the test item due to the absence of any of the described changes expected to follow a toxicity-related decrease in T4 serum levels. Hence, these changes are considered coincidental alterations within the range of normal biological variability, as only 1 animal in the intermediate and high dose group each showed T4 serum levels outside the historical range.
It is generally known that a histopathological examination of the thyroid is usually more sensitive and thus more reliable in detecting thyroid-related toxic effects of a test item than thyroid hormone levels and thyroid weight. According to the validation report of OECD Guideline 407 (referenced in OECD Guideline 408) 'thyroid histopathology was consistently the most reliable and most sensitive endpoint for the detection of thyroid modulation. Thyroid weight 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 standardised to reduce stress induced variability and to reduce analytical variability, respectively. Circulating T4 levels were the most promising of the three thyroid hormonal values'.

URINALYSIS
-Treatment and recovery period (recovery restricted to control/high dose, groups 1/4):
Daily oral treatment with 100, 300, or 1000 mg act. ingr./kg bw/day did not lead to any test item-related changes of the urinary parameters in the male and female animals compared to the control group at the end of the treatment period (test day 91, only the main study animals) and at the end of the recovery period (test day 119, groups 1 and 4 only).
No test item-related changes were noted for the specific gravity, 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 a test item-related way in male and female animals. No test item-related changes were observed in the urine colour and the microscopically analysed urine sediments.
A large amount ('+++') of erythrocytes in the centrifugal deposits and an increased haemoglobin content (250 erythrocytes/µL) were observed in the urine of 4 of 10 male animals (nos. 71, 72, 75, and 77) treated with 1000 mg act. ingr./kg bw/day at the end of the treatment period. The colour of these animals' urine was brown (no. 71), brown-yellow (nos. 72 and 75), or red (no. 77). For animal no. 77, a small injury at one claw was noticed at the end of the urine collection, which might have been a source for blood contaminating the urine sample. However, during necropsy of this animal several findings were noted (i.e. urinary bladder filled with red liquid, dilated urether, and enlarged kidneys) that also might have been the cause for the erythrocytes found in the urine sample. For the kidney and urether, respective findings were observed at histopathological examination of these organs. For animal no. 71, a minimal dilation of the pelvis of one kidney was noted during histopathological examination, however no corresponding macroscopic changes were noted during necropsy for animal no. 71, and neither macroscopic nor microscopic changes were noted in animals no. 72 and 75. In addition, no paw injuries were observed in any of these animals. However, the urine was collected overnight without continuous observation of the animals. Any small injuries which may have occurred would have been licked clean by the animals by morning, so that these injuries would not have been detected by our staff. Hence, the source of the blood contamination in the urine samples of animals no. 71, 72 and 75 cannot be determined and there is no data to either support or disaprove a causal relationship with the test item treatment.
The same findings as noted for the 4 high-dosed animals were also observed for one male control animal (no. 10, urine colour: brown). For this animal, this finding has not to be considered incidental as no test item was administered to this animal.

NEUROBEHAVIOUR
The neurological screening was performed on all main study and recovery animals (groups 1 and 4: n = 15 per group per sex, groups 2 and 3: n = 10 per group and sex) at the end of treatment (in test week 13) 1 to 2 hours after dosing, and on all recovery animals (groups 1 and 4: n = 5 per group and sex) at the end of the recovery period (in test week 17).
-Treatment and recovery period (recovery restricted to control/high dose, groups 1/4):
No test item-related and/or statistically significant influence was noted on any of the parameters examined during the functional observation tests, on the fore- and hindlimb grip strength, or on the spontaneous motility for any of the male and female animals after repeated oral treatment with 100, 300, or 1000 mg act. ingr./kg bw/day in test week 13 and in test week 17. In female animals treated with either 100, 300 or 1000 mg act. ingr./kg bw/day, a decrease in spontaneous motility was noted compared to control animals. This difference was even more pronounced at the end of the recovery period for females previously treated with 1000 mg act. ingr./kg bw/day. However, during the daily observation of the animals no corresponding clinical signs were noted, and the neurological screening revealed no differences in behaviour between the treated and control animals. Therefore, these apparent effects observed in a single test are considered to be spontaneous and not related to any toxic effect of the test item.
For all animals, all findings observed (increased number of erythrocytes, increased haemoglobin content, urine colour) are not regarded to be test item-related effects. For animals no. 71, 72 and 75 as well as the single control animal, we consider these changes most likely to be due to small injuries at the animals' claws caused by the metal grid of the funnel cages that might not have been detected at necropsy. For animal no. 77, there are two possible causes for the findings. However, due to animal no. 77 being the only high dose animal to show macroscopic and microscopic changes in the related organs, we consider these to be spontaneous and not test item-related.

ORGAN WEIGHTS
-Treatment and recovery period (recovery restricted to control/high dose, groups 1/4):
No test-item related influence was noted on the relative and absolute organ weights of the male and female animals following repeated oral treatment with 100, 300 or 1000 mg act. ingr./kg bw/day in comparison to the control animals at the end of the treatment period (test day 91, only the main study animals) and at the end of the recovery period (test day 119, groups 1 and 4 only).
Any differences noted for the relative or absolute organ weights of the test item-treated animals at the end of the treatment period or at the end of the recovery period in comparison to the control animals are not considered to be test item-related and to be within the normal range of biological variation. The increased relative brain weight noted in females treated with 300 or 1000 mg act. ingr./kg bw/day is considered a secondary effect to the reduced body weight at necropsy, as the absolute brain weight does not differ between the control group animals and the animals treated with the test item.

GROSS PATHOLOGY
-Treatment period:
The macroscopic inspection at necropsy did not reveal any test item-related changes in the organs and tissues of the male and female animals repeatedly treated orally with 100 or 300 mg act. ingr./kg bw/day at terminal sacrifice at the end of the treatment period (test day 91).
One of 10 male high dose animals (no. 77) revealed enlarged kidneys, bilateral dilated urethers and a urinary bladder filled with red liquid that are not considered test item-related. The changes in the kidneys and urethers correlated with histopathological findings.
One of 10 female high dose animals (no. 87) revealed a thickened mucosa in the cardia region of the stomach with multiple elevated foci with a diameter of 1 to 3 mm that were partly crater-like. These changes are considered to be test item-related as histopathological correlates were noted at microscopic examination.
-Recovery period (restricted to control and high dose - groups 1 and 4):
No test item-related changes were noted for the male and female animals previously treated with 1000 mg act. ingr./kg bw/day at the end of the recovery period.
A few minor macroscopic findings were noted in various organs of individual animals at terminal sacrifice or recovery sacrifice. These changes are not regarded as test item-related but to be of spontaneous nature due to their isolated occurrences. In detail, these findings were:
Enlarged kidneys, increased lobular pattern of the liver, slightly enlarged spleen, enlarged thyroids, dilated urether, urinary bladder filled with red liquid, dilated uterus, or uterus filled with clear liquid.

HISTOPATHOLOGY: NON-NEOPLASTIC
The histological examination was restricted to the main study and recovery animals of group 1 (control) and group 4 (high dose) and was performed by Global Pathology Support, The Netherlands. In order to further assess test item-related stomach changes observed for both sexes in selected group 4 animals, further histopathological evaluation of the stomachs of group 2 and 3 (low/intermediate dose) was performed in both sexes.
Histopathology evaluation of group 4 revealed test item-related changes in the non-glandular part of the stomach (i.e. the forestomach) in almost all animals for squamous cell hyperplasia and hyperkeratosis (10 of 10 males and 9 of 10 females). Furthermore, submucosal mixed inflammatory cell infiltrate was observed in the non-glandular and the glandular part of the stomach (4 of 10 animals each per sex).
Macroscopic changes noted in form of a thickened mucosa in the cardiac region of the stomach with multiple elevations for one female animal (no. 87) treated with 1000 mg act. ingr./kg bw/day were associated with the microscopic findings of squamous cell hyperplasia and hyperkeratosis of the non-glandular stomach, and mixed inflammatory cell infiltrate in the submucosa (glandular and non-glandular part).
The histopathological evaluation of the stomachs of group 2 and 3 (low/intermediate dose) did not reveal any test item-related effects.
The animals from group 4 scheduled for the 28-day recovery did not show any test item-related findings at the end of the recovery period.

OTHER FINDINGS
AUDITORY EXAMINATIONS:
-Treatment and recovery period (recovery restricted to control/high dose, groups 1/4):
There was no indication of any impairment to auditory acuity.

OESTRUS CYCLE:
The oestrus cycle stages (proestrous, oestrous, metoestrous, dioestrous) of all main study and recovery females was determined by analysis of vaginal smears taken at necropsy at terminal sacrifice (test day 91) or recovery sacrifice (test day 119). All groups showed a normal variation regarding the frequency of the different oestrus cycle stages.

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
body weight and weight gain
water consumption and compound intake

Target system / organ toxicity

Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day (actual dose received)
System:
gastrointestinal tract
Organ:
stomach
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
no

Any other information on results incl. tables

Validation report: Validation of an analytical method for the determination of Butanedioic acid, 2 (or 3)-sulfo-, 4-[2-[(1-oxo-(C12 -C18 (even numbered) and C18 unsaturated)alkyl))amino]ethyl]esters disodium salts in test item formulations with subsequent HPLC-UV detection. LPT Study No. 36867.

The aim of this study was to validate a HPLC-UV method for the quantification of Butanedioic acid, 2(or 3)-sulfo-, 4-[2-[(1-oxo(C12-C18 (even numbered) and C18unsaturated)alkyl))amino]ethyl]esters, disodium salts (also referred to as 'act.ingr.') in tap water formulations.

The analytical method applied was validated by LPT with regard to the linearity of the calibration curve as well as accuracy, precision, stability, specificity and sensitivity. The validation results are summarised in the table below.

Table 1. Summary of validation results

Parameter

Method for the determination of Butanedioic acid, 2(or 3)-sulfo-, 4-[2-[(1-oxo(C12- C18 (even numbered) and C18 unsaturated)alkyl))amino]ethyl]esters, disodium salts in test item formulation samples.

Linearity

R2 = 0.9999 (concentration range: 100 to 1000μg/mL)

Accuracy

Inaccuracy = 0.64% (mean)

Precision

Imprecision

Imprecision ≤ 1.04% CV intra-day

Imprecision = 0.68% CV inter-day

Stability

No apparent degradation of processed samples after 24h storage in the autosampler.

No apparent degradation in tap water after 42 days of storage at -20°C.

 

Sensitivity

(calculated)

Lower Limit of Quantification (LLOQ) = 24.55μg/mL

Limit of Detection (LOD) = 8.10μg/mL

Specificity

No peak interferences at the retention time of Butanedioic acid, 2(or 3)-sulfo-, 4-[2 -[(1-oxo(C12-C18 (even numbered) and C18 unsaturated)alkyl))amino]ethyl]esters, disodium salts in formulation samples at the applied concentrations.

 

The results of the validation confirm that the method employed is suitable for the determination and quantification of Butanedioic acid, 2(or 3)-sulfo-, 4-[2-[(1-oxo(C12-C18 (even numbered) and C18 unsaturated)alkyl))amino]ethyl]esters, disodium salts in tap water formulations.

Applicant's summary and conclusion

Conclusions:
Under consideration of the observations described above, in particular the reduced body weight and the substantially increased water consumption, the experimental no-observed-adverse-effect level (NOAEL) was 300 mg act. ingr./kg bw by daily oral administration.
None of the animals previously treated with 1000 mg act. ingr./kg bw/day revealed any abnormalities related to the previous treatment during the recovery period, thus all toxicological relevant changes observed at the end of the treatment period were completely reversible within the 4-week recovery period.
Executive summary:

The aim of the study was to obtain information on the toxicity of Butanedioic acid, 2(or 3)-sulfo-, 4-[2-[(1-oxo(C12-C18 (even numbered) and C18 unsaturated)alkyl)) amino]ethyl]esters, disodium salts (= 'act. ingr.') at dose levels of 100, 300, or 1000 mg/kg bw given to rats by daily oral administration via gavage for 90 days, and the reversibility of any effects after a 4-week treatment-free recovery period.

 

Treatment period:

No deaths were noted at any dose level. All animals survived until their scheduled sacrifice.

None of the male and female rats treated orally with 100, 300, or 1000 mg act. ingr./kg bw/day revealed any test item-related changes in behaviour, external appearance, consistency of faeces, body posture, and movement and coordination capabilities

No test item-related effects were noted during the observational screening and the functional tests.

The mean body weight of the male and female animals treated with 1000 mg act. ingr./kg bw/day was slightly reduced by up to 10.7% in females and up to 7.4% in males in comparison to the control group.

The body weight gain and the body weight at autopsy of both sexes were reduced accordingly. This effect is considered to be test item-related.

No toxicologically relevant test item-related influence was noted on absolute and relative food consumption

Treatment with 300 mg act. ingr./kg bw/day led to an increase of the drinking water consumption of the female animals by up to 25.9% in comparison to the control group starting in test week 1. No effect was noted for the corresponding male animals. At the high dose of 1000 mg act. ingr./kg bw/day, both the male and the female animals revealed an increase of the drinking water consumption by up to 64.0% in comparison to the control group starting in test week 1. The increased drinking water consumption is considered a test item-related effect.

No test item-related changes were noted in haematological and coagulation parameters.

No test item-related changes were noted in clinical chemistry.

No test item-related or toxicologically relevant influence was noted on the serum levels of triiodothyronine (T3), thyroxine (T4), and thyroid-stimulating hormone (TSH).

No test item-related changes were noted in urinalysis.

No test item-related changes were noted in ophtalmological and auditory examination.

At necropsy, one of 10 female animals treated with 1000 mg act. ingr./kg bw/day revealed test item-related changes in form of a thickened mucosa in the cardia region of the stomach with multiple elevated foci with a diameter of 1 to 3 mm that were partly crater-like. Histopathological correlates were noted at microscopic examination.

No test item-related changes were noted in organ weights.

Histopathological examination restricted to control and high dose group, revealed test item-related local changes in the stomach (non-glandular) of almost all animals treated with 1000 mg act. ingr./kg bw/day in form of squamous cell hyperplasia and hyperkeratosis (10 of 10 males, 9 of 10 females) and submucosal mixed inflammatory cell infiltrate (4 of 10 animals per each sex).

Histopathological stomach examination of low and intermediate dose groups revealed no local changes in the stomachs at 100 and 300 mg act. ingr./kg bw/day.

Recovery period:

None of the animals previously treated with 1000 mg act. ingr./kg bw/day revealed any abnormalities related to the previous treatment during the 4-week recovery period.

The body weight of the male and female animals recovered towards the normal range, no noteworthy difference compared to the control group was noted at the end of the recovery period.

The drinking water intake of all previously high-dosed animals was in the normal range during the recovery period.

The histomorphological examination did not reveal any findings related to the previous test item-treatment, in particular no abnormalities were noted in the stomach.

Under consideration of the observations described above, in particular the reduced body weight and the substantially increased water consumption, the experimental NOAEL was 300 mg act. ingr./kg bw by daily oral administration.

None of the animals previously treated with 1000 mg/kg bw/day revealed any abnormalities related to the previous treatment during the recovery period, thus all toxicological relevant changes observed at the end of the treatment period were completely reversible within the 4 -week recovery period.