6-[(1-oxomethyloctyl)amino]hexanoic acid

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

From 25 OCT 2011 to 18 JUL 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study in compliance with GLP and reported with a valid GLP certificate.

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
[Please provide information for all of the points below. Indicate if further information is included as attachment to the same record, or elsewhere in the dataset (insert links in 'Cross-reference' table)]

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
[Describe why the read-across can be performed (e.g. common functional group(s), common precursor(s)/breakdown product(s) or common mechanism(s) of action]

It is hypothesized that the target chemical and the following chemicals as source chemicals should exhibit comparable toxicity profiles:
-6-(isononanoylamino)hexanoic acid, compound with 2,2`,2``-nitrilotriethanol
-3,5,5-trimethylhexanoic acid
It is proposed to use the toxicity data of the mentioned source chemicals to fulfill the data requirement for the target chemical. The first chemical is a salt of the target chemical. 3,5,5-trimethylhexanoic acid is a presumed metabolite.

The underlying scientific rationale for the use of corresponding salt as source chemical is apparent. The target chemical is a weak acid due to the terminal carboxylic acid moiety and can be neutralized/dissolved in aqueous system by reaction with base such as 2,2`,2``-nitrilotriethanol. The proposed source chemical can be formally described as carboxylate of the target chemical. As the carboxylate and carboxylic acid are inter-convertible, it is apparent that source and target chemicals are inter-convertible and should exhibit comparable toxicity profile. The base 2,2`,2``-nitrilotriethanol is a well-investigated substance and is considered to be less relevant for the proposed read-across consideration.

The underlying scientific rationale for the use of 3,5,5-trimethylhexanoic acid as source chemical is based on the metabolism consideration. Upon resorption, the target chemical is expected to undergo a degradation process, resulting in the systemic release of 3,5,5-trimethylhexanoic acid, thereby providing the justification for the read-across especially for the mid- and long term toxicities such as repeated dose toxicity and reproduction toxicity.

The proposed approach applies for all exposure routes (oral/dermal/inhalation), because both the target chemical and source chemicals are expected to be bioavailable by all exposure routes: the inter-conversion between carboxylic acid and carboxylate is likely to occur prior to resorption; and the systemic release of the presumed metabolite is less dependent on exposure route.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
[Provide here, if relevant, additional information to that included in the Test material section of the source and target records]

Target chemical:
6-(Isononanoylamino)hexanoic acid; CAS: 71902-23-3
Source chemicals:
6-(isononanoylamino)hexanoic acid, compound with 2,2`,2``-nitrilotriethanol; CAS: 85702-79-0
3,5,5´-trimethylhexanoic acid; CAS: 3302-10-1

The target chemical is a mono-substituent substance, the analytical purity being >99%. The source chemicals are either the raw material (3,5,5´-trimethylhexanoic acid) or further chemically processed products (dissolved in water with 2,2`,2``-nitrilotriethanol) of the target chemical. A toxicity difference due to different impurity profiles is not likely to occur.

3. ANALOGUE APPROACH JUSTIFICATION
[Summarise here based on available experimental data how these results verify that the read-across is justified]

Justification for the use of 6-(isononanoylamino)hexanoic acid, compound with 2,2`,2``-nitrilotriethanol as source chemical:

- The given source chemical is an ionic compound that results from the neutralization reaction of the target chemical and 2,2`,2``-nitrilotriethanol. When it is dissolved in an aqueous system or in a biological fluid, an immediate dissociation occurs to give the target chemical and the base, thereby explaining the expected comparable toxicity profile to that of target chemical.
A significant toxicity contribution of 2,2`,2``-nitrilotriethanol is not expected. 2,2`,2``-nitrilotriethanol is a well investigated substance. It is of low toxicity and the available kinetic data are demonstrative of efficient elimination mechanisms in animal models.
- In order to verify the expected toxicity comparability, the given source and the target chemicals were investigated under identical testing conditions. Both substances exhibited comparable findings after 7-day oral application to rat:
-liver and kidney enlargement
-decrease of eosinophil counts
-peroxisome proliferation in the liver
The decrease of eosinophil counts is possibly a transient effect, associated with the peroxisome proliferation stimulating effect of test compounds. No such findings were present after 28-day treatment of the source chemical.
- Comparable findings were obtained in the 28-day oral toxicity study for the given source chemical and in the above mentioned two 7-day repeated oral toxicity studies. Further special histopathological investigation revealed the alpha-2µ-globulin accumulation in male kidneys and the peroxisome proliferation in liver.
- In the available skin sensitization data the given source chemical was applied using water as vehicle. The dissociation into the target chemical and 2,2`,2``-nitrilotriethanol is expected to have occurred prior to resorption, so that the animals must have been exposed to the target chemical.

Justification for the use of 3,5,5-trimethylhexanoic acid as source chemical:

- 3,5,5-trimethylhexanoic acid is the presumed metabolite. It is also the presumed metabolite of 6-(isononanoylamino)hexanoic acid, compound with 2,2`,2``-nitrilotriethanol.
This view is based on the results of the metabolites investigation in degradation samples obtained in a Zahn-Wellens test. The ß-oxidation at the terminal carboxylic acid moiety in combination with hydrolysis at amide bond could be assumed as the degradation pathway leading to 3,5,5-trimethylhexanoic acid as a stable metabolite.
Also the hydrolysis at the amide moiety is thinkable. The hydrolysis products would be then 3,5,5-trimethylhexanoic acid and 6-aminocaproic acid. The latter compound is a drug known as amicar and is expected to be far more rapidly eliminated than 3,5,5-trimethylhexanoic acid. A significant toxicity attribution of 6-aminocaproic acid cannot be derived.
One literature article was found, describing further biotransformation of 3,5,5-trimethylhexanoic acid in rat: gamma-lactone of 3,5,5-trimethylhexanoic acid is formed, which may be the ultimate toxicant for the alpha-2µ-globulin accumulation in kidneys
- The findings in the 28-day oral toxicity study on the 3,5,5-trimethylhexanoic acid are comparable to those found in the repeated dose toxicity studies of the target chemical and 6-(isononanoylamino)hexanoic acid, compound with 2,2`,2``-nitrilotriethanol:
- liver and kidney enlargement
- peroxisome proliferation in the liver
- The alpha-2µ-globulin accumulation in kidneys.


4. DATA MATRIX

Data matrix and other information see the attached read-across justification in chapter 13

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

tris(2-hydroxyethyl)ammonium 6-(3,5,5-trimethylhexanamido)hexanoate

EC Number:

701-138-0

Molecular formula:

C21H46N2O6

IUPAC Name:

tris(2-hydroxyethyl)ammonium 6-(3,5,5-trimethylhexanamido)hexanoate

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

Species and strain: Wistar Crl:WI (Han)
Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sulzfeld.
Hygienic level: SPF at arrival; standard laboratory conditions during the study
Justification of species/strain: Wistar rat as a rodent is one of the standard strains for repeat-dose toxicity studies
Number of animals: 30 male and 30 female rats (5 rats/sex/group in the four Main groups 1, 2, 3 and 4 and 5 rats/sex/group in two Recovery groups 1 and 4); Spare animals were assigned to CiToxLAB Ltd. Spare colony at the end of the study.
Age of animals: Young adult rats, 7 weeks old at starting.
Body weight: 162-185 g, males and 134-154g, females at onset of treatment
Acclimation period: 5 days
Animal health: Only healthy animals were used for the test, as certified by the veterinarian. Females were nulliparous and non-pregnant.
Room number: 241
Cage type: Type III polycarbonate
Bedding: Lignocel® Hygienic Animal Bedding produced by J. Rettenmaier & Söhne GmbH+Co.KG (Holzmühle 1, D-73494 Rosenberg, Germany),
Light: 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Temperature: 20.1-23.5 °C
Relative humidity: 39 - 61 %
Ventilation: 15-20 air exchanges/hour
Housing/Enrichment: Rodents were housed by 5 animals of the same sex and group/cage. Group housing allows social interaction and the deep wood sawdust bedding allows digging and other normal rodent activities.
The temperature and relative humidity in the animal room were recorded twice daily during the study.
Diet and water supply: The animals were provided with ssniff® SM R/M-Z+H “Autoclavable Complete Feed for Rats and Mice – Breeding and Maintenance” (Ssniff Spezialdiäten GmbH, D-59494 Soest Germany) and tap water as for human consumption, ad libitum.
The quality control analysis of the water is performed once every three months and microbiological assessment is performed monthly, by Veszprém County Institute of State Public Health and Medical Officer Service (ÁNTSZ, H-8201 Veszprém, József A.u.36., Hungary). Copies of the relevant Certificates of Analysis are retained in the archive at CiToxLAB Ltd. The diet and drinking water are routinely analysed and are considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.
Animal identification: Each animal was identified by a number unique within the study, written with indelible ink on the tail and cross-referenced to the Animal Master File at CiToxLAB Hungary Ltd. The cages were identified by cards holding information about study code, sex, dose group, cage number and individual animal numbers.
Randomization: During the acclimation period, the animals were assigned to their respective dose groups by randomization based on body weights. Animals were randomly allocated to the control and dose groups based on the most recent actual body weight; SPSS/PC+ software was used in order to verify homogeneity/variation among/within groups. Males and females were randomized separately.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

distilled

Details on oral exposure:

The test item was formulated in the vehicle at the appropriate concentrations according to the dose level and volume selected, in the Central Dispensary of CiToxLAB Hungary Ltd. Formulations were prepared fresh prior to administration to animals (Days 0 and 1) or at the appropriate frequency to allow their use according to stability assessment results, within 7 days while stored refrigerated.

Vehicle: Distilled water
Name: Humaqua, Distilled water for parenteral use
Lot/Batch No.: 2190511
Manufacturer: Teva Pharmaceutical Works.
Expiry Date: May 2014
Storage: Room temperature

The dosing solutions were administered daily starting from Day 0 for 28 consecutive days by oral gavage, using a bulb tipped gastric feeding tube
attached to a syringe. The dosing solutions were administered to all animals at a constant volume of 5 mL/kg. The actual volume to be administered was calculated and adjusted based on each animal’s most recent body weight.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Stability of the test item in the vehicle was assessed in the conditions employed on the study by the CiToxLAB Analytical laboratory, according to “Validation of the Analytical Method for the Determination of the test item.”.(CiToxLAB study code 11/100-316AN). Based on the results, the test item solution in distilled water is stable at concentration range of 10-200 mg/mL when stored 1 day at room temperature or 7 days refrigerated (at 2-8°C). The investigation was extended to 4 mg/mL.

Analysis of test item formulations for concentration and homogeneity, using validated spectrophotometric method, was performed in the Analytical Laboratory of CiToxLAB Hungary Ltd. Top, middle and bottom duplicate samples were taken from test item formulations at twice during the treatment period (during the first and last weeks of treatment). Similarly, one sample was taken in duplicate from the Group 1 (control) solution for concentration measurements.
The samples were diluted into the calibrated range with water and analyzed using UV spectrophotometry. Absorbance of the samples was measured in the reference cell at 200 nm wavelength. All formulations were found to be in the range of 97 to 110% of nominal concentration. All formulations were shown to be homogeneous. No absorbance was measured for the control samples.

Principle of the Analytical Method: Concentration of the test item in the test solutions was determined. The samples were analysed by an UV spectrophotometric method.
Apparatus
UV-VIS Spectrophotometer: U 2910 HITACHI, No.:2221-004
Balance: BP221S Sartorius, No.: 11809117
Ultrasonic bath: Elmasonic S300H, ELMA, No.: 010890105
Water purification system: MILLIPORE, DIRECT Q3, FOMNO 7334I
Refrigerator: Zanussi, No.: ZLKI-262

Duration of treatment / exposure:

28 days

Frequency of treatment:

Daily

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 20, 100, 500 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

30 male and 30 female rats (5 rats/sex/group in the four Main groups 1, 2, 3 and 4 and 5 rats/sex/group in two Recovery groups (control and high dose animals))

Control animals:

yes, concurrent vehicle

Details on study design:

Rationale for dose-selection and route of administration: The dose levels were set by the Sponsor in consultation with the Study Director, based on available data and information from previous experimental work, including the results of a preliminary dose range finding study conducted at CiToxLAB Hungary Ltd. with the test item [CiToxLAB study code 11/100-100PE: “A Dose Range Finding Toxicity Study following Oral (Gavage) Administration in Wistar Rats”].
The oral route is a possible route of exposure to the test item in humans.

- Post-exposure recovery period in satellite groups: 14 days

Positive control:

Postive control not utilised in this study.

Examinations

Observations and examinations performed and frequency:

Clinical observations and neurological assessment: Animals were inspected for signs of morbidity and mortality twice daily (at the beginning and end of each day).
General clinical observations were made daily, after treatment at approximately the same time.
Detailed clinical observations were made on all animals outside the home cage in a standard arena before the first treatment and once weekly thereafter, in the morning hours (am).
Detailed clinical observations were made on all animals outside the home cage in a standard arena once, prior to the first exposure and once weekly thereafter. Observation was performed on the skin, fur, eyes and mucous membranes, autonomic activity (lachrymation, piloerection, pupil size, respiratory pattern, occurrence of secretions and excretions), circulatory and central nervous system, somatomotor activity and behaviour pattern, changes in gait, posture and response to handling. Special attention was directed towards the observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma. No such clinical signs were noted during the study.
During the last week of treatment (Day 27), each animal was subjected to the functional observation battery, including qualitative assessment of the grip strength, and to measurements of the landing foot splay and fore/hind grip strength.
To measure the landing foot splay, the hind paws of the rat were painted with ink and the rat was dropped from a horizontal position onto the appropriate record sheet covering the examination table. The distance between the two resulting ink spots was measured.
Fore/hind grip strength measurements were conducted using a grip strength meter (Model GS3, Bioseb, Chaville, France), an instrument designed to quantify objectively rodent muscular strength, in order to identify and assess quantitatively any potential effect of test item. The rats were held appropriately such that the fore limbs are allowed to grip the support bar and gently pulled back until they release the bar; the device measures the maximum grip strength. This was performed at least 3 times for each animal on each test day. The procedure was repeated with the hind limbs with the appropriate grip support.
Sensory reactivity to different type of stimuli (e.g. auditory, visual and proprioceptive), assessment of grip strength and motor activity were conducted and the general physical condition and behaviour of animals were tested. A modified Irwin test was performed. The parameters evaluated included the following: body position, locomotor activity, respiration rate, respiration type, piloerection, head searching compulsive biting or licking, circling, upright walking, retropulsion, jumping, exophthalmos, twitches, clonic convulsions, tonic convulsions, tremor, startle, transfer arousal, spatial locomotion, gait, posture, limb position, finger approach, finger withdrawal, touch escape response, diarrhoea, diuresis, visual placing, grip strength, body tone, corneal reflex, pinna, toe pinch, grasping reflex, positional struggle, skin, mucous membrane colour, salivation, palpebral closure, lachrymation, limb tone, abdominal tone, tail pinch, righting reflex, and/or vocalisation.

Body weight measurements: Individual body weight of each animal was recorded with precision of 1g at randomization, on Day 0 and once weekly during the treatment and recovery periods, including before necropsy (fasted).

Food consumption measurements: Animal food consumption was determined by re-weighing the non-consumed diet with a precision of 1 g on Day 7 then at least weekly. The weekly and mean daily food consumption was calculated.

CLINICAL PATHOLOGY: Blood samples for clinical pathology evaluation (haematology, coagulation, and clinical biochemistry) were collected immediately prior to the scheduled necropsy, by heart puncture under pentobarbital anaesthesia (on Day 28, from the Main animals, and on Day 42 from the Recovery animals).
After an overnight period of food deprivation of animals, 3 blood samples were collected, for haematology (approximately 1.2 mL blood in tubes with K3-EDTA as anticoagulant, 1.6 mg/mL blood), for blood clotting times (approximately 1.4 mL blood for APTT and PT measurements, in tubes with sodium citrate as anticoagulant) and one to obtain serum (approximately 1 mL blood as practical in tubes with no anticoagulant) for clinical chemistry.

Haematology and blood clotting times: The following parameters were evaluated in all surviving animals: Reported in table form – detailed under Any other information.

Clinical chemistry: The following parameters were evaluated in all surviving animals: Reported in table form – detailed under Any other information.

Urinalysis: Urinalysis was performed during the last week of the study. Urine samples were collected for 16 hours during an overnight period of food deprivation. Urine samples were collected from all animals in the main and recovery groups, and animals were placed in metabolic cages during collection. The evaluation of the urine samples was performed by observation (e.g. appearance, color) or test strips as applicable. The following parameters were evaluated: Reported in table form – detailed under Any other information.

Sacrifice and pathology:

Terminal procedures and macroscopic evaluation: Gross necropsy was performed on each animal. Terminally on Day 28 (Main animals) or 42 (Recovery animals), surviving animals were euthanised under pentobarbital anaesthesia by exsanguination.
After exsanguination the external appearance was examined, cranial, thoracic and abdominal cavities were opened and the appearance of the tissues and organs were observed macroscopically. Any abnormality was recorded with details of the location, colour, shape and size, as appropriate.

Organ weight measurements: The following organs were weighed in surviving animals: Reported in table form – detailed under any other information.

Tissue preservation and microscopic evaluation: On completion of the macroscopic examination the following tissues and organs were retained from all animals: Reported in table form – detailed under Any other information.
The eyes with the optic nerves and testes with epididymides were retained in modified Davidson’s fixative, all other organs in 10% buffered formalin solution.
Additional liver samples (approximately 0.5-1 g) were taken from all animals in the Main and Recovery Groups for evaluation of peroxisome proliferation. The samples were snap frozen in liquid nitrogen and stored frozen in polypropylene bags in a deep freezer set at -70 to -90 C until further investigation (at additional cost; to be documented and reported).
Full histopathology was performed in all Groups 1 (Control) and 4 (High dose) animals and macroscopic finding of one animal from Group 3 (no. 3004, kidneys). Examination of tissues from Groups 2 (Low dose) and/or 3 (Mid dose) as well as recovery groups was limited to liver of all animals and kidneys of males. The retained tissues and organs were embedded in paraffin wax, sections were cut at 4-6μ by microtome and transferred to slides. Tissue sections were stained with
haematoxylin-eosin/phloxine and examined by light microscope.
Additional slides were prepared from male kidneys (three in the High dose and three in Control Group) for investigation on hyaline content. The following samples were investigated:
High Dose Group animal No.: 4002, 4004, 4005
Control Group animal No.: 1001, 1002, 1005
The respective paraffin blocks were sent to Principal Investigator for slide preparation and staining. The blocks were sent by courier at room temperature to the test site, to the attention of the Principal Investigator (PI).
The sample sending was coordinated by the Study Pathologist.
Blocks were sectioned at a thickness of approximately 4 microns and stained with Chromotrope-Aniline-Blue-staining for detection of hyaline droplets.
The staining was performed according to the valid CiToxLab France Standard Operating Procedure No. INST HIST_0520. Briefly, the following steps were performed:
a. deparaffinize and hydrate
b. Stain in Weigert’s hematoxylin, 2 minutes
c. Rinse in water
d. Stain in phosphomolybdic acid, 2 minutes
e. Rinse in water
f. Stain in CAB solution, 2-4 minutes
g. Rinse in running water
h. Dehydrate, clear and mount
Ready slides were sent to the Study Pathologist for quality assessment and reading.

Other examinations:

Examination of vaginal smears: Prior to necropsy, the oestrus cycle of all females was determined by taking vaginal smears, which were prepared and stained with 1% aqueous methylene blue solution. The smear was examined with a light microscope, in order to provide information regarding the stage of oestrus cycle at the time of sacrifice and assist in histological evaluation of oestrogen sensitive tissues.

Additional liver tissue samples of approximately 1-2 g were taken from the left lateral lobe of the liver, weighed, flash frozen under liquid nitrogen immediately after collection and stored frozen for future evaluation of peroxisome proliferation. The frozen liver chunks from control animals and high dose animals were processed to heavy pellets (crude mitochondrial pellet). The heavy pellet samples prepared from these livers were assayed for total protein content and the activity of CN- insensitive palmitoyl CoA oxidation (a marker of peroxisomal beta-oxidation).

Subcellular Fractionation
Liver samples were scissor-minced in ice-cold 1.15% (w/v) KCl prior to homogenisation in SET (250 mM sucrose, 5 mM EDTA, 50 mM tris-HC1, pH 7.4) buffer. Heavy pellet fractions were prepared according to Laboratory Method Sheet (LMS) Cent-001 and stored at approximately —70 °C until analysis.

Biochemical Assays
- Peroxisome proliferation: CN-insensitive paimitoyl CoA oxidation was determined spectrophotometrically in liver heavy pellet, using palmitoyl CoA as a substrate, according to LMS Spec003.
- Protein assay: The heavy pellet protein concentrations were determined in aqueous solutions using a modification of the method of Lowry et al (1951) and bovine serum albumin standards, according to LMS Spec001.

Statistics:

Data were collected using the software PROVANTIS v.7 or were recorded on the appropriate forms from the relevant SOPs of CiToxLAB Hungary Ltd., then tabulated using the Microsoft Office Word and/or Excel, as appropriate.
Numerical data obtained during the conduct of the study were subjected as appropriate to calculation of group means and standard deviations.
The statistical analysis was performed using SPSS PC+4.0 software. The heterogeneity of variance between groups was checked by Bartlett’s homogeneity of variance test. Where no significant heterogeneity is detected, a one-way analysis of variance was carried out. If the obtained result is positive, Duncan’s Multiple Range test was used to assess the significance of inter-group differences. Where significant heterogeneity is found, the normal distribution of data was examined by Kolmogorov-Smirnov test. If the data is not normal distributed, the non-parametric method of Kruskal-Wallis One-Way analysis of variance was used. If there is a positive result, the inter-group comparisons were performed using Mann-Whitney U-test.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

effects were observed in the 500 mg/kg bw/day (High dose) groups.

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

minimal periportal hepatocellular vacuolation of the liver in 3/5 males and 4/5 females of the high dose group (effect reversible within the 14 day recovery period); tubular hyaline droplets in kidney of 3/3 male high dose animals

Histopathological findings: neoplastic:

no effects observed

Details on results:

DOSE FORMULATION ANALYSIS: Test item content and homogeneity of the dosing formulations was determined twice during the treatment period (during the first and the last week of the treatment). No test item was detected in the Control solution samples as no absorbance was measured for these samples. All formulations were shown to be homogeneous. All formulations were found to be in the range of 97 to 110% of nominal concentrations. These results were considered suitable for the study purposes.

MORTALITY: There was no mortality observed during the study.

CLINICAL OBSERVATIONS: No clinical signs were observed during the study. All animals were symptom free both after a 28-day daily treatment at 20, 100 and 500 mg/kg bw/day, and during the 14 -day observation period. Occasionally slight to moderate noisy respiration was noted at 500 mg/kg (High Dose) treated females: between Days 4-7 in two female animals (no. 4505 and 4510) and on Days 14-15 in female no. 4509. This observation as considered as incidental and not test item related.

NEUROLOGICAL ASSESSMENT: There were no toxicologically significant changes in the animal behaviour, general physical condition, in the reactions to different type of stimuli, grip strength or motor activity in the control or treated groups.
Compared to the Control, no test item-related differences, or dose, or gender related response, were noted in the animals throughout all the dose groups when subjected to the modified Irwin test (functional observation battery). The behaviour of animals and their reactions to different type of stimuli or manipulations were normal.
No test item related effects or statistically significant variations were observed in the landing foot splay test. When compared to the Control, there were no toxicologically significant differences in the mean grip strength values of the forelimbs or hind limbs in the dosed animals. Slightly higher mean values (which attained statistical significance) of the fore limbs grip strength was noted in all treated males and in females for hind limbs at 20 and 500 mg/kg bw/day dose levels. However, it was considered to be biological variation without toxicological significance.

BODY WEIGHT MEASUREMENTS: There were no toxicologically significant or adverse effects observed on the animal body weights during the study. The mean body weight gains of all groups were comparable to the control means throughout the study. The mean body weight gain of the male recovery Group 4 was lower than control between Days 21 and 28 (p<0.05); however, the difference was minor and limited to one occasion only. The difference was not correlated with any changes in animal food consumption. Therefore the observed difference was considered incidental and ascribed to biological variability and not to test item administration.

FOOD CONSUMPTION: There were no adverse effects on the animal food consumption or differences between control and test item treated groups, ascribed to test item administration.

CLINICAL PATHOLOGY

Hematology: When compared to the controls, there were no differences that were considered toxicologically significant noted in the treated animals.
Variations were noted in a few parameters, on occasion attaining statistical significance during hematology evaluation following the end of the treatment period and were: slightly lower than control reticulocyte counts in the 20 and 500 mg/kg bw/day males (statistical significance for relative counts and High Dose absolute value), higher relative eosinophils counts in the 20 mg/kg bw/day dosed females (Low Dose Group). Following the 14-day recovery period slightly lower hematocrit values and hemoglobin concentration was measured at 500 mg/kg bw/day treated males.
Evaluation of the mean and individual results in comparison with the control data did not reveal any test-item related cause of the changes noted, and/or no consistent dose or gender-related response was observed. Therefore, these differences observed between the Control and treated groups were considered to be incidental or individual findings, which were not related to treatment, were generally comparable with the expected physiological range or were with no toxicological significance.

Blood clotting potential: The blood clotting parameters (Prothrombin Time and Activated Partial Thromboplastin Time) were comparable with the control values.

Clinical chemistry: When compared to the controls, following 28 days of treatment, there were no adverse effects that could be ascribed to the test item administration under the conditions of this study.
Glucose level and urea concentration were slightly decreased in males at 500 mg/kg bw/day (p<0.01), by approximately 20-30%. Slightly elevated urea concentration was recorded in females at 500 mg/kg bw/day with individual value of one animal (no. 4503) above the normal range. In this female blood glucose level and chloride concentration was well beneath the control values.
Although a test item related effect cannot be excluded, the above changes in clinical chemistry parameters were slight in magnitude and the values were within or close to the normal range.
Other parameters showed variations, however, no consistent dose or gender response was noted, the differences remained minor and were not considered toxicologically significant or related to the treatment.

Urinalysis: No test item-related effects were noted on the urinalysis parameters evaluated.
Variations occurred and consisted of minor differences to controls, unrelated to treatment and within the normal range, including slightly higher volume of collected urine and pH value in the 500 mg/kg bw/day males following the recovery period. Crystals, bacteria, epithelial, red and/or white blood cells were observed microscopically at sediment analysis in most of the animals, unrelated to treatment

TERMINAL OESTRUS CYCLE EVALUATION: Evaluation of the vaginal smears prior to necropsy both on Days 28 or 42 showed the expected distribution of the oestrus cycle phases within the normal population of female Wistar rats.

PATHOLOGY EVALUATION AND ORGAN WEIGHTS

Macroscopic evaluation: No test item related findings were noted at necropsy of the main animals on Day 28. Occasionally observed pelvic dilatation in the kidneys (males no. 3004 Mid dose and no. 4002 High dose males), dilated uterine horns (no. 4501, 4505 in High dose group) were regarded as incidental, related to the oestrus cycle or background findings.
At the end of the 14-day recovery period, no test item-related macroscopic findings were noted at necropsy examination. Bilaterally enlarged adrenals (female no. 4508, High dose), unilateral pelvic dilatation in the kidneys (male no. 4008, High dose) and dilated uterine horns (no. 4506, High dose) were regarded as incidental observations.

Organ weights: Compared to the control, the following effects were observed at 500 mg/kg
bw/day (High dose) groups:
The mean absolute and relative weights of liver were higher in males and females.
The weights of kidneys were higher in males.
Uterus weight was higher in females. However, higher uterus weights were also recorded at 20 mg/kg bw/day (Low dose).
At 500 mg/kg bw/day mean absolute liver weights were approximately 11% higher for males and 31 % higher for females, compared to controls. The relative liver weights adjusted for the brain or body weight were approximately 11-13% higher in the males and 25-32% higher in the females.
The increased liver weights were consistent with microscopic findings.
Following the recovery period, the mean liver weights were comparable with the control.
At 500 mg/kg bw/day the absolute and relative weights of kidneys were approximately 25% higher in males recorded at the end of the treatment period. All values attained statistical significance (p<0.01). Following the recovery period the kidney weights were still slightly increased, were approximately by 15% higher than control means and the differences attained statistical significance (p<0.05).
In females, no significant differences in the kidney weights were recorded at 500 mg/kg. The absolute and relative kidney weights were approximately 4-8% higher in all treated groups, compared to control. Following the recovery period, the weight of kidneys was comparable with the control.
Higher uterus weights were recorded at 20 and 500 mg/kg bw/day (Low and High Dose). The absolute weights were up to 78% higher than controls, and attained statistical significance (p<0.05). The relative weights adjusted for the brain or body weights were up to 32% higher in the Low Group (p<0.05) and up to 79% higher in the High Group (p<0.05). At 500 mg/kg bw/day, the higher uterus weights were attributable to high individual values of 3 females (no. 4501, 4509 and 4505) correlated with proliferative changes in glandular part, which were correlated to the oestrus cycle.
Following the 14-day recovery period the absolute and relative uterus weights were up to 9% higher, without statistical significance.
Other variations were noted in the absolute and/or relative organs weights (e.g. decreased heart weight at 500 mg/kg males, end of the treatment period) but were considered unrelated to treatment.
As these changes were within the historical range, had low magnitude, showed no consistent response between genders, and/or were not correlated with pathological findings, they were considered incidental and not related to treatment.
All other examined organ weights (absolute and relative to the body and brain weights) were similar in the control and test item treated groups.

Microscopic evaluation: Test-item related microscopic findings were observed in the kidney and liver at a dose level of 500 mg/kg bw.

Findings of Day 28: In the kidneys stained with Chromotrope-Aniline-Blue-method, tubular hyaline droplets were positively stained in 3 of 3 examined males from the High Dose Group. No similar findings were detected in the control kidneys.
In the kidneys from the High Dose group stained by the standard Haematoxylin-Eosin/phloxine, eosinophilic droplets were additionally diagnosed as hyaline droplets after taking into account immunohistological appearance of the representative samples from the High Dose males. The droplets were seen as having similar histomorphological features and distribution. Hyaline droplets were present with minimal (3 of 5) or mild (2 of 5) severity in the tubular cells of all High Dose males, correlated with organ weight. During histopathological examination of the males from the Mid, Low Dose and Control groups, no hyaline droplets were identified.
Minimal periportal hepatocellular vacuolation of the liver was observed in 3 of 5 High Dose males and 4 of 5 High Dose females and also correlated with organ weight changes. This test item-related hepatic change was not observed in Mid or Low dose rats by additional microscopic evaluation.

Recovery period (Day 42): After the 14 days recovery period, there was no evidence of droplets of the type seen with hyaline in the tubular cells. However, signs of regeneration process, minimal tubular regeneration were present in 4 of 5 recovery High Dose males. Tubular regeneration was characterized by increased basophilia, flat/low cuboidal to columnar cells, apoptosis, sporadic mitosis and peritubular mononuclear infiltration.
In the liver, no vacuolation was seen neither in the male, nor in female High Dose animals on Day 42.

Findings unrelated to treatment: Various microscopic findings were recorded, including minimal focal congestion/haemorrhages in the thymus, minimal multifocal mononuclear cell infiltrate in the liver and lungs pelvic dilatation in the kidneys, luminal dilatation and glandular proliferation in the uterine horns (in correlation with necropsy). Based on the low incidence and/or severity as well as distribution in the control and dosed animals, these observations were regarded as incidental or physiological regarded to the oestrous cycle, not toxicologically significant or associated with test-item administration.

OTHER FINDINGS
Administration of test material (only high dose) resulted in statistically significant increases in hepatic peroxisomal beta- oxidation of 1.9 and 1.8 fold in male and female rats respectively. For further details please see "Overall remarks"

Effect levels

Dose descriptor:

NOAEL

Effect level:

500 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

The analytical results are summarized in the table below:

Analytical occasion	Nominal concentration (mg/mL)	Measured concentrations with the 95% confidence intervals (mg/mL)	Measured concentrations in percentage of the nominal
27 October 2011 (First week)	4	3.87 +/- 0.02	97%
	20	19.6 +/- 0.03	98%
	100	100 +/- 3.4	100%
17 November 2011 (last week)	4	3.95 +/- 0.05	99%
	20	19.9 +/- 0.10	100%
	100	110 +/- 2.3	110%

 

Table: Mean values in selected clinical chemistry parameters

	Dose Group (mg/kg bw/day)
	Control	20	100	500
Males
Glucose concentration (mmol/L)	9.8	10.0	9.3	6.7**	DN
Urea concentration (mmol/L)	10.7	10.1	9.5	8.5**	DN

Remarks:

DN = Duncan’s Multiple Range Test, U = Mann-Whitney U-Test

*=p<0.05, **=p<0.01,% difference vs control

 

Table: Mean values of liver weights following the 28-day treatment

	Dose (mg/kg bw/day)
	Control	20	100	500
MALES
Liver Weight (g)Absolute	8.69	8.30	9.19	9.67	NS
	% difference	-5	6	11
Relative to Brain Weight (%)	432	441	456	488	NS
	% difference	2	6	13
Relative to Body Weight (%)	3.03	2.99	3.28	3.37*	DN
	% difference	-1	8	11
FEMALES
Liver Weight (g)Absolute	5.68	6.04	5.55	7.42**	DN
	% difference	6	-2	31
Relative to Brain Weight (%)	320	323	304	401**	DN
	% difference	1	-5	25
Relative to Body Weight (%)	3.03	3.22	3.03	4.01**	DN
	% difference	6	0	32

DN = Duncan’s Multiple range Test; NS = Not significant

*=p<0.05; **=<0.01

% difference vs control

 

Table: Mean values of kidney weights following the 28-day treatment

	Dose (mg/kg bw/day)
	Control	20	100	500
MALES
Kidney Weight (g)Absolute	2.13	2.23	2.33	2.66**	DN
	% difference	5	9	25
FEMALES
Kidney Weight (g)Absolute	1.40	1.49	1.45	1.51	NS
	% difference	6	4	8

DN = Duncan’s Multiple Range Test; NS = non significant

**=p<0.01, % difference vs control

Applicant's summary and conclusion

Conclusions:

In conclusion, under the conditions of this study, the no observed adverse effect level (NOAEL) is 500 mg/kg as effects observed in the high dose group are species specific and therfore not accounted for as adverse.

Executive summary:

The objective of this study was to assess the toxicity of the test item, following daily oral administration by gavage to Wistar Crl:WI (Han) rats for 28 consecutive days according to OECD testing guideline 407. The testing regimen contained 4 groups which obtained daily 0, 20, 100 or 500 mg test item per kg body weight.

  The control group was treated with the vehicle only (distilled water). The Main Group animals were euthanized after 28 days of daily treatment. (Start of treatment, Day 0). Recovery animals, following the 28-day daily treatment were observed for a 14-day recovery period, than subjected to necropsy.

 

Parameters monitored during the study were mortality and clinical observations, performed daily and weekly, more detailed, body weight and food consumption measurements and clinical pathology evaluation (haematology, coagulation, clinical chemistry and urinalysis) following the 28-day treatment or 14-day recovery period.

 Neurological assessment including a modified Irwin test, functional observation battery and measurements of the landing foot splay and grip strength were performed during the last week of the treatment period.

 Necropsy and macroscopic examination were performed on all animals, one day after the last dose administration, on Day 28 or following the 14-day recovery period, after the designated procedures including vaginal smear examination in females. Selected organs were weighed. Full histopathology was performed on the selected list of organs in Groups 1 (Control) and 4 (High Dose) animals. Additional histopathological examinations (Chromotrope-Aniline-Blue-staining) were performed of the kidneys of three control and three High Dose male animals. Kidneys (standard stain) of all treated males and liver from the Low, Mid and recovery High Dose females and males were also investigated.

 

Formulations were prepared in the Central Dispensary of CiToxLAB Hungary Ltd. fresh prior to administration to animals (Days 0 and 1) or at the appropriate frequency to allow their use according to stability assessment results, within 7 days while stored refrigerated. Stability of the test item in the vehicle was assessed in the conditions employed on the study by the CiToxLAB Analytical laboratory.

Based on the results, the test item solution in distilled water was stable at concentration range of 4-200 mg/mL when stored 1 day at room temperature or 7 days refrigerated (at 2-8°C).

 

Analysis of formulations (concentration and homogeneity) was performed in the Analytical Laboratory of CiToxLAB Ltd. using UV Spectrophotometry, twice during the study (during the first and last weeks of treatment). No test item was detected in the control solution samples. All formulations were shown to be homogeneous. All formulations were found to be in the range of 97 to 110% of nominal concentrations. These results were considered suitable for the study purposes.

 

No clinical signs were observed and no lethality occurred during the 28-day treatment or recovery period.

There was no treatment-related effect on motor activity or in the functional observation battery tests across groups of treated male or female animals and no findings indicative of neurotoxicity were observed. Evaluation of the vaginal smears prior to necropsy showed the expected distribution of the oestrus cycle phases within the normal population of female Wistar rats.

 

There were no toxicologically significant changes in body weight, body weight gain or animal food consumption between the control and test item treated groups.

 

Minor variations, on occasion attaining statistical significance, were noted in the clinical pathology parameters (e.g. decreased blood glucose and urea concentration in males at 500 mg/kg). However, the changes were slight in magnitude, and/or were within the physiological range. Therefore these changes were not considered toxicologically significant.

 

No test item related observation was made at necropsy of animals following the 28-day treatment or 14-day recovery period.

 

At 500 mg/kg bw/day, the weights of kidneys (absolute and relative) were increased in males (approximately 25% on Day 28) and this was associated with hyaline deposition in tubular cells.

 

No similar changes were observed in kidneys at 20 or 100 mg/kg bw/day (Low and Mid dose) or following recovery on Day 42.

At 500 mg/kg bw/day, the absolute and relative liver weights were higher than controls by approximately 30% in females and 10% in males following the 28-day treatment period, only and were associated with minimal periportal hepatocellular vacuolation of the liver (in 3 of 5 males and 4 of 5 females).

 

Conclusion: A daily oral (gavage) administration of the test item to Hanover Wistar rats for 28 days at dose level of 500 mg/kg bw/dayresulted in increased kidney weights of males associated with hyalinedeposition in tubular cells; and increased liver weights (by approximately10% and 30% in males and females, respectively) associated with minimalperiportal hepatocellular vacuolation observed in 3 of 5 males and 4 of 5 females. Based on these findings the authors of the study established a no observed adverse effect level (NOAEL) at 100 mg/kg bw/day.

In a subsequent ex-vivo enyzme analysis of liver samples taken from control animals and high dose animals the increased liver weights could be ascribed to increased hepatic peroxisomal beta-oxidation (CN-palmitoyl-CoA-oxication values 1.9 and 1.8 fold higher in male and female rats respectively). Thus confirming that the effects on organ weights can be accounted for as species specific and therefore not adverse to humans.

Finally as effects observed (i.e. elevated liver weights in male and female animals and elevated kidney weights in male animals at the end of the treatment period; the effects on the liver weights were consistent with microscopic findings in high dose group, but reversible within the 14 day recovery period) were due to species specific induction of hepatic peroxisomal beta-oxidation (liver effects) or alpha2u globulin accumulation (kidney effects in male rats) these effects wer not attributed as adverse (as not relevant for human risk assessment) and thus a NOAEL at the highest dose group was set (i..e. 500 mg/kg bw/day).