Butanedioic acid, 2-sulfo-, mono(C16-18 and C18-unsatd. alkyl) esters, ammonium sodium salts

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 April 2021 (study plan) – 03 November 2022 (final report)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Butanedioic acid,sulfo-, mono (C16-18 and C18-unsatd. alkyl) ester, ammonium sodium salt

EC Number:

604-617-1

Cas Number:

147993-66-6

IUPAC Name:

Butanedioic acid,sulfo-, mono (C16-18 and C18-unsatd. alkyl) ester, ammonium sodium salt

Test material form:

solid: flakes

Test animals

Species:

rat

Strain:

other: Crl:WI (Wistar)

Details on species / strain selection:

Wistar rat was selected due to experience of the Test Facility with this strain of rat in toxicity and reproduction toxicity studies and its known fertility.

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, (Address: Sandhofer Weg 7, D-97633, Sulzfeld, Germany) from SPF colony
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: Young adult rats, 12-13 weeks old (females/males) at start of the experiment
- Weight at study initiation: Males: 409-512 g, females: 244-298 g (at the start of the treatment). The body weights did not exceed ± 20% of the mean weight for each sex at start of treatment.
- Fasting period before study: no information
- Housing: Rodents were group-housed, up to 2 animals of the same sex and group/cage in type II, III and/or IV polycarbonate cages. During the mating and gestation, delivery, lactation period, they were paired or individually housed (with pups), respectively. SAFE 3/4-S Hygienic Animal Bedding (Batch number: 03027201208, Expiry date: 08 December 2023) and SAFE Crinklets Natural nesting material (Batch number: 05072200824 / 05072201021, Expiry date: 24 August 2023 / 21 October 2023) produced by J. Rettenmaier & Söhne GmbH+Co.KG (Address: Holzmühle 1, D-73494 Rosenberg, Germany) were used in the study. Group housing allowed social interaction. Deep wood sawdust bedding allowed digging and other normal rodent activities, while nesting material allowed normal nesting behaviour. Certified cardboard hiding tunnels (GLP Mini Fun Tunnels, Batch numbers: A/123/D and A/123/E) produced by LBS (Serving Biotechnology) Ltd. (Address: Unit 20, Gatwick Business Park, Kennel Lane, Hookwood, Surrey, RH6 0AH UK) were also provided to the animals.
- Diet (e.g. ad libitum): The animals received ssniff® SM R/M “Autoclavable complete diet for rats and mice – breeding and maintenance” (Batch numbers: 713 70882 / 187 76795, Expiry dates: 30 April 2021 / 31 August 2021) produced by ssniff Spezialdiäten GmbH (Address: Ferdinand-Gabriel Weg 16, D-59494 Soest, Germany), ad libitum.
- Water (e.g. ad libitum): The animals received tap water from the municipal supply, as for human consumption from a 400- or 500-mL bottle, ad libitum.
- Acclimation period: 19 days

DETAILS OF FOOD AND WATER QUALITY:
The food was routinely analysed and considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.
The quality control analysis of the water was performed once every three months and microbiological assessment was performed monthly, by National Institute of State Public Health and Medical Officer Service. The water was considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.8 – 25.9°C (target: 22 ± 3°C)
- Humidity (%): 30 - 61% (target: 30 - 70%)
- Air changes (per hr): 15-20 air exchanges/hour
- Photoperiod (hrs dark / hrs light): 12 hours daily, from 6.00 a.m. to 6.00 p.m.

IN-LIFE DATES: From: 13 April 2021 (first vaginal smear sampling) To: 21 June 2021 (last necropsy)

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

The time of the gavage process was prolonged, to be really sure the rat was well relaxed, and the total amount of gavage liquid was administered slowly into the stomach (‘short’ gavage to the lower oesophagus was avoided).

Vehicle:

propylene glycol

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: The test item was formulated in the selected vehicle (propylene glycol), as a visibly stable homogenous solution at the appropriate concentrations according to the dose level and volume selected in the Pharmacy of the Test Facility. The formulations were stirred with a magnetic stirrer from the preparation until completion of each treatment.
Formulations were prepared a maximum of 6 days prior to administration to animals according to stability assessment results of the analytical method validation study (Study code: 20/127-901AN). Based on those results, the test item formulation in the 1 and 200 mg/mL concentration range were stable for at least 7 days when stored at room temperature.


The calculated amount test item was weighed into a clean, calibrated glass container and then mixed properly (with ultrasonication and magnetic stirring) with the needed amount of vehicle to reach homogeneity by visual observation. During the formulation sonication was applied to aid dissolution.
VEHICLE: Propylene glycol
- Concentration in vehicle: 0, 3, 9 and 18 mg/mL for the dose levels group of 0, 15, 45 and 90 mg/kg bw/day, respectively.
- Amount of vehicle (if gavage): A constant volume of 5 mL/kg bw was administered to all animals
- Lot/batch no.: 1920944

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Sample collection was performed on three occasions (during the first and last weeks and approximately midway during the treatment period). Samples were collected immediately after formulation preparation in the Pharmacy of the Test Facility by a responsible member of the Pharmacy Department.
On each sampling occasion, top, middle and bottom duplicate samples was taken from test item formulations for concentration and homogeneity measurement, one set to analyse (which can be collected in replicates as practical) and one set as a back-up, if required for any confirmatory analyses. Similarly, duplicate samples were taken from the middle of the vehicle control formulation for concentration measurement.
After the analytical sampling, the collected formulation samples were stored at room temperature until measurement.
Analysis of control (vehicle) and test item formulations for concentration and homogeneity was performed at the Test Site for analytical work was using a validated analytical method (20/127-901AN). Representative samples of control (vehicle) and test item formulations were analysed at three times during the study (during the first and last weeks and once approximately midway during the treatment period).
Formulation samples were kept at room temperature until shipment. Samples to be analysed was shipped to the Test Site as soon as practical after collection for concentration measurements.
Analysis of the formulations for concentration and homogeneity of test item was performed using a validated analytical HPLC-MS method (High Performance Liquid chromatography–mass spectrometry) at the Test Site (Study code for method validation: 20/127-901AN).
Acceptance criteria of the concentration analysis was 100 ± 10% of the nominal concentration.
Acceptance criteria of the homogeneity was that the RSD% (relative standard deviation) of replicates (top, middle and bottom of test item formulations) must be less than 10%.
The measured concentrations of the test item in the different formulations varied between 92.1% and 105.9 % of the nominal concentrations.
All test item formulations were shown to be homogeneous. The relative standard deviation (RSD) was below 10%.
Formulations were considered to be adequately stable under the study conditions.

Duration of treatment / exposure:

Males were dosed for 28 days (14 days pre-mating and 14 days mating/post-mating period), then were euthanized and subjected to necropsy examination.
Females were dosed for 14 days pre-mating, for up to 5 days mating period, through gestation and up to and including the day before necropsy (13 days post-partum dosing). The day of birth (when parturition was complete) was defined as Day 0 post-partum.

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Remarks:

low dose

Doses / concentrations 2

Dose / conc.:

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

Remarks:

mid dose

Doses / concentrations 3

Dose / conc.:

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

Remarks:

high dose

No. of animals per sex per dose:

12 animals/sex/dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The oral route was selected as it is one of the possible routes of human exposure. The way of test item and vehicle control item administration was in compliance with the relevant OECD No. 422 guideline.
The dose levels were selected by the Sponsor in consultation with the Study Director based on the results of a Dose Range Finding (DRF) study (Study code: 20/127-220PE), with the aim of inducing toxic effects but ideally no death or suffering at the highest dose and a NOAEL at the lowest dose.
In the DRF study all the 1000 mg/kg bw/day dose group animals died or were euthanized, 2/4 female and 2/4 males in the 600 mg/kg bw/day dose group died or were euthanized: 600 mg/kg bw/day group animals were treated two days, 300 mg/kg bw/day group animals were treated eight days, then remained untreated to get information on the recovery. 1/4 male and 4/4 females died in the 300 mg/kg bw/day dose group. No animals died in the 100 mg/kg bw/day group.
There were no clinical symptoms or severe body weight decrease seen at 100 mg/kg/day. A range of symptoms (hunched back, piloerection, decreased activity, faeces liquid / soft, noisy respiration, red discharge around nose, wasted and general poor condition) were seen in Mid dose, High dose and High dose 2 animals indicating adverse effects; with no treatment effects in the Low groups.
The Low dose group had reduced growth or weight loss in the first week, with a near normal growth rate thereafter. The Mid to High dose group survivors lost significant weight in the treatment phase, and continued to have weight loss even after treatment was halted.
The Low dose group had minor haematological differences of equivocal relationship with treatment. Low dose males had no substantial effects in clinical chemistry; low dose females had changes in serum enzymes suggestive of hepatic effects, but not indicative of a severe effect. Other group data for clinical pathology was either at euthanasia or after a period without test item treatment, hence are not useful for dose setting, although no pattern was seen to suggest any specific toxic mechanism.
Test item-related bilateral enlargement of the adrenal glands (indicative of stress) and all lobes of liver was observed in High dose 2 male. Multifocal red discolouration of the glandular stomach and multifocal thickness of non-glandular stomach (indicative of gastric irritation) was observed in two Mid dose males, one High dose 2 male and one High dose 2 female. Mild to moderate periportal hepatocellular vacuolation was seen in Low dose females; it is considered as non-adverse in the context of this study, but it may possibly progress with longer exposure. Treatment-related effects were observed in the adrenal gland weights in Mid and High dose 2 males and liver weights in Low and High dose 2 females. The Low dose males had no evident changes in organ weight.
The dose of 90 mg/kg/day is selected for the High dose. Lower doses were spaced with a factor of approximately 3.
- Rationale for animal assignment (if not random): All adult/parental (P) male and female animals were sorted according to body weight by computer and divided into weight ranges. There were an equal number of animals from each weight group randomly assigned to each dose group to ensure that animals of all test groups were as nearly as practicable of a uniform weight.
This process was controlled by the software PROVANTIS v.9, to verify the homogeneity/variability between/within the groups. Males and females were randomised separately to the dose groups on the day of the first treatment (prior to the start of the treatment).
Any unused, spare animals were moved back to the stock colony after they were not needed for the study (after of treatment).
- Fasting period before blood sampling for clinical biochemistry: yes, overnight period of food deprivation, in case of females this happened after the litter had been necropsied

Positive control:

Five female and five male animals served as positive control for the Mammalian Erythrocyte Micronucleus Test (MNT).
Cyclophosphamide Dose Level: 60 mg/kg bw

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: General (routine) clinical observations were made once a day*, during the pre-treatment and treatment period in the afternoon (pm). *Note: No general clinical observations were made on the day of necropsy. Animals were inspected for signs of morbidity and mortality once per day in the pre-treatment period and twice daily in the treatment period (at the beginning and end of each working day). Any animal (including also all premature decedents) which showed clinical signs considered severe was sacrificed to prevent suffering, cannibalism and/or autolysis, and was processed in the same way as the animals subjected to terminal necropsy.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical observations were made at the start of the pre-exposure period and once before the first exposure on Day 0 (to allow for within-subject comparisons), then weekly (in the morning (am), before treatment) and before necropsy. These observations were made outside the home cage in a standard arena, at similar times as practical. Signs evaluated included changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lachrymation, piloerection, pupil size and unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g. excessive grooming, repetitive circling), difficult or prolonged parturition or bizarre behaviour (e.g. self- mutilation, walking backwards) were also recorded. Special attention was directed towards the observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma.
Pertinent behavioural changes, signs of difficult or prolonged parturition and all signs of toxicity including mortality were recorded including onset, degree and duration of signs as applicable.
On Gestation Day (GD) 13 and/or 14 the sperm positive females were examined for the presence of vaginal bleeding or “placental sign” (intrauterine extravasation of blood as an early sign of pregnancy in rat).
Furthermore, mated females were examined carefully around the time of expected delivery for any signs of difficult or prolonged parturition.

BODY WEIGHT: Yes
- Time schedule for examinations: All adult animals were weighed with accuracy of 1 g weekly during the pre-exposure period, on Day 0 (randomisation and the first day of exposure), and afterwards weekly, and at termination.
Parent females were weighed on the first day of dosing, then once a week until mating.
On Gestation Day (GD) 0, 3, 7, 10, 14, 17 and 20, on PPD (Post-partum Day) 0, 4, 7, 10 and 13, and at termination. The body weight of the female animals measured on GD3, GD10 and GD17 as well as PPD10 were additional measurements as aid for the calculation of accurate treatment volumes, these data was evaluated statistically.

FOOD CONSUMPTION AND COMPOUND INTAKE (no feeding study):
Animal food consumption was determined at randomisation by weighing the non-consumed diet with a precision of 1 g at least weekly and on Day 0. Food consumption was measured during mating. Food consumption was measured on Gestation Day (GD) 0, 3, 7, 10, 14, 17 and 20 and on PPD0, 4, 7, 10 and 13 during the lactation period).
Main daily food consumption was calculated for each interval.

FOOD EFFICIENCY: no

WATER CONSUMPTION AND COMPOUND INTAKE (no drinking water study): No
No water consumption was measured in the study.

OPHTHALMOSCOPIC EXAMINATION: No
No ophthalmoscopy was conducted in the study.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At study termination or in the case of early termination due to moribund animals, euthanasia was performed under pentobarbital anaesthesia followed by exsanguination. Blood samples were collected by cardiac puncture under pentobarbital anaesthesia, immediately prior to scheduled necropsy.
- Anaesthetic used for blood collection: Yes: Euthanimal 40% (400 mg/mL sodium pentobarbital solution) was used by intraperitoneal injection
- Animals fasted: Yes : overnight period of food deprivation, in case of females this happened after the litter had been necropsied
- How many animals: all randomly selected animals (7 males and 5-6 females/group)
- Parameters examined.
RBC Red Blood Cell (erythrocyte) count, (10^12/L) M/µL
WBC White Blood Cell (leukocyte) count, (10^9/L) K/µL
Hgb Haemoglobin concentration, (g/dL)
Hct Haematocrit (relative volume of erythrocytes) (%)
MCV Mean Corpuscular (erythrocyte) Volume (fL)
MCH Mean Corpuscular (erythrocyte) Haemoglobin, (pg)
MCHC Mean Corpuscular (erythrocyte) Haemoglobin Concentration, (g/dL)
RDW Red Cell (erythrocyte) volume (%)
Plt Platelet (thrombocyte) count (10^9/L) K/µL
MPV Mean Platelet Thrombocyte volume (fL)
RETIC % Reticulocyte count (%)
NE % Neutrophil (%)
LY % Lymphocyte (%)
MO % Monocyte (%)
BA % Basophil (%)
EO % Eosinophil (%)
LUC % Large Unstained Cells (%)
Coagulation parameters:
APTT Activated Partial Thromboplastin Time (sec)
PT Prothrombin Time (sec)
Blood smears were prepared for all selected animals but not examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At study termination or in the case of early termination due to moribund animals, euthanasia was performed under pentobarbital anaesthesia followed by exsanguination. Blood samples were collected by cardiac puncture under pentobarbital anaesthesia, immediately prior to scheduled necropsy.
- Animals fasted: Yes: overnight period of food deprivation, in case of females this happened after the litter had been necropsied
- How many animals: all randomly selected animals (7 males and 5-6 females/group)
- Parameters examined:
Glucose Blood sugar concentration (mmol/L)
T-BIL Total Bilirubin concentration (μmol/L)
Urea nitrogen Urea concentration (mmol/L)
Chol. Cholesterol concentration (mmol/L)
Creat. Creatinine concentration (μmol/L)
Phos. Phosphorus concentration (mmol/L)
Na+ Sodium concentration (mmol/L)
K+ Potassium concentration (mmol/L)
Ca++Calcium concentration (mmol/L)
Cl- Chloride concentration (mmol/L)
Tot. Prot. Total Protein concentration (g/L)
Alb. Albumin concentration (g/L)
A/G Alb/glob ration
AST/GOT Aspartate Aminotransferase activity (U/L)
ALT/GPT Alanine Aminotransferase activity (U/L)
GGT Gamma-Glutamyl transferase activity (U/L)
ALKP Alkaline Phosphatase activity (U/L)
BA Bile acids (µmol/L)

PLASMA/SERUM HORMONES/LIPIDS: yes
- Time of blood sample collection:
*Blood sampling for thyroid hormone analysis:
For thyroid hormone analysis, blood samples were taken by venepuncture (using vena sublingualis in case of adult animals) or decapitation (in case of pups) into tubes containing K3-EDTA as anticoagulant as follows:
•from up to two pups per litter on PND4 (females if possible),
•from all dams and two pups per litter on PPD 14 (females) / PND13 (pups),
•from all non-pregnant adult females at termination
•from all adult males at termination.
The collected pup blood (plasma) samples were pooled by litter.
The timing of the blood collection for thyroid hormone determination was as close as possible between animals and at the same time of the day in case of sampling on different days. Timing is documented in the raw data.
Blood samples were kept on ice from sampling until centrifugation (within 30 minutes of collection), then centrifuged rapidly (1600 g / approx. 3000 rpm, 10 minutes, 4°C). The resulting plasma was divided into three aliquots (volume target of at least 150 µL for the first and second aliquot and the remaining amount for the third aliquot and stored in an ultra-freezer (-80±10°C) until analysis.
At the first instance, samples for the PND13 pups and adult males were assessed for T4 levels.
The thyroid hormone analysis was conducted using a validated method under the control of the Contributing Scientist.
For assessment of T4 hormone measurement, a competitive immunoassay method was used. Wells of the microplate were coated with an antibody specific for T4. An enzyme-T4 conjugate was added and the reactants were mixed. A competition reaction resulted between the enzyme-T4 conjugate and the T4 (from the sample to be measured) for a limited number of antibodies immobilized on the well. After the completion of the required incubation period, the antibody-bound fraction of the enzyme-T4 conjugate was separated from the unbound enzyme-T4 conjugate via a wash step. A substrate solution was then added to the wells, the colour development was stopped after a given period of time and the absorbance was measured at 450 nm. The colour intensity was proportional to the amount of the enzyme-T4 conjugate bound in the initial step, which was inversely proportional to the amount of T4 arrived from the sample.
Each assay consisted of:
-blank samples (zero calibrator – supplied in the kit – was used as blank in triplicates),
-Six calibration standards from 0 µg/dL up to 25 µg/dL T4,
-Quality control (QC) samples,
-study samples (individual samples).
Acceptance criteria
Back-calculated results of each calibration point had to be within ± 25% of their nominal values, except of 0 level.
Results of QC samples had to be within ± 25% of their nominal values and valid results should be accepted at least two out of three results for each QC level.
- Animals fasted: Y Yes: overnight period of food deprivation, in case of females this happened after the litter had been necropsied

URINALYSIS: yes
- Time schedule for collection of urine: Urine sampling was performed prior to necropsy by placing the selected animals in metabolic cages for approximately 16 hours.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes: overnight period of food deprivation, in case of females this happened after the litter had been necropsied
- Parameters examined:
The evaluation of the urine samples was performed by using of Medi-Test URYXXON® Stick 10 Urinalysis-strips
LEU / Leukocyte
NIT / Nitrite
pH
PRO / Protein
GLU / Glucose
UBG / Urobilinogen
BIL / Bilirubin
KET / Ketones
BLD / ERY Blood/Erythrocytes
SG / Specific Gravity
SED / Sediment
VOL / Volume
Colour/Appearance

NEUROBEHAVIOURAL EXAMINATION: yes
- Time schedule for examinations: Functional Observational Battery and locomotor activity measurement was performed in the study. Five males and five females/group were randomly selected:
Assessment of any potential test item related neurotoxicity was performed during the last exposure week (males on Day 22 am, females on PPD8-12 am). Selected animals were subjected to the functional observation battery, including Irwin test and measurements of the landing foot splay and fore/hind grip strength.
In order to avoid hypothermia of pups, dams were removed from the pups for not more than approximately 30-40 minutes during FOB or 90 minutes during locomotor activity measurement (SMART).
- Dose groups that were examined: Five males and five females/group were randomly selected:
- Battery of functions tested: sensory activity / grip strength / motor activity / other:
Selected animals were subjected to the functional observation battery, including Irwin test and measurements of the landing foot splay and fore/hind grip strength.
A modified Irwin test was performed when sensory reactivity to different type of stimuli (e.g. auditory, visual and proprioceptive), assessment of grip strength and motor activity was conducted, and the general physical condition and behaviour of animals was tested. Parameters including 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 reflex, 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 were evaluated.
To measure the landing foot splay, the fore/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. This was repeated 3 times for each animal. The distance between the two resulting ink spots of the hind limbs was measured.
Fore/hind grip strength was measured 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 were allowed to grip the support bar and gently pulled back until they released the bar; the device measures the maximum grip strength. This was performed 3 times for each animal on each test day. The procedure was repeated with the hind limbs with the appropriate grip support. The results are tabulated with individual and mean data.
Locomotor activity assessment was conducted using Automatic Monitoring System of rat locomotor activity SMART v. 2.5 (Harvard Apparatus, Germany). Locomotor activity was monitored by placing each animal individually into an open-field for 1-hour observation time, when DVD recording of movement was made. Recording was made for a duration of 60 minutes, under dim-light and undisturbed conditions. The DVD was analysed with “SMART” software after all recordings were made to produce the appropriate parameters. Data was evaluated for distance travelled in 5-minute segments. The data from the 5-minute segments was presented graphically with the intention of showing plateau activity in controls and comparing the treatment groups.

IMMUNOLOGY: No

OTHER:
-Toxicokinetic blood sampling: For potential blood analysis, blood samples were taken into K2-EDTA tubes by tail vein form all the adult animals 3 hours after the last dosing. Blood samples were stored in an ultra-freezer (-80±10°C) until analysis.

-Micronucleus assay:
Five animals/sex were assigned for the positive control group for the Mammalian Erythrocyte Micronucleus Test (MNT) from the spare animals. They were treated once with 60 mg/kg bw Cyclophosphamide, administered by intraperitoneal injection approximately 24-hour prior to scheduled necropsy.

*Mammalian Erythrocyte Micronucleus Test In Bone Marrow Cells: The mammalian in vivo micronucleus test (MNT) was used for the detection of damage induced by the test substance to the chromosomes or the mitotic apparatus of erythroblasts by analysis of erythrocytes as sampled from bone marrow of animals, usually rodents.
The purpose of the micronucleus test was to identify substances that cause cytogenetic damage which results in the formation of micronuclei containing lagging chromosome fragments or whole chromosomes. When a bone marrow erythroblast develops into a polychromatic erythrocyte, the main nucleus is extruded; any micronucleus that has been formed may remain behind in the otherwise anucleated cytoplasm. Visualisation of micronuclei is facilitated in these cells because they lack a main nucleus. An increase in the frequency of micronucleated polychromatic erythrocytes in treated animals is an indication of induced chromosome damage.
The purpose of this test as part of the study was to determine whether the test item causes genotoxic effects resulting in the formation of micronuclei in erythrocytes as sampled from the bone marrow of treated animals.
Three sets of bone marrow smears for MNT were prepared from all the animals, including the vehicle control and the positive control groups.
The bone marrow was collected from the right femur of the rats immediately after euthanasia (the left femur of all the dosed animals was used for routine histopathology, the left femur of positive control animals was discarded) and flushed with foetal bovine serum (5 mL) using a syringe and needle. Cells were concentrated by a gentle centrifugation.

*micronucleus analysis: From the preserved slides three sets of slides were stained with 10 % Giemsa solution for approximately 18-20 minutes. Slides were thoroughly rinsed with distilled water, and then air-dried at room temperature for at least 12 hours. After staining, cover slips were mounted on them. Spare slides were retained for possible future staining in case of uncertain results.
The stained slides were given a unique code number at the Test Facility by a person who was not involved in the analysis. The code labels were cover all unique identification markings on the slides to ensure that they were scored without bias.
Smears of the cell pellet were made on standard microscope slides. Slides were then air-dried at room temperature for approximately 24 hours. Dried slides were fixed in methanol for a minimum of 5 minutes and allowed to air-dry.
The coded slides were shipped for evaluation to the Principal Investigator.
At least 4000 polychromatic (immature) erythrocytes (PCEs) were scored per animal to assess the micronucleated cells. The frequency of micronucleated cells was expressed as numbers of micronucleated cells based on the first 4000 PCEs counted in the optic field.
The proportion of immature among total (immature + mature) erythrocytes was determined for each animal by counting a total of at least 1000 cells (immature erythrocytes, PCEs plus mature normochromatic erythrocytes, NCEs), in which the number of micronuclei were recorded in both types of erythrocytes.
The analysis were conducted under the control of the Principal Investigator in compliance with Good Laboratory Practice according to the United Kingdom GLP Compliance Regulations 1999 (SI 1999 No. 3106, as amended 2004, SI No. 0994) that corresponds the OECD GLP, ENV/MC/CHEM (98) 17 and own SOPs. These Principles were in conformity with other international GLP regulations.
When slide reading has been performed, the original raw data (record sheets) for the analysis and the microscope slides were shipped back to the Test Facility (Charles River Laboratories Hungary Kft.) for evaluation and then archiving. After the in-life phase of the study was ended, the Principal Investigator issued a Work Phase Report.
Criteria for Identification of Micronucleated Erythrocytes:
°A bluish mauve strongly coloured uniform round or oval particle in the cell.
°The particle should be large enough for the colour to be recognisable, and it should be located inside the cells. Areas with micronucleus-like particles outside the cells should not be used for analysis.
°During focusing, the particle should stay uniform in colour /light refraction and shape within a large interval and focus in the same plane as the erythrocyte.
°The unit of damage is deemed to be the cell, and therefore cells with two or more micronuclei will be counted as single micronucleated cells.
The Micronucleus Test is considered acceptable if it meets the following criteria:
°The frequencies of micronucleated polychromatic erythrocytes found in the negative and /or solvent controls falls within the range of historical laboratory control data.
°The positive control item should produce biologically relevant increases in the number of micronucleated polychromatic erythrocytes.
°Each treated and control group should include at least 5 analysable animals.
°The appropriate number of cells has been analysed.
Interpretation of Results
The frequencies of micronucleated polychromatic erythrocytes in animals in the test and positive control groups will be compared to the values found in the negative control group.
Cytotoxicity of the samples was expressed by the PCE/NCE ratio.
The test item will be considered to have shown genotoxic activity in this study if the following criteria are met:
°increases in the frequency of micronucleated polychromatic erythrocytes are observed in treated animals compared to the corresponding negative control
°the increases are dose-related
°the increases are statistically significant.
The historical control range for this laboratory was also considered when evaluating the biological significance of small increases.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
-method of euthanasia
At study termination or in the case of early termination due to moribund animals, euthanasia was performed under pentobarbital anaesthesia followed by exsanguination.
Euthanimal 40% (400 mg/mL sodium pentobarbital solution) was used by intraperitoneal injection.

-unscheduled euthanasia
Gross necropsy was performed on each animal irrespective of the date of death, including the animal found dead or euthanized pre-terminally in extremis. No organ weight measurement was performed, but tissue or organ samples were retained for those animals. Organ weighs of one Low dose female (#2510) was recorded due to oversight.

-scheduled euthanasia
Surviving animals were euthanized at termination. Gross necropsy was performed on each animal. Weight of selected organs were measured, and selected organs and tissues were retained.

-necropsy:
Gross necropsy was performed on each adult animal irrespective of the date of death. Terminally (one day after the last treatment), animals were sacrificed under pentobarbital anaesthesia by exsanguination; anaesthetic product was diluted for pups’ euthanasia as required.
After exsanguination the external appearance was examined, cranium, 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. Special attention was paid to the organs of the reproductive system.

-organ weights:
At the time of termination, body weight and weight of the following organs of all adult males were determined:
•With a precision of 0.01 g: uterus (including cervix), testes, epididymides, prostate, seminal vesicles with coagulating glands, brain, heart, kidneys, liver, spleen and thymus
•With a precision of 0.001 g: adrenals, ovaries, thyroids with parathyroids
Testes and epididymides were weighed individually. Individual and/or paired absolute organ weight was reported for each animal and adjusted for the body and brain weights. Paired organ weights as applicable were summarised. Relative organ weight (to body and brain weight) was calculated and reported.

-tissue collection and preservation
The weighed organs and all organs showing macroscopic lesions of all adult animals were preserved. The eyes with the optic nerve, testes and epididymides were retained in modified Davidson’s fixative, all other organs in 10% buffered formalin solution.
In addition, on completion of the macroscopic examination the following tissues and organs were retained from all surviving animals:
Gross findings
Adrenals
Animal identification (Fixation and preservation only.)
Aorta (Aorta thoracic and abdominal.)
Brain (7 section according to the NTP recommendations)
Epididymis
Eye with the optic nerve (If applicable, parathyroids and optic nerves was examined histologically only if present in routine sections.)
Oesophagus
Femur with marrow
Heart (Section including both ventricles and atria, septum with papillary muscle.)
Kidney
Large intestine (Caecum, colon and rectum.)
Extraorbital lachrymal gland
Harderian gland
Liver (Liver, 3 lobes, left lateral, right medial, caudate.)
Lungs with bronchi (Lungs of euthanized animals was infused with formalin; 3 lobes, left, right cranial, right caudal.)
Lymph node (Mandibular and mesenteric.)
Ovary
Oviduct
Pancreas
Pituitary
Prostate
Salivary gland (including mandibular, sublingual and parotid glands)
Sciatic nerve
Seminal vesicle with coagulating gland
Skin, subcutis with mammary gland (inguinal)
Skeletal muscle (quadriceps)
Small intestine (Duodenum, ileum and jejunum with Peyer’s patches.)
Spinal cord (Transverse sections, 3 levels – cervical, thoracic and lumbar.)
Spleen
Sternum with marrow
Stomach
Testis
Thymus
Thyroid with parathyroid gland (If applicable, parathyroids and optic nerves was examined histologically only if present in routine sections)
Tongue
Trachea
Urinary bladder
Uterus (Horns, body and cervix.)
Vagina
Additionally, thyroid glands from all adult animals and one male and one female PND13 pup from each litter were preserved in 10% buffered formalin solution. In this case, the thyroid weight (pooled) was determined after fixation. Trimming was done very carefully and only after fixation to avoid tissue damage.

HISTOPATHOLOGY: yes
The retained tissues and organs required for histopathology (below) were embedded in paraffin wax; sections were cut at 4-6 µm by microtome and transferred to slides. Tissue sections were stained with haematoxylin-eosin/phloxine and examined by light microscope.
For the adult animals, detailed histological examination was performed as follows:
• on the selected list of retained tissues and organs (as above) in the Control and High dose groups (5 animals/sex/group), - number of animals was notified by a Memo
• any animals found dead or euthanized pre-terminally during the study in all groups,
• all macroscopic findings (abnormalities), except of minor order from all animals,
• on the retained reproductive organs (testes, epididymides, prostate gland, seminal vesicles with coagulation gland for males and uterus, cervix, ovary, oviduct and vagina for females) of all animals of the Control and High dose groups, and of all males that failed to sire, total intrauterine mortality animals and all females that failed to deliver healthy pups (this was notified by Memo).
Special attention was paid to evaluation of the stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure. Detailed histological examination of the ovaries covered the follicular, luteal, and interstitial compartments of the ovary, as well as the epithelial capsule and ovarian stroma.
Special attention was paid to the organ weight, appearance and histopathology of immune-system tissues for any evidence of immunotoxicity (spleen, thymus, lymph nodes, bone marrow, and blood smears if examined).
Special attention was paid to the central and peripheral nervous system tissues for any evidence of neurotoxicity.
Additional histopathology examination on the liver of all animals (Control, Low, Mid and High dose) and on the prostate and seminal vesicle on all the males (Control, Low, Mid and High dose) was performed because it was deemed necessary by the Pathologist and the Study Director.

Statistics:

see "Any other information on materials and method incl. tables"

Results and discussion

Results of examinations

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No test item related clinical signs were observed in the study.
Liquid faeces were recorded during the lactation period (Day 52) for one Low dose female (#2503) and on Day 51 for a Mid dose female (#3505).
Red liquid in the vulva was observed for one High dose female (#4509) on Day 33 and 34. Piloerection was observed for this animal on Day 16.
Noisy respiration was observed for one Control female (#1503) on Day 6 and 7.
Hunched back and piloerection were observed for one Control female (#1510) form Day 17 to Day 20.
These findings were all considered as being incidental, not related to the test item administration.

Mortality:

mortality observed, treatment-related

Description (incidence):

No test item related mortality was observed in the study.
One Control male (#1009) was found dead on Day 28. No clinical sings were recorded prior to death. The cause of death could not be determined.
One High dose male (#4004) was preterminally euthanised on Day 14. Hunched back, piloerection, and wasted were observed prior to euthanasia. The cause of the death was the poor clinical condition, which was probably treatment related, but no cause of the clinical effects was evident from the macroscopic or histological evaluation. One Low dose female (#2510) was preterminally euthanised on PND0 because of prolapsed vagina, considered to be unrelated to treatment. Moderate paleness (whole body) and piloerection were recorded prior to death.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Test item related adverse effect was observed on body weight gain parameters in High dose (90 mg/kg bw/day) males and females and Mid dose females (45 mg/kg bw/day). No test item related adverse effect on body weight or body weight gain was detected in Mid dose (45 mg/kg bw/day) male or Low dose (15 mg/kg bw/day) animals (males or females).
In the first week, High dose males lost 8.5% of their bodyweight, with weight gain thereafter. This High dose body weight loss, in the first week, was considered as a test item related effect.
No effect on body weight parameters was seen in Mid and Low dose males.
In case of females, the observed body weight or body weight gain values were comparable to the control level at the end of the pre-mating period in all dose groups. The Mid and High dose females showed reduced body weight / body weight gain at the end of the gestation period (body weight was lower by 7.8% (p<0.05) and 16.7% (p<0.01), body weight gain by 21.8% (p<0.01) and 42.2% (p<0.01)).
In case of High dose females, statistically significantly lower body weight parameters were recorded by the end of the lactation period (by 19.5% (p<0.01) lower body weight and by 87.4% (p<0.01) lower body weight gain), with virtually no weight gain at all during the lactation period. The body weight gain from Day 0 to PPD 13 was statistically significantly lower for Mid dose and High dose females than Control (by 14.8% (p<0.05) and 70.6% (p<0.01)). Body weight data from PND 7 in the High dose were outside of the historical control range. These body weight effects were considered as being a test item related adverse effect.
No effect on body weight parameters was seen in Low dose females.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Test item related adverse effects were observed on food consumption in High dose males and females (90 mg/kg bw/day) and Mid dose females (45 mg/kg bw/day) during the treatment period; Mid and Low dose males and Low dose females were not affected.
Statistically significantly decreased (p<0.01) food consumption was observed in the High dose male group (90 mg/kg bw/day) at an average of ~30% below control for the 4-week period. The other groups (Mid and Low) were unaffected.
Statistically significantly decreased food consumption was observed in the Mid (p<0.05) and High dose (p<0.05 and p<0.01) female groups during gestation and lactation periods. Premating the female food intakes were not significantly different to control (all ~11-15g/day); during gestation although there were statistical differences, all groups were within about 15% of the controls; during lactation normal control food intake goes up dramatically (from about 35g/day initially, up to ~70g/day by Days 10-13) but the High dose group had no normal increased food intake and remained at the pre-lactation range of generally <20g/day, the Mid dose group were generally within 15-20% of the control values, which is a relatively slight effect. Together with the body weight data, the observed values were considered as a test item related adverse effect, but at the High dose the very low food intake could not support a normal high-energy-requirement for the level of lactation needed for pup growth.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

There were statistically significantly changes in the male dosed groups sporadically, but all the data were within the historical control range therefore they were not considered as test item related effects. There was statistically significantly decreased reticulocyte relative (%) and mean cell volume observed in the High dose female group and the values were outside of the historical control range, hence it was considered as test item related effect.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

There were test item related effects observed in the High dose male and female groups on clinical chemistry parameters. There were no significant changes or biologically relevant effects on the serum chemistry that could be ascribed to the test item administration in the Low dose group for both sexes.
Statistically significantly decreased cholesterol concentration was observed in Mid dose male and High dose males and females (p<0.01), the data were outside of the historical control range. But this fact was most probably related to the significantly reduced food consumption of animals during the gestation and lactation periods, not considered as being a direct adverse effect.
Statistically significantly decreased total protein (p<0.01) was measured in the High dose male group (possibly related to food intake) but the data were within the historical control range.
Statistically significantly increased A/G ratio (p<0.05) was measured in the High dose male group, the data were outside of the historical control range, hence it was considered as test item related effect but probably related to food intake. There was statistically significantly increased (p<0.01) bile acid concentration observed in the High dose male group, the data were outside of the historical control range, therefore it was considered as a possible test item related effect. Statistically significantly increased chloride concentration (p<0.01) and decreased phosphorus concentration (p<0.05) was observed in the High dose female group, but the data were within the historical control range.
Alanine aminotransferase (ALT/GPT) activity was significantly increased in the Mid dose males (p<0.01) and High dose males (p<0.01) and females (p<0.01) when compared to control animals. In case of High dose females, the observed values were outside of the historical control range. Furthermore, increased Alkaline phosphate (ALKP) activity was recorded in the Mid dose males and females (p<0.01) and High dose males and females (p<0.01), the observed female mean values were within of the historical control range, the High dose male value was outside of the historical control range.
Statistically significantly increased urea concentration (p<0.01) was observed in the Mid dose female groups, which was outside of the historical control range but without dose response it was not considered as test item related effect. Statistically significantly increased potassium and calcium concentration (p<0.05) and was observed in the Mid dose female groups, which were within the historical control range.
Observed values of alanine aminotransferase (ALT/GPT), A/G (albumin/globulin) ratio and bile acid concentration in case of the High dose males were considered as test item related but none were of a magnitude to be considered clearly adverse. These changes were probably related to the low food intakes and/or the histopathology findings in the liver.

Endocrine findings:

no effects observed

Description (incidence and severity):

In the parameters measured in adults and pups, there were no effects on thyroids or other endocrine systems.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

No test item-related changes were observed in the urinalysis parameters in male and female animals of any dose groups when compared to control.
Statistically significantly increased urinary blood (p<0.01) was observed in the High dose male group but in the absence of any histopathological kidney changes are not considered as a clear adverse effect of the test item. Statistically significantly decreased specific gravity (p<0.01) and protein level (p<0.05) was observed in the High dose female group. The specific gravity data were outside of the historical control range. Statistically significantly increased urine crystals (p<0.01) were observed in the High dose female group.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

There were no changes in animal behaviour, general physical condition or in the reactions to different type of stimuli in the control or test groups.
There was no effect of treatment noted in the Irwin test or during the assessment of grip strength. There was statistically significantly increased (p<0.05) landing splay (Hind paws) observed in the High dose male group, but the data were within the historical control range therefore these changes did not consider as test item related effects. There was statistically significantly increased (p<0.05) landing splay (Fore paws) observed in the Low and Mid dose male groups.
All dose groups of males and females had comparable locomotor activity to the Control. There was statistically significantly increased LMA distance (10-15 min) was observed in the Low dose male group without dose response. In all cases, the initial activity was high, with reduced activity in each 5-minute period to an approximate plateau by about 20-30 minutes. The test item did not increase or decrease the normal locomotor activity, all treated groups had a profile of activity the same as historical control data.

Immunological findings:

no effects observed

Description (incidence and severity):

In the parameters measured in adults and pups, there was no evidence for immunological effects in immunocompetent organs or tissues.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Parental Males:
Test item-related changes were observed in the organ weights of the liver of Mid and High dose male animals and in the prostate and seminal vesicles in High dose males compared to controls.
Statistically significantly decreased kidney (absolute (p<0.05) and brain related (p<0.01)) weight was observed in the High dose male group, but not when adjusted for body weight hence it was considered as not a test item related effect.
Statistically significantly increased liver (absolute, body and brain related (p<0.01)) weight was observed in the High dose male group and body related liver weight in the Mid dose group. The absolute and relative to body weight of the liver in the High dose group were outside of the historical control range and these statistically significant weight changes were correlated with macroscopic and microscopic findings therefore they were considered as test item related effect.
Statistically significantly decreased prostate gland (absolute, body and brain related (p<0.05)) weight was observed in the High dose male group, the data were within the historical control range, but based on the histopathological changes, the weight difference is considered to be adverse but probably a result of low food intake and ~10% body weight loss in the first 7 days of the study.
The relative to brain weight of the seminal vesicle decreased by -19.8% in High dose males compared to controls. The data were within the historical control range, but based on the histopathological changes, the weight difference is considered to be adverse but probably a result of low food intake and ~10% body weight loss in the first 7 days of the study.
The other statistically significant organ weight changes were within the historical control range and without histological relevance and were considered as incidental.

Parental Females:
Statistically significant organ weight changes were seen in the brain, heart, kidney, spleen, uterus and ovary. The data did not show dose-relation and/or were without histological relevance therefore they were considered as incidental.
Statistically significantly decreased body weight of the High dose females was observed, the data were outside of the historical control range.
Statistically significantly increased brain weight related to body was observed in the High dose female group. The data were considered to be due to lower body weight and were without histopathology findings, it was not considered as test item related adverse effect.
Statistically significantly decreased absolute, body and brain related (p<0.01) weight of the heart was measured in the High dose female group and statistically significantly decreased relative to brain and absolute weight was measured in the Mid dose group. The absolute and brain related values were outside of the historical control range. However, in the absence of any histopathological cardiac changes, the weight difference is not considered to be adverse.
Statistically significantly decreased kidney (absolute (p<0.01) and brain related (p<0.01)) weight was observed in the High dose female group, but not for the body weight relative mean. Since these rats were smaller, lower kidney weights are expected, also there were no histopathological changes, hence this kidney weight difference is not considered to be adverse.
Other statistical differences did not show dose-relation, were a reflection of lower body weight at the High dose and/or were without histological relevance and were considered as incidental.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

-NON-PREGNANT FEMALES / Parental Generation
Two females (Low dose #2512 and High dose #4505) were non pregnant during the study.
At necropsy no test item-related changes were seen in these females or their male pair, in the female #4505 the ovaries were small, the uterine horns were dilated and were considered as incidental, in female #2512 no macroscopic findings were noted. In both males (#2012 and #4005) the mandibular lymph nodes were dark red and were considered as procedure related (due to sublingual blood sampling).
- FOUND DEAD ANIMAL / Parental Generation
One Control male (#1009) died on Day 28 of the study. The cause of death could not be determined.
Macroscopic Findings:
The lungs, the thymus and the liver were dark red, focal red discoloration was seen in the glandular mucosa of the stomach.
-PRE-TERMINAL EUTHANASIA / Parental Generation
One Low Dose female (#2510) was preterminally euthanised on Day 37 (after giving birth) due to prolapsed vagina. One High Dose Male (#4004) was preterminally euthanised on Day 14 of the study due to weak general condition.
Macroscopic Findings
Prolapsed vagina and invagination of the uterine horn was seen at necropsy for the Low dose female; there were no significant findings in the male.
-TERMINAL EUTHANASIA / Parental Generation
Macroscopic Findings:
Test item-related pale discoloration of the liver were observed in 3/11 High dose males and in 7/7 High dose females. The prostate and seminal vesicles were small in 1/11 High dose male, considered as test item-related; seminal vesicles were also small in 1/12 Low dose males, of uncertain relationship with treatment.
All other changes were considered procedure related, incidental or a common background.

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

-NON-PREGNANT FEMALES / Parental Generation
Microscopically in the #4505 female ovarian atrophy and dilatation of uterine horns were seen and considered to be incidental, in the male pair (#4005) of this female, minimal multifocal hepatocellular vacuolation, and minimal decreased secretion in the prostate and seminal vesicle were present (similarly to the terminal animals) which were considered as test item-related. Other findings in these two animals were considered procedure related, incidental or a common background.
In the #2512 female no microscopic findings were noted, in the male pair (#2012) of this female sinusoidal erythrocytosis was present in the mandibular lymph nodes, as procedure related (due to sublingual blood sampling).
-FOUND DEAD ANIMAL / Parental Generation
Microscopic Findings: Microscopically congestion/haemorrhage in the lungs and thymus; these were considered as agonal, or incidental.
-PRE-TERMINAL EUTHANASIA / Parental Generation
Microscopic Findings: No test item-related changes were observed microscopically for the Low dose female. In case of the High dose male the mandibular lymph nodes were dark red, the thymus was small, on the glandular gastric mucosa white and red foci were present at necropsy, microscopically and were considered as not test item related. The cause of termination of the male was considered to be treatment related clinical signs, but there was no evidence of pathological changes that could indicate the aetiology of the adverse clinical signs.
-TERMINAL EUTHANASIA / Parental Generation
Microscopic Findings: Test item-related multifocal/diffuse (mainly multifocal) hepatocellular vacuolation was detected in 10/11 High dose, in 4/12 Mid dose males and in 7/7 High dose and in 7/12 Mid dose females with minimal to marked severity (typically minimal/mild severity in males and moderate in the females), correlated with organ weight changes and necropsy findings.
In High dose males, in the prostate decreased secretion was seen in 7/11 cases, in the Mid dose in 2/12 cases with minimal/mild severity. Decreased secretion was detected in the seminal vesicles in 4/11 High dose males (correlated with organ weight changes) and were considered as test item related. Decreased secretion was detected in the seminal vesicles in 1/9 Low dose male as well and was considered as incidental. Similar findings in secondary sex organs are common when animals have a significant body weight loss in the first week of treatment.
In the kidney of all examined High dose males, minimal multifocal eosinophilic droplets/globules were seen in the renal tubules and were considered as test item related. These hyaline droplets are most likely to be Alpha 2u globulin which is a low molecular weight protein produced by the liver and excreted through kidney in male rats only; in the context of human safety evaluation, this rat-specific change is considered to be non-adverse.
All other findings were considered as incidental or background and not to represent adverse effects of the test item.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

Mammalian erythrocyte micronucleus test:
All groups treated with the test item which had a higher average number of micronuclei compared with the corresponding negative control group were tested for statistical significance using the Kruskal Wallis test. None were statistically significant, giving a negative response. Thus, the test item gave a negative response in all treatment groups, there was no evidence of any genotoxicity.
The positive and negative control results were also compared, but although the average number of micronuclei was slightly increased in the males, and almost double in the females, neither gave a statistically significant increase. However, the positive control animals were treated separately from the main experiment and therefore this outcome does not invalidate the study. The positive control values are within the historical control range (males 1 – 43, females 5 – 162). It was also noted that the ratio of PCE:NCE/1000 erythrocytes was also lower than the corresponding negative control group, indicating cytotoxicity in the positive control animals. This is generally associated with cell cycle delay and can result in cells with micronuclei having insufficient time to develop into PCEs in the positive controls.

Details on results:

-mortality and morbidity:
No test item related mortality was observed in the study.
One Control male (#1009) was found dead on Day 28. No clinical sings were recorded prior to death. The cause of death could not be determined.
One High dose male (#4004) was preterminally euthanised on Day 14. Hunched back, piloerection, and wasted were observed prior to euthanasia. The cause of the death was the poor clinical condition, which was probably treatment related, but no cause of the clinical effects was evident from the macroscopic or histological evaluation. One Low dose female (#2510) was preterminally euthanised on PND0 because of prolapsed vagina, considered to be unrelated to treatment. Moderate paleness (whole body) and piloerection were recorded prior to death.
-clinical observations:
No test item related clinical signs were observed in the study.
Liquid faeces were recorded during the lactation period (Day 52) for one Low dose female (#2503) and on Day 51 for a Mid dose female (#3505).
Red liquid in the vulva was observed for one High dose female (#4509) on Day 33 and 34. Piloerection was observed for this animal on Day 16.
Noisy respiration was observed for one Control female (#1503) on Day 6 and 7.
Hunched back and piloerection were observed for one Control female (#1510) form Day 17 to Day 20.
These findings were all considered as being incidental, not related to the test item administration.
-body weight and body weight gain:
Test item related adverse effect was observed on body weight gain parameters in High dose (90 mg/kg bw/day) males and females and Mid dose females (45 mg/kg bw/day). No test item related adverse effect on body weight or body weight gain was detected in Mid dose (45 mg/kg bw/day) male or Low dose (15 mg/kg bw/day) animals (males or females).
In the first week, High dose males lost 8.5% of their bodyweight, with weight gain thereafter. This High dose body weight loss, in the first week, was considered as a test item related effect.
No effect on body weight parameters was seen in Mid and Low dose males.
In case of females, the observed body weight or body weight gain values were comparable to the control level at the end of the pre-mating period in all dose groups . The Mid and High dose females showed reduced body weight / body weight gain at the end of the gestation period (body weight was lower by 7.8% (p<0.05) and 16.7% (p<0.01), body weight gain by 21.8% (p<0.01) and 42.2% (p<0.01)).
In case of High dose females, statistically significantly lower body weight parameters were recorded by the end of the lactation period (by 19.5% (p<0.01) lower body weight and by 87.4% (p<0.01) lower body weight gain), with virtually no weight gain at all during the lactation period. The body weight gain from Day 0 to PPD 13 was statistically significantly lower for Mid dose and High dose females than Control (by 14.8% (p<0.05) and 70.6% (p<0.01)). Body weight data from PND 7 in the High dose were outside of the historical control range. These body weight effects were considered as being a test item related adverse effect.
No effect on body weight parameters was seen in Low dose females.
-food consumption:
Test item related adverse effects were observed on food consumption in High dose males and females (90 mg/kg bw/day) and Mid dose females (45 mg/kg bw/day) during the treatment period; Mid and Low dose males and Low dose females were not affected.
Statistically significantly decreased (p<0.01) food consumption was observed in the High dose male group (90 mg/kg bw/day) at an average of ~30% below control for the 4-week period. The other groups (Mid and Low) were unaffected.
Statistically significantly decreased food consumption was observed in the Mid (p<0.05) and High dose (p<0.05 and p<0.01) female groups during gestation and lactation periods. Premating the female food intakes were not significantly different to control (all ~11-15g/day); during gestation although there were statistical differences, all groups were within about 15% of the controls; during lactation normal control food intake goes up dramatically (from about 35g/day initially, up to ~70g/day by Days 10-13) but the High dose group had no normal increased food intake and remained at the pre-lactation range of generally <20g/day, the Mid dose group were generally within 15-20% of the control values, which is a relatively slight effect. Together with the body weight data, the observed values were considered as a test item related adverse effect, but at the High dose the very low food intake could not support a normal high-energy-requirement for the level of lactation needed for pup growth.
-neurological assessment:
There were no changes in animal behaviour, general physical condition or in the reactions to different type of stimuli in the control or test groups.
There was no effect of treatment noted in the Irwin test or during the assessment of grip strength. There was statistically significantly increased (p<0.05) landing splay (Hind paws) observed in the High dose male group, but the data were within the historical control range therefore these changes did not consider as test item related effects. There was statistically significantly increased (p<0.05) landing splay (Fore paws) observed in the Low and Mid dose male groups.
All dose groups of males and females had comparable locomotor activity to the Control. There was statistically significantly increased LMA distance (10-15 min) was observed in the Low dose male group without dose response. In all cases, the initial activity was high, with reduced activity in each 5-minute period to an approximate plateau by about 20-30 minutes. The test item did not increase or decrease the normal locomotor activity, all treated groups had a profile of activity the same as historical control data.
-Haematology
There were statistically significantly changes in the male dosed groups sporadically, but all the data were within the historical control range therefore they were not considered as test item related effects. There was statistically significantly decreased reticulocyte relative (%) and mean cell volume observed in the High dose female group and the values were outside of the historical control range, hence it was considered as test item related effect.
-Clinical chemistry
There were test item related effects observed in the High dose male and female groups on clinical chemistry parameters. There were no significant changes or biologically relevant effects on the serum chemistry that could be ascribed to the test item administration in the Low dose group for both sexes.
Statistically significantly decreased cholesterol concentration was observed in Mid dose male and High dose males and females (p<0.01), the data were outside of the historical control range. But this fact was most probably related to the significantly reduced food consumption of animals during the gestation and lactation periods, not considered as being a direct adverse effect.
Statistically significantly decreased total protein (p<0.01) was measured in the High dose male group (possibly related to food intake) but the data were within the historical control range.
Statistically significantly increased A/G ratio (p<0.05) was measured in the High dose male group, the data were outside of the historical control range, hence it was considered as test item related effect but probably related to food intake. There was statistically significantly increased (p<0.01) bile acid concentration observed in the High dose male group, the data were outside of the historical control range, therefore it was considered as a possible test item related effect. Statistically significantly increased chloride concentration (p<0.01) and decreased phosphorus concentration (p<0.05) was observed in the High dose female group, but the data were within the historical control range.
Alanine aminotransferase (ALT/GPT) activity was significantly increased in the Mid dose males (p<0.01) and High dose males (p<0.01) and females (p<0.01) when compared to control animals. In case of High dose females, the observed values were outside of the historical control range. Furthermore, increased Alkaline phosphate (ALKP) activity was recorded in the Mid dose males and females (p<0.01) and High dose males and females (p<0.01), the observed female mean values were within of the historical control range, the High dose male value was outside of the historical control range.
Statistically significantly increased urea concentration (p<0.01) was observed in the Mid dose female groups, which was outside of the historical control range but without dose response it was not considered as test item related effect. Statistically significantly increased potassium and calcium concentration (p<0.05) and was observed in the Mid dose female groups, which were within the historical control range.
Observed values of alanine aminotransferase (ALT/GPT), A/G (albumin/globulin) ratio and bile acid concentration in case of the High dose males were considered as test item related but none were of a magnitude to be considered clearly adverse. These changes were probably related to the low food intakes and/or the histopathology findings in the liver.
-Urinalysis
No test item-related changes were observed in the urinalysis parameters in male and female animals of any dose groups when compared to control.
Statistically significantly increased urinary blood (p<0.01) was observed in the High dose male group but in the absence of any histopathological kidney changes are not considered as a clear adverse effect of the test item. Statistically significantly decreased specific gravity (p<0.01) and protein level (p<0.05) was observed in the High dose female group. The specific gravity data were outside of the historical control range. Statistically significantly increased urine crystals (p<0.01) were observed in the High dose female group.
-thyroid hormone analysis
There was no test item related effect on the thyroid hormone concentration levels recorded in the PND13 pups and for the males.
The terminal body weight of the pups was lower compared to the Control therefore the thyroid gland weights of the PND13 pups were lower as well, but the thyroid weight adjusted for body weight was unaffected. There was statistically significantly decreased only for the absolute pup thyroid gland weight compared to the Control. In summary, there were considered to be no effects on the thyroid hormone levels or on the thyroid gland weights in the PND13 pups that were ascribed to the test item.
Statistically significantly decreased T4 concentration was measured in the Low dose group but the data were within the historical control range.
-organ weights:
Parental Males
Test item-related changes were observed in the organ weights of the liver of Mid and High dose male animals and in the prostate and seminal vesicles in High dose males compared to controls.
Statistically significantly decreased kidney (absolute (p<0.05) and brain related (p<0.01)) weight was observed in the High dose male group, but not when adjusted for body weight hence it was considered as not a test item related effect.
Statistically significantly increased liver (absolute, body and brain related (p<0.01)) weight was observed in the High dose male group and body related liver weight in the Mid dose group. The absolute and relative to body weight of the liver in the High dose group were outside of the historical control range and these statistically significant weight changes were correlated with macroscopic and microscopic findings therefore they were considered as test item related effect.
Statistically significantly decreased prostate gland (absolute, body and brain related (p<0.05)) weight was observed in the High dose male group, the data were within the historical control range, but based on the histopathological changes, the weight difference is considered to be adverse but probably a result of low food intake and ~10% body weight loss in the first 7 days of the study.
The relative to brain weight of the seminal vesicle decreased by -19.8% in High dose males compared to controls. The data were within the historical control range, but based on the histopathological changes, the weight difference is considered to be adverse but probably a result of low food intake and ~10% body weight loss in the first 7 days of the study.
The other statistically significant organ weight changes were within the historical control range and without histological relevance and were considered as incidental.
Parental Females
Statistically significant organ weight changes were seen in the brain, heart, kidney, spleen, uterus and ovary. The data did not show dose-relation and/or were without histological relevance therefore they were considered as incidental.
Statistically significantly decreased body weight of the High dose females was observed, the data were outside of the historical control range.
Statistically significantly increased brain weight related to body was observed in the High dose female group. The data were considered to be due to lower body weight and were without histopathology findings, it was not considered as test item related adverse effect.
Statistically significantly decreased absolute, body and brain related (p<0.01) weight of the heart was measured in the High dose female group and statistically significantly decreased relative to brain and absolute weight was measured in the Mid dose group. The absolute and brain related values were outside of the historical control range. However, in the absence of any histopathological cardiac changes, the weight difference is not considered to be adverse.
Statistically significantly decreased kidney (absolute (p<0.01) and brain related (p<0.01)) weight was observed in the High dose female group, but not for the body weight relative mean. Since these rats were smaller, lower kidney weights are expected, also there were no histopathological changes, hence this kidney weight difference is not considered to be adverse.
Other statistical differences did not show dose-relation, were a reflection of lower body weight at the High dose and/or were without histological relevance and were considered as incidental.
-pathology evaluation:
NON-PREGNANT FEMALES / Parental Generation
Two females (Low dose #2512 and High dose #4505) were non pregnant during the study.
At necropsy no test item-related changes were seen in these females or their male pair, in the female #4505 the ovaries were small, the uterine horns were dilated and were considered as incidental, in female #2512 no macroscopic findings were noted. In both males (#2012 and #4005) the mandibular lymph nodes were dark red and were considered as procedure related (due to sublingual blood sampling).
Microscopically in the #4505 female ovarian atrophy and dilatation of uterine horns were seen and considered to be incidental, in the male pair (#4005) of this female, minimal multifocal hepatocellular vacuolation, and minimal decreased secretion in the prostate and seminal vesicle were present (similarly to the terminal animals) which were considered as test item-related. Other findings in these two animals were considered procedure related, incidental or a common background.
In the #2512 female no microscopic findings were noted, in the male pair (#2012) of this female sinusoidal erythrocytosis was present in the mandibular lymph nodes, as procedure related (due to sublingual blood sampling).

FOUND DEAD ANIMAL / Parental Generation
One Control male (#1009) died on Day 28 of the study. The cause of death could not be determined.
Macroscopic Findings
The lungs, the thymus and the liver were dark red, focal red discoloration was seen in the glandular mucosa of the stomach.
Microscopic Findings
Microscopically congestion/haemorrhage in the lungs and thymus; these were considered as agonal, or incidental.

PRE-TERMINAL EUTHANASIA / Parental Generation
One Low Dose female (#2510) was preterminally euthanised on Day 37 (after giving birth) due to prolapsed vagina. One High Dose Male (#4004) was preterminally euthanised on Day 14 of the study due to weak general condition.
Macroscopic Findings
Prolapsed vagina and invagination of the uterine horn was seen at necropsy for the Low dose female; there were no significant findings in the male.
Microscopic Findings
No test item-related changes were observed microscopically for the Low dose female. In case of the High dose male the mandibular lymph nodes were dark red, the thymus was small, on the glandular gastric mucosa white and red foci were present at necropsy, microscopically and were considered as not test item related. The cause of termination of the male was considered to be treatment related clinical signs, but there was no evidence of pathological changes that could indicate the aetiology of the adverse clinical signs.

TERMINAL EUTHANASIA / Parental Generation
Macroscopic Findings
Test item-related pale discoloration of the liver were observed in 3/11 High dose males and in 7/7 High dose females. The prostate and seminal vesicles were small in 1/11 High dose male, considered as test item-related; seminal vesicles were also small in 1/12 Low dose males, of uncertain relationship with treatment.
All other changes were considered procedure related, incidental or a common background.
Microscopic Findings
Test item-related multifocal/diffuse (mainly multifocal) hepatocellular vacuolation was detected in 10/11 High dose, in 4/12 Mid dose males and in 7/7 High dose and in 7/12 Mid dose females with minimal to marked severity (typically minimal/mild severity in males and moderate in the females), correlated with organ weight changes and necropsy findings.
In High dose males, in the prostate decreased secretion was seen in 7/11 cases, in the Mid dose in 2/12 cases with minimal/mild severity. Decreased secretion was detected in the seminal vesicles in 4/11 High dose males (correlated with organ weight changes) and were considered as test item related. Decreased secretion was detected in the seminal vesicles in 1/9 Low dose male as well and was considered as incidental. Similar findings in secondary sex organs are common when animals have a significant body weight loss in the first week of treatment.
In the kidney of all examined High dose males, minimal multifocal eosinophilic droplets/globules were seen in the renal tubules and were considered as test item related. These hyaline droplets are most likely to be Alpha 2u globulin which is a low molecular weight protein produced by the liver and excreted through kidney in male rats only; in the context of human safety evaluation, this rat-specific change is considered to be non-adverse.
All other findings were considered as incidental or background and not to represent adverse effects of the test item.

-mammalian erythrocyte micronucleus test
All groups treated with the test item which had a higher average number of micronuclei compared with the corresponding negative control group were tested for statistical significance using the Kruskal Wallis test. None were statistically significant, giving a negative response. Thus, the test item gave a negative response in all treatment groups, there was no evidence of any genotoxicity.
The positive and negative control results were also compared, but although the average number of micronuclei was slightly increased in the males, and almost double in the females, neither gave a statistically significant increase. However, the positive control animals were treated separately from the main experiment and therefore this outcome does not invalidate the study. The positive control values are within the historical control range (males 1 – 43, females 5 – 162). It was also noted that the ratio of PCE:NCE/1000 erythrocytes was also lower than the corresponding negative control group, indicating cytotoxicity in the positive control animals. This is generally associated with cell cycle delay and can result in cells with micronuclei having insufficient time to develop into PCEs in the positive controls.

Effect levels

Key result

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Target system / organ toxicity

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Any other information on results incl. tables

Validation of Analytical Method for the Determination of Butanedioic acid, sulfo-, mono (C16- 18 and C18-unsatd. alkyl) esters, ammonium sodium salts (CAS 147993-66-6)

from Gavage Formulation

The purpose of this study was to validate the developed analytical method under the study code: FPBSTUDY-229-MD1 (CRL-VES study code 20/127-901ANE) for the quantitative determination of Butanedioic acid, sulfo-, mono (C16-18 and C18-unsatd. alkyl) esters, ammonium sodium salts from gavage formulations.

 

The procedure was found to be suitable for the analysis of the test item from gavage formulations. The validation parameters met the requirements.

Summary of the validated method parameters is presented in the table below.

Table. Results of the method validation

Validated parameter	Acceptance criteria	Measured values
Specificity	Interference in blank samples must be less than 20.0% of the lowest concentration of the calibration curve.	Interfering components conform to less than 20% of the 10 ng/mL (LOQ) criteria
Repeatability (7 injections)	Areas and retention times of reinjected sample should be RSD < 5.0%	0.17% RSD for Retention time of test item 2.45% RSD for Area of test item
Detector response in 10 – 200 ng/mL concentration range	Correlation coefficient is above of 0.99	R^2 ≥ 0.9962
	Re-calculated concentration values at LOQ level are in 80.0 – 120.0% of the nominal concentrations	91.7 – 119.5
	Re-calculated concentration values above LOQ level are in 85.0 – 115.0% of the nominal concentrations	87.4 – 106.4%
Limit of Quantification (3 injections)	Re-calculated concentration values at LOQ level are in 80.0 – 120.0% of the nominal concentrations	10 ng/mL in calibration solution 0.1 mg/mL in formulation (10000x dilution) 91.7 – 115.7%
	CV% < 20.0%	11.9 %
Recovery and precision
Concentration mg/mL		1	60	200
Recovery (%)	90.0 – 110.0%	104.9	102.8	102.3
Precision (%)	Less than 10.0% CV	0.31	1.86	1.70
Stability
Stability of analytical sample in autosampler for 18h 24 min (%)	Considered stable if the measured concentration is 90 – 110% of the initial concentration	98.3
Concentration (mg/mL)		1	60	200
Stability at 20±5°C – 7 days (%)	Considered stable if the measured concentration is 90 – 110% of the initial concentration	98.7	99.6	101.8
Stability under transportation conditions 20±5°C – 16 days (%)		100.6	101.9	101.8

Applicant's summary and conclusion

Conclusions:

The NOAEL for systemic toxicity of the parental generation was considered to be
15 mg/kg bw/day. (based on a strong effect on body weight and food consumption in the High dose animals, and a slight but statistically significant effect in the Mid dose group).
No test item-related effect, thus no evidence of genotoxic activity was seen in the Mammalian Erythrocyte Micronucleus Test.

Executive summary:

The purpose of this OECD No. 422 study was to obtain information on the possible toxic effects of the test item Butanedioic acid, sulfo-, mono (C16-18 and C18-unsatd. alkyl) esters, ammonium sodium salts following repeated (daily) administration by oral gavage to Wistar (Crl:WI) rats at 3 dose levels. A control group received the vehicle only (propylene glycol).

The study also comprised a reproductive/developmental toxicity screening test, intended to provide initial information on possible effects on male and female reproductive performance such as gonadal function, mating behaviour, conception, pregnancy, parturition and also on the development of the F1 offspring from conception to Post-natal Day (PND) 13.

The dose levels were selected by the Sponsor in consultation with the Study Director based on the results of a Dose Range Finding (DRF) study. Based on the results of the DRF study, 90 mg/kg bw/day was selected as the High dose for this study. Five female and five male animals served as positive control for the Mammalian Erythrocyte Micronucleus Test (MNT).

Experimental design:

Group Number	Group designation	Dose level (mg/kg bw/day)	Concentration (mg/mL)	Dose volume (mL/kg bw)	Animal numbers
					Male	Female
1	Control	0	0	5	1001-1012	1501-1512
2	Low Dose	15	3		2001-2012	2501-2512
3	Mid Dose	45	9		3001-3012	3501-3512
4	High Dose	90	18		4001-4012	4501-4512

 

Gr. No.	Group Designation	Cyclophosphamide Dose Level (mg/kg bw)	Cyclophosphamide Conc. (mg/mL)	Dose volume (mL/kg bw)	Animal Numbers
					Male	Female
5	Positive control MNT	60	12	5	5	5

 

Parameters measured during the study included twice a day mortality checking, daily routine and weekly detailed observation of clinical signs, weekly body weight and food consumption measurements and clinical pathology evaluation (including haematology, coagulation, clinical chemistry and urinalysis).

Neurological assessment (Functional Observation Battery (FOB) including measurements of the landing foot splay, grip strength as well as locomotor activity measurement) was performed during the last week of the treatment for each sex. In addition, the reproductive performance, pregnancy, parturition and postpartum/lactation period were monitored in the adult animals, and viability, clinical signs and development were evaluated in their F1 offspring until PND13.

At termination (Day 28 for males, PPD (Post-partum Day) 14 for females), necropsy with macroscopic examination was performed. Weights of selected organs were recorded, and representative tissues/organs were sampled and preserved in appropriate fixatives from the adult animals or F1 animals. The thyroxine (T4) levels in the PND (Post-natal Day) 13 pups and parental males were also determined.

For the adult animals, a detailed histological examination was performed on the selected list of retained organs of 5 animals/sex in the Control and High dose groups, the retained reproductive organs of all Control and High dose animals, the found dead animal or all organs with relevant macroscopic changes, and the male / female mating pair where no liveborn pups were achieved.

 

RESULTS

In summary, under the conditions of this study the daily administration of Butanedioic acid, sulfo-, mono (C16-18 and C18-unsatd. alkyl) esters, ammonium sodium salts by oral gavage to Wistar rats at dose levels of 15, 45 or 90 mg/kg bw/day (Low, Mid and High dose groups, respectively) did not result in test item related mortality or clinical signs in the majority of animals. One High dose male had clinical signs of weakness and general poor condition, it was terminated early, but there was no pathology found that could indicate the aetiology of the test item effects; however the early termination is ascribed to treatment.

Test item related adverse effect was observed on body weight, body weight gain parameters and food consumption in High dose (90 mg/kg bw/day) males and females. Test item related adverse effect was observed on body weight, body weight gain parameters and food consumption in Mid dose (45 mg/kg bw/day) females. In males, the High dose animals lost ~8.5% body weight in the first week of the study but gained weight thereafter; lower food intake in the High dose female group during lactation would have resulted in lower milk production (associated with loss of all pups in this group).

There were no changes in animal behaviour, general physical condition or in the reactions to different type of stimuli in test item treated groups when compared to control.

Statistically significant haematological changes comprised decreased reticulocytes (%) in the High dose female group with the value was outside of the historical control range, which was considered a test item related effect. Statistically significant clinical chemistry changes included increased alanine aminotransferase, ALT/GPT ratio and bile acid concentration in case of the High dose males were considered as test item related but none were of a magnitude to be considered clearly adverse. These changes were probably related to the low food intakes and/or the histopathology findings in the liver. No test item-related changes were observed in the urinalysis parameters in male and female animals of any dose groups when compared to control.

Eleven females in the High dose group were pregnant. However, one female had no live born pups and four pregnant females had no delivery (100% of the embryos was resorbed). High dose pups who were live born were dead in three days after birth the latest. It was considered as test item related adverse effect.

There were severe test item related effects on pup mortality or survival of the pups (F1 generation) in the High dose groups; slight but similar tendency was observed in the Mid dose group which was considered to be adverse.

In summary for the reproductive effects, the changes in High dose males were most probably secondary to significant body weight loss during the study. However, in females a significant reduction in implantations was not associated with body weight or food intake effect and as such is probably a reprotoxic effect at the High dose. Death/resorption of embryos/foetuses at the High dose were treatment related, reduced numbers of pups born, relatively high numbers of pups born dead and the death of all High dose pups by Day 3, were considered as treatment related. During gestation, the lower body weight gain at the High dose was probably a reflection of the presence of fewer foetuses. It is not considered likely that the lower body weight of the dams caused to foetal losses. During gestation all High dose pups died, and the dams had a food intake similar to females with no litter; it is not possible to be sure if the low food intake caused low milk production and pup death, or if the lack of pups resulted in less milk production hence a lower food intake, although this was clearly a treatment related effect. In the Mid dose, there were statistically significant differences suggesting a similar trend to the High dose, a dose response could be observed from the data on these parameters.

Test item-related pale discoloration of the liver were observed in 3/11 High dose males and in 7/7 High dose females. The prostate and seminal vesicles were small in 1/11 High dose male, considered as test item-related; seminal vesicles were also small in 1/12 Low dose males, of uncertain relationship with treatment.

Test item-related changes were observed in the parental organ weights of the liver of Mid (~14%) and High dose male animals (~35%). Multifocal/diffuse hepatocellular vacuolation at 90 mg/kg bw/day dose level both in males and females were observed, ascribed to test item, but not considered as adverse. Reduced weights of the prostate and seminal vesicles in High dose males, compared to controls, was probably secondary to the ~8.5% body weight loss in the first week of the study. No adverse test item related changes were observed in the female animal organ weights.

In the parameters measured in adults and pups, there was no evidence for any neurotoxicity and no effects on thyroids or other endocrine systems, and no evidence for immunological effects in immunocompetent organs or tissues.

No test item-related effect, thus no evidence of genotoxic activity was seen in the Mammalian Erythrocyte Micronucleus Test.

The NOAEL for systemic toxicity of the parental generation was considered to be  15 mg/kg bw/day. (based on a strong effect on body weight and food consumption in the High dose animals, and a slight but statistically significant effect in the Mid dose group).

The NOAEL for reproductive effects of the parental generation: 45 mg/kg bw/day (based on marked adverse findings on pregnancy seen only in the High dose group, whereas statistical differences in the Mid dose were consistent with historical control ranges).

The NOAEL for pups’ (F1 generation) development and survival: 15 mg/kg bw/day (based on the death of all High dose pups and based on Mid dose group statistically significant mortality data).