N-(2,3-dihydro-2-oxo-1H-benzimidazol-5-yl)-3-hydroxy-4-[[2,5-dimethoxy-4-[(methylamino)sulphonyl]phenyl]azo]naphthalene-2-carboxamide

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012 to 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study (OECD 422), performed under GLP

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Butyl 2-[[3-[[(2,3-dihydro-2-oxo-1H-benzimidazol-5-yl)amino]carbonyl]-2-hydroxy-1-naphthyl]azo]benzoate

EC Number:

250-800-0

EC Name:

Butyl 2-[[3-[[(2,3-dihydro-2-oxo-1H-benzimidazol-5-yl)amino]carbonyl]-2-hydroxy-1-naphthyl]azo]benzoate

Cas Number:

31778-10-6

Molecular formula:

C29H25N5O5

IUPAC Name:

butyl 2-({2-hydroxy-3-[(2-oxo-2,3-dihydro-1H-benzimidazol-5-yl)carbamoyl]-1-naphthyl}diazenyl)benzoate

Test material form:

solid: nanoform, no surface treatment

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Species/strain: healthy Wistar rats, Crl: WI(Han) (Full Barrier)
- Source: Charles River, 97633 Sulzfeld, Germany (The animals were derived from a controlled full-barrier maintained breeding system (SPF). According to Art. 9.2, No. 7 of the German Act on Animal Welfare [7] the animals were bred for experimental purposes.)
- Sex: male and female; the female animals were non-pregnant and nulliparous
- Age at study initiation: males: 10-11 weeks old, females: 10-11 weeks old.
- Weight at study initiation (allocation of the animals to the experimental groups): males: 272 - 306 g (mean: 290.65 g, +/-20% = 232.52 – 348.78 g)
females: 177 - 208 g (mean: 190.68 g, +/- 20% = 152.54 – 228.81 g)
- Housing: Full barrier in an air-conditioned room. The animals were kept individually in IVC cages (except during the mating period when one female will be paired with one male), type III H, polysulphone cages on Altromin saw fibre bedding (lot no. 261111)
- Diet: Altromin 1324 maintenance diet for rats and mice (lot no. 2355), ad libitum
- Water: tap water, sulphur acidified to a pH of approximately 2.8 (drinking water, municipal residuecontrol, microbiological controls at regular intervals), ad libitum
- Certificates of food, water and bedding are filed at BSL BIOSERVICE
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22+/-3
- Humidity (%): 55+/-10
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

VEHICLE
- Justification for use and choice of vehicle (if other than water): The vehicle was selected based on the test item’s characteristics (stability and
homogeneity) of the test item in the vehicle
- Concentration in vehicle: 25 mg/ml, 75 mg/ml, 250 mg/ml
- Amount of vehicle (if gavage): 4 ml/kg body weight
- Lot/batch no. (if required): Batch No.: MKBF8603V; Manufacturer: Sigma

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: maximum 14 days
- Proof of pregnancy:sperm in vaginal smear referred to as[day 0 of pregnancy
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how):individually

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Each dosing concentration was analysed for nominal concentration. Stability and homogeneity of the test item in the vehicle were analysed for the for the LD, MD, and HD dosing formulation. Samples for the nominal concentration verification were taken in study week 1 (first week of pre mating period), 3 (first week of mating), 5 (gestation) and 7 (gestation/lactation) of control and all treatment groups (16 samples in total). Samples for homogeneity were taken from the top, middle and bottom of HD, MD, and LD preparation in study week 1 and 5 (18 samples in total).
All formulation samples were analyzed on the day of sample collection at BSL BIOSERVICE Scientific Laboratories GmbH according to a phase plan which was amended to the study plan.

Duration of treatment / exposure:

Females:
Maximum exposure of 54 days in total (for meales at least 14 days of pre-mating, up to 14 days of mating, 22 days of gestation and 4 days of post-partum).
Males:
28 days

Frequency of treatment:

The test item is orally administered daily, i.e. 7 days per week in graduated doses

Details on study schedule:

- Age at mating of the mated animals in the study: [12-13] weeks

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 100, 300 and 1000 mg/kg body weight
Basis:
actual ingested

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The highest dose level was chosen with the aim of inducing toxic effects, but no death or severe suffering. Thereafter, a descending sequence of dose levels was selected with a view to demonstrate any dosage related response and NOAEL. The doses were selected on the basis of data from a Dose Range Finding Study.
- Rationale for animal assignment (if not random): random

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once a day

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once a week

BODY WEIGHT: Yes
- Time schedule for examinations: The body weight was recorded once before the assignment to the experimental groups, on the first day of administration and weekly during the treatment period as well as at the end of the study. During pregnancy, females were weighed on gestation days (GD) 0, 7, 14 and 20 and within 24 hours of parturition (day 0 post-partum) as well as day 4 post-partum along with pups. Any animals prematurely sacrificed were weighed prior to the sacrifice.

FOOD CONSUMPTION
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No
Food consumption was measured weekly on the corresponding days of the body weight measurements after the beginning of the dose administration. Food consumption was not measured during the mating period in males and females and the post-mating period in males.

OTHER: Functional Observations
Multiple detailed behavioural observations were made in the week before the first treatment and during the last week of the treatment in 5 randomly selected males /group and in the week before the first treatment as well as on day 3 of the lactation period in 5 randomly selected females / group outside the home cage using a functional observational battery of tests

Sperm parameters (parental animals):

Parameters examined in all male parental generations: testis weight, epididymis weight, sperm motility

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no


PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross external anomalies, weight gain

GROSS EXAMINATION OF DEAD PUPS:
yes, for external abnormalities

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving male animals were sacrificed after the completion of the mating period (total dosing period: 28 days) on study day 29 or 30
- Maternal animals: All surviving female animals were sacrificed on post-natal day 4 along with pups

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.]

HISTOPATHOLOGY / ORGAN WEIGHTS
The wet weight of the organs of 5 sacrificed males and 5 females randomly selected from each group was recorded as soon as possible: liver uterus with cervix, kidneys thymus, adrenals, thyroid/parathyroid glands, testes, spleen, epididymides, brain, prostate, seminal vesicles and coagulating glands, pituitary gland, ovaries, heart
Paired organs were weighed separately. Organ weights of animals found dead or euthanised for animal welfare reasons were not recorded. In addition reproductive organs of all animals were weighed.
A full histopathology was carried out on the preserved organs and tissues of 5 randomly selected male and female animals of the control and high dose groups which were sacrificed at the end of the treatment period. These examinations were extended to animals of all other dosage groups for treatmentrelated changes that were observed in the high dose group. Only organs and tissues of the other dosage groups showing changes in the high dose group were examined. Testes, epididymides, ovaries, uterus with cervix, vagina, accessory sex organs (prostate, seminal vesicle with coagulating gland) and all organs showing gross lesions were examined in all animals. For the testes, a detailed qualitative examination was made; taking into account the tubular stages of the spermatogenic cycle for the evaluation of additional hematoxylin- PAS (Periodic Acid Schiff) stained slides.

Haematology
Haematological parameters from five randomly selected males and females of each group were examined at the end of the treatment prior to or as part of the sacrifice of the animals. Blood from the abdominal aorta of the animals was collected in EDTA-coated tubes. The following haematological parameters were examined: haemoglobin content (Hb), red blood cell count (RBC), reticulocytes (Re), platelet count (PLT), white blood cells (WBC), neutrophils (Neu), lymphocytes (Lym), monocytes (Mono), eosinophils (Eos), basophils (Baso).

Blood Coagulation
Coagulation parameters from five randomly selected males and females of each group were examined at the end of the treatment prior to or as part of the sacrifice of the animals. Blood from the abdominal aorta of the animals was collected in citrate tubes. The following coagulation parameters were examined: prothrombin time (PT), activated partial thromboplastin time (aPTT)

Clinical Biochemistry
Parameters of clinical biochemistry from five randomly selected males and females of each group were examined at the end of the treatment prior to or as part of the sacrifice of the animals. Blood from the abdominal aorta of the animals was collected in serum separator tubes. The following parameters of clinical biochemistry were examined: alnine aminotransferase (ALAT), aspartate-aminotransferase (ASAT), alkaline phosphatase (AP), creatinine (Crea), total protein (TP), albumin (Alb), urea, total cholesterol (Chol), glucose (Gluc), sodium (Na), potassium (K), total bile acids (TBA)

Urinalysis
A urinalysis was performed with samples collected from 5 randomly selected males prior to or as part of the sacrifice of the animals. Additionally, urine colour/ appearance were corded. The following parameters were measured using qualitative indicators (Heiland Urine Stripes URI 10SL): specific gravity, nitrite, ph-value (pH), protein, glucose, ketone bodies (ketones), urobilinogen (ubg), bilirubin, blood, leukocytes

Postmortem examinations (offspring):

GROSS NECROPSY
- Gross necropsy consisted of external examinations

Statistics:

A statistical assessment of the results of the body weight, food consumption, parameters of haematology, blood coagulation and clinical biochemistry and absolute and relative organ weights were performed for each gender by comparing values of dosed with control animals of the main groups using a one-way ANOVA and a post-hoc Dunnett Test. These statistics were performed with GraphPad Prism 5.01 software.

Reproductive indices:

Viability index, copulation index, fertility index, and delivery index were calculated.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Based on the histopath. evaluation discolourations were considered to be most probably unrelated to the treatment. No test item-related histopath. findings were noted in the reproductive organs and in the other organs evaluated in this study.

Other effects:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
No mortalities were recorded at any dose levels.
No clinical symptoms were recorded for the male animals. For female animals, individual findings could be recorded but none of them can be attributed to the test item.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
In females, a slightly increased body weight gain could be mentioned for MD and HD groups between GD 14 and GD20. Furthermore, between GD 0 and GD 20 this slight increase in body weight gain for MD and HD group could be mentioned, too. This increased body weight gain was slight, not dose-dependent and not statistically significant. Hence, it is not assumed to be test item related. Throughout the whole treatment period of male animals, body weights were within the normal range of variation for this strain and no relevant difference could be mentioned for treatment groups when compared to the C group.
No considerable effect of the test item on food consumption was found in any of the groups of male animals and in female animals.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
The sperm motility analysis, performed at the end of the treatment period, revealed no test- item related effect in any of the treatment groups compared to the control group. No test item related influence on weights of testes or epididymides were found.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
No test item related influence couldbe mentioned for reproductive performance.

ORGAN WEIGHTS (PARENTAL ANIMALS)
In male animals, mean weight of hearts of LD group was significantly increased (p<0.05) when compared to C group (C: 0.984 g; LD: 1.082 g). This increase was very slight and no increased weight could be found in MD and HD group. Hence, this increased weight is considered to be not test item related. All other organs of treatment groups of male animals were comparable in weight to the C group – absolute and relative organ weights. For female animals, no biological relevant difference was detected when comparing absolute and relative organ weights of treatment groups with C group.

GROSS PATHOLOGY (PARENTAL ANIMALS)
Few specific gross pathological changes were recorded for the male and female animals but just discolouration was increased in treatment groups. Discoloured red mesenteric lymph (4/10 LD; 2/10 MD; 4/10 HD) nodes as well as Ileum (1/10 C; 2/10 LD; 4/10 HD) could be found in treatment groups of male animals. Based on the histopathological evaluation of some of these changes, they were considered to be most probably unrelated to the treatment. A toxicological relevance seems unlikely.

HISTOPATHOLOGY (PARENTAL ANIMALS)
One female of the control group was recorded not to be pregnant at terminal sacrifice. However, histopathological evaluation of this animal revealed one single implantation site in the uterus, indicating a recent pregnancy. At terminal sacrifice, red discolorations of ileum and/or mesenteric lymph node were observed in a number of test item-treated males, without dose-relationship. Based on the histopathological evaluation of a representative subset, these changes were considered to be most probably due to congestion (ileum) and intrasinusoidal blood resorption (mesenteric lymph node), although a test item relationship cannot be fully excluded. No test item-related histopathological findings were noted in the reproductive organs and in the other organs evaluated in this study.

OTHER FINDINGS (PARENTAL ANIMALS)
Urinalysis
The urinalysis performed in male and female animals revealed no test- item related effect in any of the treatment groups compared to the control group.

Clinical Biochemistry
At the end of the treatment period values of SGOT (ASAT) were slightly but dose dependently decreased in treatment groups of female animals. However a slight decrease is commonly not indicative for adverse effects. In addition the deviation from the control is statistically not significant and values are within historical C data. Therefore the occurrence was either considered not to be test item related and/or not to be of toxicological relevance.. Values of cholesterol were slightly but dose-dependently increased in treatment groups of female animals when compared to C group. However, since the C value was relatively low when compared with our historical control data, this is not assumed to be of toxicological relevance. Besides, all parameters of clinical chemistry of male and female animals were within the normal range of variation for this strain and no relevant difference could be mentioned between treatment groups and C group.

Haematology and Coagulation
At the end of the treatment period PLT values were slightly increased in treatment groups of male animals when compared to C group. A test item relation is unlikely. Since this increase was only slight and since no statistical significance could be mentioned, this is not assumed to be biological relevant.
In male animals, percentage of neutrophils were statistically significant increased in LD group and percentage of lymphocytes was significantly decreased (p<0.01) in LD group. However, this is assumed to be occurred incidentally since this pattern was not found in MD or HD group.
Besides, all haematological parameters of male and female animals were within the normal range of variation. Blood coagulation was not affected by the test item. The increase in HD group of female animals is not considered to be biologically relevant since it is not statistically significant and a high standard deviation could be found.

Precoital Interval and Duration of Gestation
No treatment-related effect was observed during the precoital interval or during the duration of gestation when compared with the control group. The values were comparable between the groups. Only one pregnant female of the C group, which had 1 implantation site, did not deliver any pub. All other pregnancies resulted in normal births. Successful mating resulted in 9 pregnancies in the control and 10 pregnancies in low, medium and high dose group animals, respectively.

Pre- and Post-Natal Data
The group mean number of corpora lutea, number of implantation sites, number of live pups born on PND 0, and percentage of pre-implantation loss remained unaffected due to the treatment with test item when compared with the control group. Although there was a slight decrease in post-implantation loss in the LD, MD and HD groups compared to the controls, this is not attributed to the test item but to 2 C animals No. 43 and 47 which had a high incidental percentage of post-implantation loss. No test item relation can be mentioned.

Effect levels (P0)

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed up to 1000 mg/kg bw/day, the highest dose level tested.

Results: F1 generation

General toxicity (F1)

Clinical signs:

not examined

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Details on results (F1)

Litter Data
At PND 0, number of live pubs was increased in HD group when compared to C group (p<0.05). Furthermore, the number of total pubs as well as the number of male pubs was slightly but not statistically significant increased in HD group. This confirms the higher body weight gain which was mentioned for the HD group during pregnancy. A very slight increased amount of total pubs could be also mentioned for MD group which also confirms the higher amount of body weight gain during pregnancy. This is not assumed to be test item related – neither for MD nor for HD animals. At PND 0, a slightly increased sex ratio towards the males could be mentioned for HD group. However, this belongs mostly due to animal No. 78 which has born 10 males and 1 female and the other female animals had comparable sex ratios. Hence, this is not assumed to be test item related.
The pattern found on PND 4 was similar.

Litter Weight Data
Total litter weight and male litter weight was slightly increased at PND 0 and PND 4. This mainly belongs to the increased amount of pubs and not on individual pub weights. Hence, this increase is not considered to be test item related. Litter mean weights on PND 0 and PND4 was comparable among all groups.

Pup External Findings
No treatment-related gross external findings were observed in any of the treated groups. Pub No. 5 from Dam No. 69 could not be found on PND4 which can be probably attributed to cannibalism. However, since this was not found for any other female animals, this is not assumed to be test item related.

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed up to 1000 mg/kg bw/day, the highest dose level tested.

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Applicant's summary and conclusion

Conclusions:

A combined repeated dose oral toxicity and reproduction/ developmental toxicity screening test in male and female rats was performed (according to OECD 422, GLP compliant). The test item was administered orally via gavage in vehicle (corn oil) at dosages of 100, 300, and 1000 mg/kg body weight/day, animals in control groups received the vehicle only. Test item was administered to male rats for 28 days and to female rats for 14 days prior to pairing, through the pairing and gestation periods until the F1 generation reached day 4 post partum (i.e. 54 days).
No test item related effects of toxicological relevance were found at any dose level tested. Thus, the NOAEL for general toxicity in males and females and for reproduction/developmental toxicity is considered to be >= 1000 mg/kg body weight.

Executive summary:

The aim of this study was to provide information on the possible health hazards after repeated dose administration in male and female Wistar rats. It further provides information on possible effects on male and female reproductive performance, development of the conceptus and parturition.

The test item was administered daily in graduated doses to 3 groups of test animals. Animals of an additional control group were handled identically as the dose groups but received corn oil, the vehicle used in this study. The 4 groups comprised 10 male and 10 femaleWistarrats.

During the period of administration, the animals were observed each day for signs of toxicity. Animals that died were examined macroscopically and at the conclusion of the test, surviving animals were sacrificed and observed macroscopically.

Body weight and food consumption were measured weekly, except for food consumption measurements which were not taken during the mating period in female animals and the mating and post-mating period in male animals.

Haematological and clinical biochemistry evaluations were performedon blood samples collected at terminal sacrifice fromfive males and five randomly selected females from each group. Urinalysis was performed on samples collected at terminal sacrifice fromfive randomly selected males from each group.

Epididymal sperm motility was evaluated in all male animals.

Functional observations including sensory reactivity to different stimuli, grip strength, motor activity assessments and other behaviour observations were performed in the week before the treatment and at the end of the study.

After 14 days of treatment to both male and female, animals were mated (1:1) for a maximum of 14 days. The subsequent morning onwards the vaginal smears of females were checked to confirm the evidence of mating. After the confirmation of the mating, females were separated and housed individually. Each litter was examined as soon as possible after delivery of the dam to establish the number and sex of pups, stillbirths, live births, runts and the presence of gross abnormalities. Live pups were counted, sexed and litters weighed within 24 hours of parturition and on day 4 post-partum.

The males were sacrificed after completion of the mating period on treatment days 29 and 30 and the females along with their pups were sacrificed on post natal day 4. Non-pregnant females were sacrificed on day 26.

Pups sacrificed on postnatalday 4 and those found dead, were carefully examined for gross external abnormalities.

A full histopathological evaluation of the tissues was performed on high dose and control animals. Organs showing gross alterations were also examined histopathologically. The examinations of these organs were extended to animals of the medium and low dose groups if treatment-related changes were observed in high dose groups.

 The following doses were evaluated:

Control:                       0        mg/kg body weight

Low Dose:                   100     mg/kg body weight

Medium Dose:             300     mg/kg body weight

High Dose:                  1000   mg/kg body weight

The test item formulation was prepared freshly on each day of administration. The test item was suspended in corn oil and administered daily during 14 days of pre-mating and 14 days of mating in both male and female animals, during the gestation period and up to post-natal day 3 in females. Males were dosed for 28-30 days. Dose volumes were adjusted individually based on weekly body weight measurements. Theadministration volume was 4 mL/kg body weight.

1.1.           Summary Results

No mortalities were recorded at any dose levels.

No clinical symptoms which were related to the test item could be recognized.

No relevant effects were observed in any of the parameters of the functional observation battery before and at the end of the treatment period. There were no biologically relevant differences in body temperature between the groups.

No test item related change in body weight could be found for male and female animals during the treatment period.

No considerable effect of the test item on food consumption was found in any of the groups of male animals and in female animals.

Litter data was not affected due to treatment with the test item.

Litter weight data was not affected due to treatment with the test item.

No treatment-related effect was observed during the precoital interval or during the duration of gestation when compared with the control group.

No test item related change could be found for pre- and post-natal data.

No test item related influence could be found for viability index, copulation index, fertility index, and delivery index.

No significant effect on survival of the pups from PND 0 to PND 4 was observed in any treatment group when compared with controls.

No treatment-related gross external findings were observed in any of the treated groups.

At the end of the treatment period PLT values were slightly increased in treatment groups of male animals when compared to C group. A test item relation is unlikely.

In male animals, percentage of neutrophils were statistically significant increased in LD group and percentage of lymphocytes was significantly decreased (p<0.01) in LD group. However, this is assumed to be occurred incidentally since this pattern was not found in MD or HD group.

Besides, all haematological parameters of male and female animals were within the normal range of variation.

Blood coagulation was not affected by the test item. The increase in HD group of female animals is not considered to be biologically relevant since it is not statistically significant and a high standard deviation could be found.

At the end of the treatment period values of SGOT (ASAT) were slightly but dose-dependently decreased in treatment groups of female animals. Since a slight decrease is commonly not indicative for adverse effects and since all values are within historical C data findings were considered not to be of toxicological relevance.

Values of cholesterol were slightly but dose-dependently increased in treatment groups of female animals when compared to C group. However, since the C value was relatively low when compared with our historical control data, this is not assumed to be of toxicological relevance.

Besides, all parameters of clinical chemistry of male and female animals were within the normal range of variation for this strain and no relevant difference could be mentioned between treatment groups and C group.

The urinalysis performed in male and female animals revealed no test- item related effect in any of the treatment groups compared to the control group.

The sperm motility analysis, performed at the end of the treatment period, revealed no test- item related effect in any of the treatment groups compared to the control group.

Discoloured red mesenteric lymph (4/10 LD; 2/10 MD; 4/10 HD) nodes as well as Ileum (1/10 C; 2/10 LD; 4/10 HD) could be found in treatment groups of male animals. A toxicological relevance seems unlikely.

Based on the histopathological evaluation of a representative subset, these changes were considered to be most probably unrelated to the treatment. No test item-related histopathological findings were noted in the reproductive organs and inthe other organs evaluated in this study.

In male animals, mean weight of hearts of LD group was significantly increased (p<0.05) when compared to C group (C: 0.984 g; LD: 1.082 g). This is considered to be not test item related.

The mean recoveries observed in LD, MD and HD groups were 105.0%, 98.3%, and 88.8% of the nominal concentration, respectively. Homogeneity of formulation samples was determined in study week 1 and 5 for all dose groups. The mean recoveries observed for LD group were 103.9% and 97.1%, for MD group 101.6% and 95.6%, and for HD group 99.5% and 85.3% of the nominal value. The coefficients of variation of the different sampling locations (top, middle, bottom) were in LD group 7.3% and 4.9%, in MD group 1.7% and 2.3%, and in HD group 1.0% and 0.5%.

Under the conditions of this study, no adverse effects were found in males or females up to the highest dose level of 1000 mg/kg bw/day (i.e NOAEL >=.1000 mg/kg bw/day)