Dimethyl 2-[[1-[[(2,3-dihydro-2-oxo-1H-benzimidazol-5-yl)amino]carbonyl]-2-oxopropyl]azo]terephthalate

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study (OECD 422, GLP-compliant)

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

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3650 (Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Tocixity Screening Test)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

2-[(4-chloro-2-nitrophenyl)azo]-N-(2,3-dihydro-2-oxo-1H-benzimidazol-5-yl)-3-oxobutyramide

EC Number:

235-462-4

EC Name:

2-[(4-chloro-2-nitrophenyl)azo]-N-(2,3-dihydro-2-oxo-1H-benzimidazol-5-yl)-3-oxobutyramide

Cas Number:

12236-62-3

Molecular formula:

C17H13ClN6O5

IUPAC Name:

2-[(4-chloro-2-nitrophenyl)diazenyl]-3-oxo-N-(2-oxo-2,3-dihydro-1H-benzimidazol-5-yl)butanamide

Test material form:

solid: bulk

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Animals: Rat, RccHanTM: WIST(SPF)
- Rationale: Recognized by international guidelines as a recommended test system.
- Source: Harlan Laboratories, Inc., Maasheseweg 87c, 5800 AN Vernay / Netherlands
- Number of Animals: 44 males (11 per group) and 44 females (11 per group)
- Age (at Start of Treatment): 11 weeks
- Body Weight Range (at Start of Treatment): 301 to 362 g (males), 216 to 247 g (females)
- Identification: Parent animals had cage card and individual animal number (ear tattoo), pups were individually tattooed with Indian ink on day 1 post partum
- Randomization: Performed after at least three days of acclimatization using a computer-generated random algorithm. Body weights (recorded on the day of allocation) were taken into consideration in order to ensure similar mean body weights in all groups.
- Accommodation: In groups of five in Makrolon type-4 cages with wire mesh tops up to the day of randomization and afterwards individually in Makrolon type-3 cages with wire mesh tops and sterilized standard softwood bedding (‘Lignocel’ J. Rettenmaier & Söhne GmbH & CoKG, 73494 Rosenberg / Germany, imported by Provimi Kliba SA, 4303 Kaiseraugst / Switzerland) with paper enrichment (ISO-BLOX from Harlan Laboratories B.V., Netherlands), batch/lot nos. 02105111001, 02105111201, 02105120301 and 6960C.CS-100099). During the pre-pairing period, cages with males were interspersed amongst those holding females to promote the development of regular estrus cycles.
- Diet: Pelleted standard Harlan Teklad 2018C (batch no. 80/11) rodent maintenance diet (Provimi Kliba SA, 4303 Kaiseraugst / Switzerland) was available ad libitum.
- Water: Community tap-water from Füllinsdorf was available ad libitum in water bottles.
- Acclimatization: Under test conditions after health examination. Only animals without any visible signs of illness were used for the study.

ENVIRONMENTAL CONDITIONS
Standard laboratory conditions, continuously monitored.
- Temperature (°C): 22 +/- 3
- Humidity (%): 30 to 70
- Air changes (per hr): . Air-conditioned with 10 - 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12/12 (with at least eight hours music during the light period)

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

DOSE FORMULATIONS
The dose formulations were prepared weekly using the test item as supplied by the Sponsor.
The test item was weighed into a glass beaker on a tared precision balance and approximately 80% of the vehicle was added (w/v). Using a magnetic stirrer, a homogeneous suspension was prepared. Having obtained a homogeneous mixture, the remaining vehicle was added. Separate formulations were prepared for each concentration.
Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.

STORAGE OF DOSE FORMULATIONS
Dose formulations were stored at room temperature (20 +/- 5 °C) in glass beakers.
Based upon the results of stability analyses performed within the non-GLP Harlan Laboratories study (Dose Range-Finding Study for a Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test in the Han Wistar Rat), dose formulations were stable for at least 8 days if stored at room temperature.

TREATMENT
- Method: Oral, by gavage
- Rationale for Method: Administration by gavage is a common and accepted route of exposure for this type of studies.
- Frequency of Administration: Once daily
- Target Dose Levels: 0 mg/kg/day (control group), 100 mg/kg/day (group 2), 300 mg/kg/day (group 3) and 1000 mg/kg/day (group 4)
- Rationale for Dose Level Selection: The dose levels were selected based on a previous non-GLP dose range-finding toxicity study in Han Wistar rats, Harlan Laboratories Study D33711, using dose levels of 0, 100, 300 and 1000 mg/kg/ day, where no adverse effects were observed up to and including the highest dose level.
- Dose Volume: 10 mL/kg body weight
- Dose Concentrations: 0 mg/mL/day (control group), 10 mg/mL/day (group 2), 30 mg/mL/day (group 3) and 100 mg/mL/day (group 4).
- Duration of Acclimatization Period: 7 days.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

METHOD
On the first treatment day samples from the control group as well as three samples (top, middle and bottom) of about 0.5 g of each concentration were taken prior to dosing for analysis of concentration and homogeneity. Samples of about 0.5 g of each concentration were taken from the middle only to confirm stability (8 days). During the last week of the treatment, samples were taken from the middle to confirm concentration. The aliquots for analysis of dose formulations were frozen (-20 +/- 5 °C) and delivered on dry ice to the responsible for formulation analysis (Harlan Laboratories Ltd., Itingen / Switzerland) and stored there at -20 +/- 5 °C until analysis.
The samples were analyzed by UV-VIS spectroscopy following an analytical procedure developed at Harlan Laboratories. The test item was used as the analytical standard.

RESULTS
Blank samples showed no significant absorbance and, therefore, it was confirmed that only highly purified water was applied within the control experiment.
The application formulations investigated during the study were found to comprise test material in the range of 93.1% to 105.6% and, thus, the required content limit of +/-20% with reference to the nominal content was met. The homogeneous distribution of test item in the preparations was approved because single results found did not deviate more than 5.5% (<15%) from the corresponding mean.
The test item was found to be stable in application formulations when kept eight days at 20 +/- 5 °C due to recoveries which met the variation limit of 10% from the time-zero (homogeneity) mean.

In conclusion, the results indicate the accurate use of the test item and highly purified water as vehicle during this study. Application formulations were found to be homogeneously prepared and stable over a storage period of eight days (20 +/- 5 °C).

Duration of treatment / exposure:

MALES: 40 days
FEMALES: Approximately 7 weeks

Frequency of treatment:

Once daily

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 100, 300, 1000 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

11

Control animals:

yes, concurrent vehicle

Details on study design:

MALES
- Acclimatization: 7 days
- First Test Item Administration: Day 1 of pre-pairing
- Pre-Pairing: 14 days
- Pairing: 14 days maximum
- Treatment Ends: On day before sacrifice
- Blood Sampling: After 28 days of Treatment
- Necropsy: After treatment for 39 days, when no longer needed for assessment of reproductive effects

FEMALES
- Acclimatization: 7 days
- First Test Item Administration: Day 1 of pre-pairing
- Pre-Pairing: 14 days
- Pairing: 14 days maximum
- Gestation: Approximately 21 days
- Treatment Ends: On day 4 post partum
- Blood Sampling: Day 5 post partum
- Necropsy: On day 5 post partum (pups on day 4 post partum)

Positive control:

Not required

Examinations

Observations and examinations performed and frequency:

VIABILITY/MORTALITY: Twice daily

CLINICAL SIGNS
Daily cage-side clinical observations (once daily, during acclimatization and up to day of necropsy). Additionally females were observed for signs of difficult or prolonged parturition, and behavioral abnormalities in nesting and nursing.

FOOD CONSUMPTION
Males: on days 1 - 4, 4 - 8, 8 - 11 and 11 - 14 during pre-pairing period and weekly during after pairing period.
Females: on days 1 - 4, 4 - 8, 8 - 11 and 11 - 14 during pre-pairing period; on days 0 - 7, 7 14 and 14 - 21 during gestation period and on days 1 - 4 of during lactation period.
No food consumption was recorded during the pairing period.

BODY WEIGHTS: Recorded daily from treatment start to day of necropsy.

DETAILED CLINICAL OBSERVATIONS
Detailed clinical observations were performed outside the home cage in all animals. In males, it was performed once prior to the first administration of the test item and weekly thereafter. In females, it was performed once prior to the first administration of the test item, weekly during the pre-pairing and pairing periods and on days 0, 6, 13 and 20 of the gestation period.
Animals were observed for the following: changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies or bizarre behavior were also reported.

FUNCTIONAL OBSERVATIONAL BATTERY
At one time during the study (males shortly before the scheduled sacrifice and females on day 3 or 4 post partum) relevant parameters were performed with five P generation males and five P generation females from each group. This FOB assessment was conducted following the daily dose administration. Animals were observed for the following:
- Cage-side observations: faeces-balls, urine and posture as well as resistance to removal.
- Hand-held observations: muscle tone, constituation, skin, pupile size, palpebral closure, lacrimation, salivation, reaction to handling and general abnormalities.
- Open field observations: level of ambulatory activity including rearing (one minute evaluation), unusual body movements (e.g. spasms, convulsions), gait evaluation, behavior, hair coat, respiration, quantity of faeces-balls and urine.
- Reflexes: blinking, palpebral closure, pinna reflex, extensor thrust response, paw pinch, responsiveness to sharp noise, righting reflex and hearing ability (Preyer’s reflex).
- Measurements / Counts: hind limb / fore limb grip strength, landing foot splay, rectal temperature.

Any abnormal findings were recorded and, where appropriate, graded in severity.

Additionally, locomotor activity was measured quantitatively for the same animals. Activity was measured with an Activity Monitor AMS-0151 (FMI, Germany). Activity of the animals (based on beam count) was recorded for 6-minute intervals over a period of 30 minutes. These data and the total activity over 30 minutes were reported.

CLINICAL LABORATORY INVESTIGATIONS
Blood samples were obtained on the day of the scheduled necropsy from 5 males from each group. Blood samples from 5 lactating females from each group were obtained on day 5 post partum. Blood samples were drawn sublingually from all animals under light isoflurane anesthesia. The animals were fasted for approximately 18 hours before blood sampling but allowed access to water ad libitum. The samples were collected early in the working day to reduce biological variation caused by circadian rhythms.

The following hematology parameters were determined:
- Erythrocyte count
- Hemoglobin
- Hematocrit
- Mean corpuscular volume
- Red cell volume distribution width
- Mean corpuscular hemoglobin
- Mean corpuscular hemoglobin concentration
- Hemoglobin concentration distribution width
- Leukocyte count, total
- Differential leukocyte count
- Platelet count
- Prothrombin time (= Thromboplastin time)
- Activated partial Thromboplastin time

The following clinical biochemistry parameters were determined:
- Glucose
- Urea
- Creatinine
- Bilirubin, total
- Cholesterol, total
- Triglycerides
- Aspartate aminotransferase
- Alanine aminotransferase
- Alkaline phosphatase
- Gamma-glutamyl-transferase
- Bile acids
- Sodium
- Potassium
- Chloride
- Calcium
- Phosphorus
- Protein, total
- Albumin
- Globulin
- Albumin/Globulin ratio

URINALYSIS
The following urinalysis parameters were determined in five males of each group, which are allocated to the blood analysis, during the last week of the study using timed urine volume collection:
- Volume (18 hours)
- Specific gravity (relative density)
- Color
- Appearance
- pH
- Nitrite
- Osmolality
- Protein
- Glucose
- Ketones
- Urobilinogen
- Bilirubin
- Blood/Blood cells

Sacrifice and pathology:

TERMINATION AND NECROPSY

Males were sacrificed after treatment for 39 days, when no longer needed for the assessment of reproductive effects. Dams were sacrificed on day 5 post partum. If birth did not occur on the expected date (day 21 post coitum), the dam was sacrificed on day 25 post coitum.

All animals sacrificed were subjected to a detailed macroscopic examination. Specimens of abnormal tissue were fixed in neutral phosphate buffered 4% formaldehyde solution. At the scheduled sacrifice, all animals were sacrificed by an injection of sodium pentobarbital. All P generation animals were exsanguinated. All parent animals were examined macroscopically for any structural changes. For the parent animals, special attention was directed at the organs of the reproductive system.The number of implantation sites and corpora lutea was recorded for all dams with litters. The uteri of non-pregnant females were placed in a solution of ammonium sulfide to visualize possible hemorrhagic areas of implantation sites.

SEMINOLOGY AND SPERMATID COUNT
Sperm analysis was performed on the first 5 males per group.

Motility:
At necropsy of adult males an epididymal sperm sample was obtained from the left cauda epididymidis of each male. The sample was diluted with a pre-warmed (about 35 °C) physiological medium, and shortly after being obtained, one hundred sperm were counted microscopically for determination of percentage of not motile, stationary motile and progressively motile sperm.

Morphology:
A second sperm sample from the left cauda epididymidis was used for morphological assessment after fixation and Eosin staining. 500 sperm per sample were evaluated microscopically and classified into the following categories:
A: Normal, complete sperm
B: Normal head only (tail detached)
C: Complete sperm, misshapen hook
D: Complete sperm, abnormally curved hook
E: Complete sperm, reversed head
F: Abnormal head only (tail detached)

Morphological sperm evaluation was performed only for group 1 and 4 males. In the absence of a treatment-related effect the slides for the group 2 and 3 males were not evaluated.

Sperm, Spermatid Count:
The left caudal epididymis and left testis were taken for determination of homogenization-resistant spermatids and caudal epididymal sperm reserve. These tissues were frozen at -20 +/- 5 °C pending evaluation. For evaluation the weighed tissues were placed in Triton-X-100 solution and homogenized with a blender (Ultra Turrax) and an ultrasonic water bath. Sperm or spermatid heads were counted microscopically using a modified Neubauer chamber. These evaluations were performed in the first instance only for group 1 and 4 males. In the absence of a treatment-related effect the remaining frozen tissues were not evaluated.

ORGAN WEIGHTS
At the scheduled sacrifice, testes and epididymides from all parental males were weighed separately. In addition, from 5 males and 5 females sacrificed at the end of the study which were selected from each group, the following organs were trimmed from any adherent tissue, as appropriate, and their wet weight taken.
- Adrenal glands (weighed as pairs)
- Brain
- Heart
- Kidneys (weighed as pairs)
- Liver
- Thymus
- Spleen

TISSUE PRESERVATION
The following tissues from all parental males were preserved in neutral phosphate buffered 4% formaldehyde solution:
- Prostate
- Seminal vesicles with coagulating gland
- Testes (in Bouin’s fixative)*
- Epididymides (in Bouin’s fixative)*
*From the first five males in each group which were used for sperm analysis, only the right testis and right epididymis were preserved for histopathological examination.

The following tissues from all parental females were preserved in neutral phosphate buffered 4% formaldehyde solution:
- Ovaries

In addition, from 5 males and 5 females per group selected for organ weights, the following tissues were preserved in neutral phosphate buffered 4% formaldehyde solution:
- Gross lesions
- Brain (representative regions including cerebrum, cerebellum and pons)
- Spinal chord
- Small and large intestines (incl. Peyer’s patches)
- Stomach
- Liver
- Kidneys
- Adrenals
- Spleen
- Heart
- Thymus
- Thyroids, and parathyroids if possible
- Trachea and lungs (preserved by inflation with fixative and then immersion)
- Uterus (with vagina)
- Urinary bladder
- Lymph nodes (mesenterial, mandibular)
- Peripheral nerve (sciatic)
- Bone marrow

HISTOTECHNIQUE
All organ and tissue samples to be examined by the study pathologist were processed, embedded and cut at an approximate thickness of 2 - 4 micrometers and stained with hematoxylin and eosin. Additionally, the testis was stained by PAS-hematoxylin. Special stains were used at the discretion of the study pathologist.

HISTOPATHOLOGY
Slides of all organs and tissues listed collected at terminal sacrifice from the animals of the control and high-dose groups were examined by the study pathologist. The same applied to all occurring gross lesions.
Special emphasis was made on the stages of spermatogenesis and histopathology of interstitial cell structure.
Histological examination of ovaries was carried out on any females that did not give birth. In addition, microscopic examination of the reproductive organs of all infertile males was made, where necessary.
A histopathology peer review was performed. A histopathology phase report was provided by the principal investigator which was included in the report.

Other examinations:

MATING, GESTATION, LACTATION
During the pairing period, females were housed with sexually mature males (1:1) until evidence of copulation was observed. The females were removed and housed individually if the daily vaginal smear was sperm positive, or a copulation plug was observed. The day on which a positive mating was determined (copulation plug or sperm) was designated day 0 post coitum. For a female which did not mate during the 14-day pairing period, a second pairing of this female with a male in the same group, which had already mated successfully, was performed. All dams were allowed to give birth and rear their litters (F1 pups) up to day 4 post partum. Day 0 was designated as the day on which a female had delivered all her pups.

REPRODUCTIVE AND OFFSPRING VIABILITY INDICES
From the on-line recorded reproduction data, the following parameters were calculated: fertility indices, mean precoital time, post-implantation losses, mean litter size, pup sex ratios and viability indices.

LITTER OBSERVATIONS
The litters were examined for litter size, live births, still births and any gross anomalies. The sex ratio of the pups was recorded. Pups were weighed individually (without identification) on days 0 (if possible), 1 and 4 post partum.

POSTMORTEM EXAMINATION OF OFFSPRING
Pups were sacrificed on day 4 post partum. All animals were sacrificed by by an injection of sodium pentobarbital and subjected to a detailed macroscopic examination. Specimens of abnormal tissue were fixed in neutral phosphate buffered 4% formaldehyde solution. Pups found dead during the study, except those excessively cannibalized, were examined macroscopically.

Statistics:

The following statistical methods were used to analyze food consumption, body weights and reproduction data:
- Means and standard deviations of various data were calculated.
- The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.
- Fisher's exact-test was applied when the variables could be dichotomized without loss of information.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Red stained faeces were noted in all males and females in dose groups; this was due to the staining properties of the test item

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Red stained faeces were noted in all males and females in dose groups; this was due to the staining properties of the test item

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

In males at the dose level of 1000 mg/kg bw/day, lower body weight gain during the pre-pairing period

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Locomotor activity was not affected and functional observational battery gave no indication of a test item-related effect.

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Details on results:

1. IN-LIFE DATA OF PARENTAL ANIMALS
VIABILITY / MORTALITY
All animals survived scheduled study period.

DAILY CLINICAL SIGNS OR OBSERVATIONS
Red stained feces was noted in all males and females in all dose groups starting from day 2 of the treatment until completion of the study with dose-related intensity of discoloration. This observation was due to staining properties of the test item.
No further test item-related clinical signs or observations were noted in males or females at any dose level.
Incidentally, in one male (no. 16) at the dose level of 100 mg/kg bw/day chromodacryorrhea was noted during the study (starting on day 1 of the pre-pairing period) and eye reduced in size was noted in the same animal from day 13 of the pre-pairing period.
No further test item-related findings were noted at any dose level.

FINDINGS AT DETAILED WEEKLY CLINICAL OBSERVATIONS
No test item-related findings were noted during detailed weekly clinical observations.
The only findings noted were chromodacryorrhea and eye reduced in size in male no. 16 at the dose level of 100 mg/kg bw/day recorded already during the daily clinical observations.

FUNCTIONAL OBSERVATIONAL BATTERY
No test item-related findings were noted during the functional observational battery tests in males or females at any dose level.
Statistically significantly lower body temperature was noted in both sexes. In males mean body temperature was 37.9 °C and 37.8 °C at the high- and mid-dose levels, respectively, compared to 38.4 °C in the control group. In females, 38.5 °C was noted at the high-dose level, compared to 38.9 °C in the control group. The differences noted in males and females were only minor, not clearly dose dependent and all values remained in the historical control range. For these reasons, changes in body temperature were considered not to be test item-related.
No further findings were noted during functional observational battery in males or females at any dose level except for the eye findings in male no. 16 at 100 mg/kg bw/day.

LOCOMOTOR ACTIVITY
No effects on locomotor activity were noted in males or females at any dose level.
Mean beam counts during the 30 minutes of measurement at the dose levels of 0, 100, 300 and 1000 mg/kg bw/day were respectively: 1255, 1137, 1219 and 1209 in males and 924, 882, 1002 and 1050 in females.

FOOD CONSUMPTION OF MALES: No effects on food consumption were noted in males at any dose level.
Mean differences in food consumption at the dose levels 100, 300 and 1000 mg/kg bw/day were respectively: +1.1%, -3.4% and -3.4% during the pre-pairing period and -1.5%, -3.1% and -3.8% during the after pairing period ( percentages refer to the respective values in the control group).

FOOD CONSUMPTION OF FEMALES: No test item-related effects on food consumption were noted in females at any dose level.
Incidentally, statistically significantly higher food consumption was noted at the dose level of 100 mg/kg bw/day during lactation period. In the absence of an effect in females at the dose levels of 300 and 1000 mg/kg bw/day, this difference was considered not to be related to the treatment.
Mean differences in food consumption at the dose levels 100, 300 and 1000 mg/kg bw/day were respectively: +5.9%, +2.7%, and +1.1% during the pre-pairing period, +7.0%, +2.5% and +2.9% during the gestation period and +23.2%, -5.9% and +11.0% during the lactation period (percentages refer to the respective values in the control group).

BODY WEIGHTS OF MALES
At the dose level of 1000 mg/kg bw/day, a slightly lower body weight gain if compared to the controls was noted during the pre-pairing period. Mean body weight gain within this period was +10%, compared to +13% in the control group. The difference in body weight gain was statistically significant during the most days starting from day 3 until the end of the pre-pairing period. This effect was considered to be test item-related. During the pairing and after pairing periods, body weight gain was similar at all dose levels.
No significant changes in body weights were noted in males at any time during the study.
Because the lower body weight gain at the high-dose level was reversible despite treatment continued and did not result in any significant changes in body weights, this finding was considered not to be adverse.
No significant changes in body weight gain or body weights were noted in males at the dose levels of 100 and 300 mg/kg bw/day.
Mean differences in body weight gain at the dose levels of 0, 100, 300 and 1000 mg/kg bw/day were respectively: +13%, +13%, +11% and +10% during the pre-pairing period, +10%, +10%, +9% and +9% during the pairing period and +7%, +6%, +7% and +6% during the after pairing period (percentages refer to the body weight change within the respective period).

BODY WEIGHTS OF FEMALES
Body weights and body weight gain of females were not affected by the treatment with the test item at any dose level.
On individual days some statistically significantly changed values of body weight gain were noted at the low-, mid- and high-dose levels. The changes did not follow a dose dependency and were therefore not related to the treatment.
Mean differences in body weight gain at the dose levels of 0, 100, 300 and 1000 mg/kg bw/day were respectively: 5%, 7%, 6% and 7% during the pre-pairing period, 48%, 56%, 47% and 54% during the gestation period and 3%, 8%, 4% and 5% during the lactation period (percentages refer to the body weight change within the respective period).

2. CLINICAL LABORATORY INVESTIGATIONS
HEMATOLOGY
No test item-related effects on hematology parameters were noted in males or females at any dose level.
In males, statistically significant changes of several parameters: higher distribution width of red cell volume (RDW) at the low-dose level and higher distribution width of hemoglobin concentration (HDW) at the low- and mid-dose levels occurred in the absence of an effect at the high dose and therefore were considered not to be test item-related.
In females, at the low-dose level, statistically significantly higher platelets count was noted in the absence of any increase of this value at the mid- and high-dose levels and therefore it was not test item-related.
No further changes of hematology parameters were noted in males or females at any dose level.

CLINICAL BIOCHEMISTRY
No test item-related effects on biochemistry parameters were noted in males or females at any dose level.
In males, at the mid-dose level, statistically significantly lower concentration of triglycerides was noted. In the absence of dose dependency, this finding was not test item-related.
In females at the low dose level, following statistically significant changes were noted: higher concentration of cholesterol, higher concentration of globulin, and lower globulin to albumin ratio. These changes were not dose-dependent and therefore they were considered not to be test item-related.
No further changes of biochemistry parameters were noted in either males or females at any dose level.

URINALYSIS
No changes in urine parameters were noted in males at any dose level.

3. TERMINAL FINDINGS - PARENTAL ANIMALS
SEMINOLOGY AND SPERMATID COUNT
In all dose groups, statistically significant changes in motility of sperms were noted. Following values were assessed in sperm samples at the dose levels of 0, 100, 300 and 1000 mg/kg bw/day respectively: 81.1%, 64.6%, 57.3% and 47.5% of progressive sperms (changes were statistically significant in all dose groups), 3.7%, 11.3%, 6.0% and 11.9% of stationary sperms (changes were statistically significant at the dose levels of 1000 and 100 mg/kg bw/day) and 15.2%, 24.1, 36.7 and 40.6% of not motile sperms (changes were statistically significant at the dose levels of 1000 and 300 mg/kg bw/day). These changes might be test item-related. However no significant dose dependent trend indicated by probability values of <0.05 was determined for any of these changes when performing a linear regression analysis (least squares).

No further changes were noted during sperm analysis. At the high-dose level, all morphological categories of sperms were represented with similar frequency to that in the control group whereas sperm count was similar to the respective control values in samples from both testis and epididymidis.

ORGAN WEIGHTS
No changes in absolute organ weights or organ weights to body weights and to brain weights ratios were noted in males or females at any dose level.

MACROSCOPICAL FINDINGS
Type and distribution of findings noted during macroscopical examination of males or females did not indicate any test item-related effect.

HISTOPATHOLOGY FINDINGS
Under the conditions of this experiment, treatment with test item did not cause pathological findings. All findings recorded were within the range of normal background lesions which may be recorded in animals of this strain and age.

Effect levels

1

Key result

Dose descriptor:

NOAEL

Remarks:

P (for general toxicity)

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.

2

Dose descriptor:

NOAEL

Remarks:

P (for reproduction)

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.

3

Dose descriptor:

NOAEL

Remarks:

F1 (for development)

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.

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

1. REPRODUCTION, BREEDING AND PUP DATA

SUMMARY OF PERFORMANCE

P Animals Breeding for F1 Litters

Group (mg/kg/day)	1 (0)	2 (100)	3 (300)	4 (100)
Female numbers	45-55	56-66	67-77	78-88
Number of females paired	11	11	11	11
Number of females mated	11	11	11	11
Number of non pregnant females (A)	3	1	3	0
Numbers of pregnant females, which did not deliver any pups (B)	0	0	0	1
Number of females which reared their pups until day 4 post partum	8	10	8	10

(A)  Female Nos. 45, 46, 55, 62, 74, 75 and 77.

(B)  Female No. 85 had implantations only.

 

MATING PERFORMANCE AND FERTILITY

 Mating performance and fertility were not affected by the treatment at any dose level.

All females in groups 2, 3 and 4 mated within the first pairing period. In group 1, one female (no. 54) was mated during the second pairing period.

 Mean (median) precoital times were 4.5 (3), 2.5 (3), 4.0 (2) and 2.6 (3) days at the dose levels of 0, 100, 300 and 1000 mg/kg bw/day, respectively.

 Seven females were not pregnant: three in the control group and in the mid-dose level and one in the low-dose level. Consequently, fertility indexes (number of females pregnant as percentages of females paired) and conception rate (number of females pregnant as percentages of females mated) were 72.7%, 90.9%, 72.7% and 100.0% at the dose levels of 0, 100, 300 and 1000 mg/kg bw/day, respectively.

 

One female at the high dose level had one implantation site but delivered no pups. Consequently, gestation index (number of females with living pups as percentages of females pregnant) was 100% in the control group and at low- and mid-dose levels and 90.9% at the high-dose level.

 

CORPORA LUTEA COUNT

 No test item-related effects on corpora lutea count were observed at any dose level.

Mean number of corpora lutea per dam was 16.0, 17.2, 16.3 and 18.4 in order of ascending dose levels.

 

DURATION OF GESTATION

 No effects on duration of gestation were observed at any dose level.

 Mean duration of gestation was 21.6, 21.6, 21.5 and 21.7 days, in order of ascending dose level.

 

IMPLANTATION RATE AND POST-IMPLANTATION LOSS

 No effects on implantation rate and post-implantation loss were observed at any dose level.

 In order of ascending dose levels, mean number of implantations per dam was 12.6, 14.9, 12.6 and 14.0 whereas mean incidence of post-implantation loss per dam was 1.5, 0.8, 0.6 and 0.5 per dam.

 

LITTER SIZE AT FIRST LITTER CHECK

 No effects on litter size were noted at any dose level.

 During the first litter check, one dead pup was found in a litter at the dose level of 1000 mg/kg bw/day. Because of isolated occurrence, this finding was considered to be incidental.

 Mean number of living pups per dam at first litter check was 11.1, 14.3, 12.0 and 13.5 in order of ascending dose levels.

 Birth index (number of pups born alive as a percentage of implantations) was 88.1%, 94.8%, 95.0% and 96.4% at the dose level of 0, 100, 300 and 1000 mg/kg bw/day.

 Birth index at the dose level of 1000 mg kg bw/day was statistically significantly higher than the respective control value. This was considered to be a result of biological variability.

 

POSTNATAL LOSS DAYS 0 - 4 POST PARTUM

 No test item-related effects on postnatal loss were noted at any dose level.

 In the control group one pup was missing on day 4, at the low-dose level one pup was missing on day 2, at the mid dose level three pups (from two litters) were missing on day 2 and at the high dose level no postnatal loss was noted in any litter.

 Mean postnatal loss per dam during four days of lactation was 0.1%, 0.1%, 0.4% and 0.0% at the dose level of 0, 100, 300 and 1000 mg/kg bw/day, respectively. Consequently, viability index (number of pups alive at termination on day 4 p.p. as a percentage of pups born alive) was 98.9%, 99.3%, 96.9% and 100% in order of ascending dose levels.

 

EXTERNAL EXAMINATION AT FIRST LITTER CHECK AND DURING LACTATION

 No test item-related findings were noted in pups during first litter check and during lactation at any dose level.

 Incidentally, one pup in the control group was found with a wound and missing tail tip, two further pups, each one at the low- and mid-dose levels, had a wound at first litter check. These findings were also noted during the remaining lactation period.

 

SEX RATIOS

 Pups sex ratio was not affected by exposure to the test item at any dose level.

 At first litter check, percentages of male pups were 56%, 48%, 51% and 56% at the dose levels of 0, 100, 300 and 1000 mg/kg bw/day.

 

 BODY WEIGHTS TO DAY 4 POST PARTUM

 Body weights and body weight gain of pups were not affected by the treatment with the test item at any dose level.

Mean body weights of pups on day 1 post partum were: 6.4 g, 6.1 g, 6.4 and 6.2 g and mean differences in body weights during lactation were +49.9%, +43.6%, +47.8% and +42.6%, at the dose levels of 0, 100, 300 and 1000 mg/kg/day, respectively.

 

At the dose levels of 100 and 1000 mg/kg bw/day, slightly not statistically significantly lower body weight gain of pups was noted. This effect was considered to be due to a higher number of pups at these dose levels which was supported by observation that reduction of body weight gain was more pronounced in litters of higher size. Therefore this effect was considered not to be test item-related.

 

MACROSCOPICAL FINDINGS

 No test item-related findings were noted at macroscopic examination of pups at any dose level.

 Incidentally, in the control group one pup had a sore in the thoraco-dorsal region, one further pup in this group had a missing tail tip. These findings were already recorded during the in life phase. At the high-dose level, one pup had a watery cyst in the left kidney.

 No further findings were noted in pups at any dose level.

 

Applicant's summary and conclusion

Conclusions:

This study is a valid investigation of the toxicological effects resulting from repeated oral-gavage administration of the test item to rats (according to OECD 422, GLP compliant). The test item was administered in vehicle (highly purified water) at dosages of 0, 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.
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.

All animals survived the scheduled study period.
During the treatment, faeces stained red with dose-dependent intensity of discoloration were noted in all males and females receiving test material. This observation was due to staining properties of the test item.

No effects on food consumption were noted in males at any dose level. Body weight gain was slightly but statistically significantly reduced in males at the dose level of 1000 mg/kg bw/day during the pre-pairing period. No differences in body weight gain were noted at any dose level during the remaining study period. Body weights of males in all dose groups were similar to the respective control values during the entire study period. Because lower body weight gain at the high-dose levels was reversible and did not cause significant changes in body weights, this effect was considered not to be adverse.

Food consumption, body weights and body weight gain of females were not affected by the treatment at any dose level.

No further test item-related observations were noted in males or females at any dose level during the live part of the study.

Terminal examinations revealed changes in motility of sperms in all dose groups. Statistically significant decrease in mean count of progressive sperms was noted at the dose levels of 100, 300 and 1000 mg/kg bw/day, statistically significant increase in mean count of stationary sperms was noted at the dose levels of 100 and 1000 mg/kg bw/day and statistically significant increase in mean count in not motile sperms was noted at the dose levels of 300 and 1000 mg/kg bw/day. However a significant dose dependent trend indicated by probability values of <0.05 was not established for any of these changes when performing a linear regression analysis (least squares).
No further effects on male reproductive system were noted during the study. Sperm morphology and sperm count at the high-dose level was similar to the control values. Weights of male reproductive organs, macroscopical and histopathological examination of testes and epididymides gave no indication of any treatment-related effect. Further, no indication of effects on reproduction was noted within this study up to and including the highest dose level. For this reason, changes in motility of sperms were considered not to be adverse in this study.


Reproduction and development were not affected by the treatment. Mating performance, fertility, duration of gestation, corpora lutea count, implantation rate, post implantation and postnatal loss or litter size were similar in the control and all dose groups. There were no test item-related findings in pups noted during the first litter check, the first 4 days post partum or during the necropsy, pups body weights and body weight gain were not affected by the treatment at any dose level.

Based on these results, the NOAEL (No Observed Adverse Effect Level) for general toxicity in males and females and for reproduction/developmental toxicity in this study was considered to be 1000 mg/kg bw/day, the highest dose level tested.

Executive summary:

The purpose of this study was to generate preliminary information concerning the effects of the test itemon the possible health hazards likely to arise from repeated exposure over a relatively limited period of time. In addition it provides information on possible effects on male and female reproductive performance such as gonadal function, mating behavior, conception, development of the conceptus and parturition.

 

The test item was administered to male rats for 39 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.

 

The following dose levels were applied:

Group 1:                        0 mg/kg body weight/day (control group)

Group 2:                    100 mg/kg body weight/day

Group 3:                    300 mg/kg body weight/day

Group 4:                   1000 mg/kg body weight/day

 

A standard dose volume of 10 mL/kg body weight with a daily adjustment to the actual body weight was used. Control animals were dosed with the vehicle alone (highly purified water).

 

The following results were obtained:

 MORTALITY AND GENERAL TOLERABILITY OF PARENTAL ANIMALS

 All animals survived the scheduled study period. Feces stained red with dose-dependent intensity of discoloration were noted in all males and females in all dose groups starting from day 2 of the treatment until completion of the study. This observation was due to staining properties of the test item.

 No further test item-related clinical signs or observations were noted in males or females at any dose level.

 

FUNCTIONAL OBSERVATIONAL BATTERY IN PARENTAL ANIMALS

 No test item-related findings were noted during the functional observational battery tests in males or females at any dose level.

 

FOOD CONSUMPTION OF PARENTAL ANIMALS

 No effects on food consumption were noted in males or females at any dose level.

 

BODY WEIGHTS OF PARENTAL ANIMALS

 In males at the dose level of 1000 mg/kg bw/day, a slight but statistically significant lower body weight gain was noted during the pre-pairing period. No differences in body weight gain were noted at any dose level during the remaining study period. Body weights of males in all dose groups were similar to the respective control values during the entire study period. Because reduction in body weight gain at the high-dose levels was reversible and did not cause significant changes in body weights, this effect was considered not to be adverse.

 Body weights and body weight gain of females were not affected by the treatment at any dose level.

 

CLINICAL LABORATORY INVESTIGATIONS IN PARENTAL ANIMALS

 No test item-related effects on hematology and clinical biochemistry parameters were noted in males or females at any dose level.

 No changes in urine parameters were noted in males at any dose level.

 

REPRODUCTION AND BREEDING DATA

 Mating performance, fertility, corpora lutea count, duration of gestation, implantation rate and post-implantation loss, litter size or postnatal loss were not affected by the treatment with the test item.

 

SEMINOLOGY AND SPERMATID COUNT IN PARENTAL MALES

Effects on sperm motility which might be test item-related were noted in all dose groups. Mean count of progressive sperms was statistically significantly reduced at the dose levels of 1000, 300 and 100 mg/kg bw/day, mean count of stationary sperms was statistically significantly increased at the dose levels of 1000 and 100 mg/kg bw/day and mean count of not motile sperms was statistically significantly increased at the dose level of 1000 and 300 mg/kg bw/day. But a significant dose dependent trend couldn’t be established.

In the absence of any findings during necropsy or histopathological examination of male reproductive organs as well as in the absence of any effects on reproduction, the differences in sperm motility were considered not to be adverse.

ORGAN WEIGHTS OF PARENTAL ANIMALS

 No changes in absolute organ weights or organ weights to body weights and to brain weights ratios were noted in males or females at any dose level.

 

MACROSCOPICAL FINDINGS AND HISTOPATHOLOGICAL EXAMINATIONS OF PARENTAL ANIMALS

 Type and distribution of findings noted during macroscopical examination did not indicate any test item-related effect.

 Treatment with the test item did not cause pathological findings. All findings recorded were within the range of normal background lesions which may be recorded in animals of this strain and age.

 

FINDINGS IN PUPS AT FIRST LITTER CHECK AND DURING LACTATION

 No test item-related findings were noted in pups during first litter check and during lactation at any dose level.

 Pups sex ratio was not affected by the exposure to the test item at any dose level.

 

PUP WEIGHTS TO DAY 4 POST PARTUM

 Body Weights and body weight gain of pups were not affected by the treatment with the test item at any dose level.

 

MACROSCOPICAL FINDINGS OF PUPS

 No test item-related findings were noted at macroscopic examination of pups at any dose level.

 

CONCLUSION

Based on these results, the NOAEL (No Observed Adverse Effect Level) for general toxicity in males and females and for reproduction/developmental toxicity in this study was considered to be 1000 mg/kg bw/day, the highest dose level tested.