Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose toxicity study with reproduction / developmental toxicity screening
Type of information:
experimental study
Adequacy of study:
key study
Study period:
9.6.2015-5.10.2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
The study was performed according to the recommended Guidelines (OECD Guidelines for Testing of Chemicals, No.422: “Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test” (adopted 22 March 1996)) and GLP. The study was also designed to be compatible with Commission Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH). Deviations from the study plan are not considered to have affected the scientific purpose or integrity of the study or the results obtained.
Cross-reference
Reason / purpose for cross-reference:
reference to same study
Reference
Endpoint:
screening for reproductive / developmental toxicity
Remarks:
combined repeated dose toxicity study with reproduction / developmental toxicity screening
Type of information:
experimental study
Adequacy of study:
key study
Study period:
9.6.2015-5.10.2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
The study was performed according to the recommended Guidelines (OECD Guidelines for Testing of Chemicals, No.422: “Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test” (adopted 22 March 1996)) and GLP. The study was also designed to be compatible with Commission Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH). Deviations from the study plan are not considered to have affected the scientific purpose or integrity of the study or the results obtained.
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
Adopted 22 March 1996
Deviations:
yes
Remarks:
No analysis was carried out to determine the stability of the test item formulation. Deviations from the target range for relative humidity. Necropsy details for 2 animals not recorded. For histopathology examinations some tissues were not processed.
GLP compliance:
yes
Remarks:
Certificate is as an attachment to the study report
Limit test:
no
Species:
rat
Strain:
Wistar
Details on species / strain selection:
Wistar Han™:RccHan™:WIST strain rats. The rat was selected for this study as it is a readily available rodent species historically used in safety evaluation studies and is acceptable to appropriate regulatory authorities.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories UK Ltd., Oxon, UK.
- Age at study initiation: Approximately 12 weeks
- Weight at study initiation: males 288-328 g, females 173-203 g
- Fasting period before study: No fasting
- Housing: Initially, all animals were housed in groups of three in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding. During the pairing phase, animals were transferred to polypropylene grid floor cages suspended over trays lined with absorbent paper on a one male: one female basis within each dose group. Following evidence of successful mating, the males were returned to their original cages. Mated females were housed individually during gestation and lactation in solid floor polypropylene cages with stainless steel mesh lids and softwood flakes.
- Diet (e.g. ad libitum): Pelleted diet (Rodent 2018C Teklad Global Certified Diet, Harlan Laboratories U.K Ltd., Oxon, UK.), free access to food
- Water (e.g. ad libitum): Tap water, free access to water
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25 °C
- Humidity (%): 30 to 70% with deviations from the target range up to 85.36%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
arachis oil
Remarks:
Arachis oil BP was used because the test item did not dissolve/suspend in distilled water.
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The test item was prepared at the appropriate concentrations as a suspension in Arachis oil BP. Due to the complex nature of the test item and its limited solubility in organic and aqueous media, a substance specific quantitative method of analysis could not be developed and no stability determined. The test item was formulated within two hours of it being applied to the test system; it is assumed that the formulation was stable for this duration. The concentration of test item in the formulations was determined using a gravimetric technique. Formulations were prepared daily.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Arachis oil BP was used because the test item did not dissolve/suspend in distilled water.
- Concentration in vehicle: 0, 25, 75 and 250 mg/mL
- Amount of vehicle (if gavage): 4 mL/kg treatment volume. The volume of test and control item administered to each animal was based on the most recent scheduled body weight and was adjusted at weekly intervals.
Details on mating procedure:
- M/F ratio per cage: animals were paired on a 1 male: 1 female basis within each dose group
- Length of cohabitation: maximum of fourteen days
- Proof of pregnancy: Vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy.
Cage tray-liners were checked each morning for the presence of ejected copulation plugs and each female was examined for the presence of a copulation plug in the vagina. A vaginal smear was prepared for each female and the stage of estrus or the presence of sperm was recorded. The presence of sperm within the vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating (Day 0 of gestation).
- After successful mating each pregnant female was caged: Following evidence of mating (designated as Day 0 post coitum) the males were returned to their original cages and females were transferred to individual cages. Mated females were housed individually during the period of gestation and lactation.
- Any other deviations from standard protocol: no deviations
Analytical verification of doses or concentrations:
yes
Remarks:
Samples of the test item formulation were analyzed gravimetrically for the concentration of the test item.
Details on analytical verification of doses or concentrations:
Due to the complex nature of the test item and its limited solubility in organic and aqueous media, a substance specific quantitative method of analysis could not be developed. The concentration of test item in the formulations was determined using a gravimetric technique. Therefore formulations were prepared daily.

Samples of the test item formulation were taken and analyzed for concentration of the test item. The results indicate the accurate use of the test item and Arachis Oil BP as vehicle during the study, and that the prepared formulations were within acceptable ranges for the purpose of this study. The formulations were found to be homogeneously prepared. The analytical procedure had acceptable recoveries of test item in the vehicle. The method of analysis was validated and proven to be suitable for use.
Duration of treatment / exposure:
The test item was administered by gavage to groups of rats (parental generation) for up to eight weeks (including a two week pre-pairing phase, pairing, gestation and early lactation for females).
Frequency of treatment:
The test item was administered daily.
Details on study schedule:
- Only animals of parental generation mated
- Age at mating of the mated animals in the study: 14-16 weeks
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
P, m/f
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
P, m/f
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Remarks:
P, m/f
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
P, m/f
No. of animals per sex per dose:
Animals from the parental generation
Control group (vehicle alone): 12 males, 12 females
Dose level 100 mg/kg bw/day: 12 males, 12 females
Dose level 300 mg/kg bw/day: 12 males, 12 females
Dose level 1000 mg/kg bw/day: 12 males, 12 females
See Table 1 in "Any other information on results incl. tables" for further information
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were chosen based on the results of previous toxicity work (Fourteen day repeated dose oral (gavage) range-finding toxicity study in the rat, see Attached background material).

In the range-finding toxicity study, the test item was administered by gavage to three groups, each of three male and three female Wistar Han™:RccHan™:WIST strain rats, for fourteen consecutive days, at dose levels of 250, 500 and 1000 mg/kg bw/day. A control group of three males and three females was dosed with vehicle alone (Arachis oil BP). Clinical signs, body weight change, dietary intake and water consumption were monitored during the study. All animals were subjected to gross necropsy examination.

Results:
Mortality -There were no unscheduled deaths on the study.
Clinical Observations - There were no clinical signs apparent during the study.
Body Weight- There was no adverse effects on body weight development for either sex at 1000, 500 or 250 mg/kg bw/day.
Food Consumption - There was no obvious effect on food consumption or food conversion efficiency for either sex at 1000, 500 or 250 mg/kg bw/day.
Water Consumption - There was no consistent effect on water consumption for either sex at 1000, 500 or 250 mg/kg bw/day.
Necropsy - There were no macroscopic abnormalities were detected.

The oral administration of the test item to rats by gavage, at dose levels of 250, 500 and 1000 mg/kg bw/day was well tolerated. Based on the results from this study a dose level of 1000 mg/kg bw/day was considered to be suitable as a high dose level for the OECD 422 study together with 100 and 300 mg/kg bw/day as the low and intermediate dose levels, respectively.

- Rationale for animal assignment: The animals were randomly allocated to treatment groups using a stratified body weight randomization procedure and the group mean body weights were then determined to ensure similarity between the treatment groups. Animals were paired on a 1 male: 1 female basis within each dose group.
Positive control:
No positive control
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: No

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily clinical observations. All animals were examined for overt signs of toxicity, ill-health and behavioral change immediately before dosing, soon after dosing, and one hour after dosing during the working week, at weekends and public holidays (except for females during parturition where applicable).

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were recorded on Day 1 (prior to dosing) and then weekly for males until termination and weekly for females until pairing. During pairing phase females were weighed daily until mating was confirmed. Body weights were then recorded for females on Days 0, 7, 14 and 20 post coitum, and on Days 1 and 4 post partum. Body weights were also recorded at terminal kill.

FOOD CONSUMPTION:
- During the pre-pairing period, weekly food consumption was recorded for each cage of adults. This was continued for males after the mating phase. For females showing evidence of mating, food consumption was recorded for the periods covering post coitum Days 0-7, 7-14 and 14-20. For females with live litters, food consumption was recorded on Days 1 and 4 post partum.

WATER CONSUMPTION: Yes
- Time schedule for examinations: Water intake was observed daily by visual inspection of water bottles for any overt changes.

OTHER: HAEMATOLOGY, CLINICAL CHEMISTRY, NEUROBEHAVIOURAL EXAMINATION

HAEMATOLOGY:
- Time schedule for collection of blood: Hematological investigations were performed on five males and five females selected from each test and control group prior to termination (Day 42 for males and Day 4 post partum for females).
- Anaesthetic used for blood collection: No data
- Animals fasted: No
- How many animals: 10
- Parameters checked in table 2 were examined: Hemoglobin (Hb), Erythrocyte count (RBC), Hematocrit (Hct), Erythrocyte indices (mean corpuscular hemoglobin (MCH), mean corpuscular volume (MCV), mean corpuscular hemoglobin concentration (MCHC)), Total leukocyte count (WBC), Differential leukocyte count (neutrophils (Neut), lymphocytes (Lymph), monocytes (Mono), eosinophils (Eos), basophils (Bas)), Platelet count (PLT), Reticulocyte count (Retic) (Methylene blue stained slides were prepared but reticulocytes were not assessed). Prothrombin time (CT) was assessed by ‘Innovin’ and Activated partial thromboplastin time (APTT) was assessed by ‘Actin FS’.

CLINICAL CHEMISTRY:
- Time schedule for collection of blood: Blood chemical investigations were performed on five males and five females selected from each test and control group prior to termination (Day 42 for males and Day 4 post partum for females).
- Animals fasted: No
- How many animals: 10
- Parameters checked in table 3 were examined: Urea, Calcium (Ca++), Glucose, Inorganic phosphorus (P), Total protein (Tot.Prot.), Aspartate aminotransferase (ASAT), Albumin, Alanine aminotransferase (ALAT), Albumin/Globulin (A/G) ratio (by calculation), Alkaline phosphatase (AP), Sodium (Na+), Creatinine (Creat), Potassium (K+), Total cholesterol (Chol), Chloride (Cl-), Total bilirubin (Bili), Bile acids

NEUROBEHAVIOURAL EXAMINATION:
- Time schedule for examinations: Prior to the start of treatment and at weekly intervals thereafter, all animals were observed for signs of functional/behavioral toxicity. Functional performance tests were also performed on five selected males (during Week 6,on completion of the pairing phase) and females (at Day 4 post partum) from each dose level, prior to termination, together with an assessment of sensory reactivity to various stimuli.
- Battery of functions tested: Forelimb/Hindlimb grip strength, motor activity and sensory reactivity to auditory, visual and
proprioceptive stimuli (Grasp response, Touch escape, Vocalization, Pupil reflex, Toe pinch, Blink reflex, Tail pinch, Startle reflex, Finger approach)

-Behavioral Assessments (Parameters observed: Gait, Hyper/Hypothermia, Tremors, Skin color, Twitches, Respiration, Convulsions , Palpebral closure, Bizarre/Abnormal/Stereotypic behavior, Urination, Salivation , Defecation, Pilo-erection, Transfer arousal, Exophthalmia, Tail elevation, Lachrymation)
Oestrous cyclicity (parental animals):
Estrous cyclicity not determined. After pairing the animals (male and female) for a period of up to fourteen days, cage tray-liners were checked each morning for the presence of ejected copulation plugs and each female was examined for the presence of a copulation plug in the vagina. A vaginal smear was prepared for each female and the stage of estrus or the presence of sperm was recorded. The presence of sperm within the vaginal smear and/or vaginal plug in situ
was taken as positive evidence of mating (Day 0 of gestation).
Sperm parameters (parental animals):
Parameters examined in P male parental generations: testis weight, epididymis weight, sperm reading score, weight of prostate and seminal vesicles

Histopathology: Sections of testes from all control and 1000 mg/kg bw/day males, any animals dying during the study, and any animals which failed to mate were stained and examined. Detailed qualitative examination of the testes was undertaken, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment-related effects such as missing germ cell layers or types, retained spermatids, multinucleated or apoptotic germ cells and sloughing of spermatogenic cells into the lumen. Any cell-or stage-specificity of testicular findings was noted.

Samples of the following tissues from control and 1000 mg/kg bw/day dose group animals, any animals dying during the study, and any animals which failed to mate were prepared as paraffin blocks, sectioned and stained for subsequent microscopic examination:
Seminal vesicles, epididymides, testes
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no
- termination of all surviving offspring on Day 5 post partum

PARAMETERS EXAMINED
The following parameters were examined in offspring of the parental generation: number of pups and postnatal mortality (Day 0 post partum, recorded daily and reported on Days 1 and 4 post partum), sex of pups (Days 1 and 4 post partum), stillbirths and live births (Day 0 post partum), presence of gross anomalies (following termination on Day 5 post partum), weight gain (on Days 1 and 4 post partum), physical abnormalities (Day 1 post partum), other: clinical condition (from birth to Day 5 post partum).

During the lactation phase, daily clinical observations were performed on all surviving offspring, together with litter size and offspring weights and assessment of surface righting reflex. Pregnant females were allowed to give birth and maintain their offspring until Day 5 post partum. Litter size, offspring weight and sex, surface righting and clinical signs were also recorded during this period.

GROSS EXAMINATION OF DEAD PUPS:
All offspring, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded. Possible cause of death was not determined for pups born or found dead.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals. The male dose groups were killed on Day 43 or Day 44.
- Maternal animals: All surviving animals. At Day 5 post partum, all females were killed. Any females which failed to achieve pregnancy or produce a litter were killed on or after Day 25 post coitum.

GROSS NECROPSY
- For all females, the uterus was examined for signs of implantation and the number of uterine implantations in each horn was recorded. The corpora lutea were also counted.

All adult animals and offspring, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

On the day of necropsy, necropsy details for Male 11 from the Control dose group and Female 42 from the 100 mg/kg bw/day dose group were not recorded on the data capture system, in error. The required organ weights for both animals were recorded. As there were sufficient data available for adequate interpretation of results, this deviation from the Study Plan was considered not to have affected the scientific integrity of the study or the results obtained.

HISTOPATHOLOGY / ORGAN WEIGHTS

-The following organs were dissected free from fat and weighed before fixation from five selected males and five selected females from each dose group: Adrenals, Prostate, Brain, Seminal vesicles, Epididymides, Spleen, Heart, Testes, Kidneys, Thymus, Liver, Thyroid (weighed post-fixation with Parathyroid), Ovaries, Uterus (weighed with Cervix), Pituitary (post fixation)

The following tissues were weighed from all remaining animals: Prostate, Seminal vesicles, Epididymides, Testes, Ovaries, Uterus (weighed with Cervix), Pituitary (post fixation)

Samples of the following tissues were removed from five selected males and five selected females from each dose group:
Adrenals, Muscle (skeletal), Aorta (thoracic), Ovaries, Bone & bone marrow (femur including stifle joint), Pancreas, Bone & bone marrow (sternum), Pituitary, Brain (including cerebrum, cerebellum and pons), Prostate, Caecum, Rectum, Coagulating gland, Salivary glands (submaxillary), Colon, Sciatic nerve, Duodenum, Seminal vesicles, Epididymides, Skin, Esophagus, Spinal cord (cervical, mid-thoracic and lumbar), Eyes, Gross lesions, Spleen, Heart, Stomach, Ileum (including peyer’s patches), Thyroid/parathyroid, Jejunum, Trachea, Kidneys, Testes, Liver, Thymus, Lungs (with bronchi), Urinary bladder, Lymph nodes (mandibular and mesenteric), Uterus/Cervix, Mammary gland, Vagina.

The tissues from five selected control and 1000 mg/kg bw/day dose group animals, any animals dying during the study, and any animals which failed to mate or did not achieve a pregnancy were prepared as paraffin blocks, sectioned and stained for subsequent microscopic examination.

Samples of the following tissues were preserved from all remaining animals: Ovaries, Pituitary, Prostate, Coagulating gland, Seminal vesicles, Epididymides, Gross lesions, Testes, Uterus/Cervix, Mammary gland, Vagina

Of these tissues from the remaining control and 1000 mg/kg bw/day animals and animals which did not achieve a pregnancy were also processed.

In addition, sections of testes from all control and 1000 mg/kg bw/day males were also stained and examined. Detailed qualitative examination of the testes was undertaken.

The following tissues were not processed due to technician error at necropsy and/or at post fix: The lungs with bronchi from animal numbers 32, 41, 56, 58, 72, 82 and 91 as gross lesions, and the colon (animal 1), vagina (animal 89) and pituitary (animal 91). The additional missing tissues recorded in the histopathology examination data can be due to the actual processing and trimming of the tissues. While it is accepted that this deviation was less than ideal, overall it is considered that this deviation had no adverse impact on the scientific purpose of the study as the remaining examined tissues showed no treatment related findings.
Postmortem examinations (offspring):
SACRIFICE
- At Day 5 post partum, all surviving offspring were killed and examined macroscopically.

GROSS NECROPSY
- All offspring, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded, following termination on Day 5 post partum.

HISTOPATHOLOGY / ORGAN WEIGTHS
- For offspring no organ weights recorded, no histopathology performed.
Statistics:
Where considered appropriate, quantitative data was subjected to statistical analysis to detect the significance of intergroup differences from control; statistical significance was achieved at a level of p<0.05. Statistical analysis was performed on the following parameters:

Grip Strength, Motor Activity, Body Weight, Body Weight Change, Food Consumption during gestation and lactation, Pre-Coital Interval, Gestation Length, Litter Size, Litter Weight, Sex Ratio, Corpora Lutea, Implantation Sites, Implantation Losses, Viability Indices, Offspring Body Weight, Offspring Body Weight Change, Offspring Surface Righting, Hematology, Blood Chemistry, Absolute Organ Weights, Body Weight-Relative Organ Weights.

Data were analyzed using the decision tree from the ProvantisTM Tables and Statistics Module. Data not analyzed by the Provantis data capture system were assessed separately using the R Environment for Statistical Computing. For a more detailed description of the statistical analysis performed, see the field "Any other information on materials and methods incl. tables".
Reproductive indices:
Mating Performance and Fertility:
The following parameters were calculated from the individual data during the mating period of the parental generation:
i. Pre-coital Interval, calculated as the time elapsing between initial pairing and the observation of positive evidence of mating.
ii. Fertility Indices. For each group the following were calculated:
Mating Index (%) = (Number of animals mated / Number of animals paired) x 100
Pregnancy Index (%) = (Number of pregnant females / Number of animals mated) x 100

Gestation and Parturition Data:
The following parameters were calculated from individual data during the gestation and parturition period of the parental generation:
i. Gestation Length, calculated as the number of days of gestation including the day for observation of mating and the start of parturition.
ii. Calculated for each group: Parturition Index (%) = (Number of females delivering live offspring / Number of pregnant females) x 100
Offspring viability indices:
For litter responses, the standard unit of assessment was considered to be the litter, therefore values were first calculated for each litter and the group mean was calculated using their individual litter values. Group mean values included all litters reared to termination (Day 5 of age).

Implantation Losses (%)
Group mean percentile pre-implantation and post-implantation loss were calculated for each female/litter as follows:
Pre–implantation loss (%) = ((Number of corpora lutea - Number of implantation sites) / Number of corpora lutea) x 100
Post–implantation loss (%) = ((Number of implantation sites - Total number of offspring born) / Number of implantation sites) x 100

Live Birth and Viability Indices
The following indices were calculated for each litter as follows:
Live Birth Index (%) = (Number of offspring alive on Day 1 / Number of offspring born) x 100
Viability Index (%) = (Number of offspring alive on Day 4 / Number of offspring alive on Day 1) x 100

Sex Ratio (% males)
Sex ratio was calculated for each litter value on Days 1 and 4 post partum, using the
following formula: (Number of male offspring / Total number of offspring) x 100
Clinical signs:
no effects observed
Description (incidence and severity):
There were no clinical signs of toxicity related to the test item (see Table 2 in 'Any other information on results incl. tables').

One 1000 mg/kg bw/day male showed signs of increased salivation on Day 1 and one 300 mg/kg bw/day female showed signs of generalised fur loss on Days 35 to 42. These observations were considered to be of no toxicological importance.
Mortality:
no mortality observed
Description (incidence):
There were no unscheduled deaths on study (see Table 2 in 'Any other information on results incl. tables').
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There was no adverse effect on body weight development for animals of either sex treated with 100, 300 or 1000 mg/kg bw/day (see tables 3 and 4 in 'Any other information on results incl. tables').

There were no adverse effects detected at 100, 300 or 1000 mg/kg bw/day for males throughout the study and for females during pre-mating, gestation or lactation phase.

At 300 or 1000 mg/kg bw/day actual body weight values for females on Day 1 of lactation were slightly higher than controls achieving statistical significance for the later. This was considered to be due to the slightly lower litter size for these females and as the actual body weight was higher than controls it was deemed not to be an adverse effect.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Not a feeding study; administration by oral gavage. However, there were no effects detected in food consumption for animals of either sex treated with 100, 300 or 1000 mg/kg bw/day when compared with controls.
Food efficiency:
no effects observed
Description (incidence and severity):
There were no effects detected in food efficiencies for animals of either sex treated with 100, 300 or 1000 mg/kg bw/day when compared with controls.
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Not a drinking water study. However, visual inspection of water residues did not indicate any effect of treatment on water intake throughout the study.
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
There were no treatment-related effects detected in the hematological parameters measured (see Table 5 in 'Any other information on results incl. tables').
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
No toxicologically significant changes were detected in blood chemical parameters.

Treated males from all dose groups showed statistically significant reductions in inorganic phosphorus and a statistically significant increase in chloride concentrations. All affected parameters showed no dose relationship and all individual values were within the background control ranges. As there were no histopathology findings these observations were considered to be of no toxicological significance.

Males and females treated with 1000 mg/kg bw/day showed statistically significant increases in glucose levels with these females also showing a statistically significant reduction in Albumin/Globulin ratio. At 300 or 1000 mg/kg bw/day, females also showed a statistically significant reduction in bilirubin, but without any dose relationship. All individual values were within background control ranges and in the absence of histopathology correlates these findings were deemed to be of no toxicological significance.

For results in tabular form, see Table 6 in 'Any other information on results incl. tables'.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Behavioral Assessments: No treatment-related effects were detected.

One female treated with 1000 mg/kg bw/day showed signs of hunched posture and pilo erection on Day 4 post partum. This was considered incidental and of no toxicological importance.

Functional Performance Tests: There were no treatment-related changes in the functional performance.

Males treated with 1000 mg/kg bw/day showed a statistically significant reduction in the hind limb grip strength during test 1 and an increase in grip strength during hind limb test 3. Due to the lack of any consistency, these findings were considered to be incidental. Females treated with 1000 or 300 mg/kg bw/day showed a statistically significant reductions during the hind limb strength test 3. These females showed comparable values to control during the preceding
two grip strength tests and in the absence of any signs of neurotoxicity, this finding was deemed likely to be incidental.

Sensory Reactivity Assessments: There were no treatment-related changes in sensory reactivity.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Histopathological findings, including examination of the brain (incl. cerebrum, cerebellum and pons): no findings were noted which could be related to treatment with the test item (see attached Illustration in section "Overall remarks, attachments").

Organ weight findings, including the brains: there were no treatment-related effects in the organ weights measured (see Table 7 in 'Any other information on results incl. tables').
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
No findings were noted which could be related to treatment with the test item (see attached Illustration in section "Overall remarks, attachments"). Findings observed were considered to be within the normal range of background alterations that are seen in untreated animals of this age and strain.
Histopathological findings: neoplastic:
not specified
Reproductive function: oestrous cycle:
not specified
Description (incidence and severity):
There were no treatment-related differences in fertility. One control female and one female treated with 1000 mg/kg bw/day were non-pregnant following positive evidence of mating.
Reproductive function: sperm measures:
not specified
Description (incidence and severity):
There were no treatment-related differences in fertility. One control female and one female treated with 1000 mg/kg bw/day were non-pregnant following positive evidence of mating.
Reproductive performance:
no effects observed
Description (incidence and severity):
No effects observed in reproductive performance (see tables 9 and 10 in 'Any other information on results incl. tables').

No treatment-related effects were detected in mating performance. Females 45 and 47 treated with 100 mg/kg bw/day, showed signs of mating after 12 and 14 days; respectively, whilst all remaining females mated within 5 days after pairing.

There were no treatment-related differences in fertility. One control female and one female treated with 1000 mg/kg bw/day were non-pregnant following positive evidence of mating.

There were no differences in gestation lengths. The distribution for treated females was comparable to controls as gestation lengths were between 22 and 24½ days.
No treatment related adverse effects observed.
Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: NOEL = highest dose tested.
Remarks on result:
other: No treatment related adverse effects observed.
Clinical signs:
no effects observed
Description (incidence and severity):
There were no clinical signs of toxicity observed in offspring, or any macroscopic abnormalities detected at terminal kill. There was no effect of treatment on surface righting reflex on Day 1 post partum.
Mortality / viability:
no mortality observed
Description (incidence and severity):
No effect on viability of offspring (see table 11 in 'Any other information on results incl. tables').

In total ten, twelve, twelve and eleven females from control, 100, 300, and 1000 mg/kg bw/day dose groups, respectively, gave birth to a live litter and successfully reared young to Day 5 of age. The following assessment of litter response is based on all litters reared to termination on Day 5 of lactation/age.

There was no effect of treatment on the mean number of corpora lutea, implantation sites, and pre- or post implantation losses. Of the litters born, litter size at birth and subsequently on Days 1 and 4 post partum for the 300 and 1000 mg/kg bw/day dose groups was slightly lower than controls. There was, however, no dose relationship and these intergroup differences were not statistically significant, and this finding was considered to be incidental. There was also no effect of treatment with the test item on sex ratio. One control female had only one live pup at birth and showed a total litter loss on Day 3 post partum.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Offspring (F1) body weights and litter weights at birth and on Days 1 and 4 post partum were generally comparable to controls (see table 11 in 'Any other information on results incl. tables'). Any minor intergroup differences were considered to be due to differences in litter sizes and as such, of no relationship to maternal treatment with the test item.
Food efficiency:
not specified
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no macroscopic abnormalities detected at terminal kill.
Histopathological findings:
not examined
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Dose descriptor:
other: No dose descriptor determined for F1 generation
Generation:
F1
Based on:
other: No dose descriptor determined for F1 generation
Sex:
not specified
Remarks on result:
not measured/tested
Reproductive effects observed:
no

Table 1. Allocation of animals to treatment groups

Treatment Group Dose Level Treatment Volume Concentration Animal Numbers
(mg/kg bw/day) (mL/kg) (mg/mL) Male Female
Control 0 4 0 12 (1-12) 12 (13-24)
Low 100 4 25 12 (25-36) 12 (37-48)
Intermediate 300 4 75 12 (49-60) 12 (61-72)
High 1000 4 250 12 (73-84) 12 (85-96)

The numbers in parentheses ( ) show the individual animal numbers allocated to each treatment

group.

Table 2. Summary Incidence of Daily Clinical Observations

Day Numbers Relative to Start Date
Group 1 Group 2 Group 3 Group 4
0(Control) 100 mg/kg bw/day 300 mg/kg bw/day 1000 mg/kg bw/day
Sex: Male
Scheduled kill
Number of Animals 12 12 12 12
Days from - to  43 44 43 44 43 44 43 44
Increased salivation
Number of Animals . . . 1
Days from - to  . . . 1 1
Sex: Female 
Littering
Number of Animals . 1 . 1
Days from - to  . 41 41 . 41 41
Not Observed Due to Littering
Number of Animals . 1 . 1
Days from - to  . 41 41 . 41 41
Scheduled kill
Number of Animals 12 12 12 12
Days from - to  42 46 42 55 42 46 42 46
Generalised fur loss
Number of Animals . . 1 .
Days from - to  . . 35 42 .

. No data available

Table 3. Group Mean Body Weight Values

Body Weights (g)
Day Numbers Relative to Start Date
Group (Sex) 1 8 15 22 29 36 43
1(M) Mean 309.9 322.5 342.7 351.1 367.8 381.4 392.0
S.D. 6.9 9.9 14.4 14.5 15.4 19.5 20.9
N 12 12 12 12 12 12 12
2(M) Mean 309.2 322.4 340.8 350.8 366.0 382.4 391.0
S.D. 7.0 7.8 9.6 15.1 17.2 19.8 19.8
N 12 12 12 12 12 12 12
3(M) Mean 309.7 324.5 343.5 354.1 374.0 391.2 398.9
S.D. 12.6 17.4 19.6 21.1 22.0 22.7 23.0
N 12 12 12 12 12 12 12
4(M) Mean 310.8 324.3 343.9 355.3 372.8 389.5 397.2
S.D. 10.6 13.0 15.8 18.5 21.5 21.0 20.8
N 12 12 12 12 12 12 12
1(F) Mean 188.6 196.0 206.3
S.D. 5.9 7.4 6.5
N 12 12 12
2(F) Mean 188.1 194.7 206.2
S.D. 7.3 9.7 7.8
N 12 12 12
3(F) Mean 188.7 196.8 206.1
S.D. 4.7 7.6 8.8
N 12 12 12
4(F) Mean 190.7 200.6 211.6
S.D. 7.7 9.1 10.5
N 12 12 12
Day Numbers
Gestation Lactation
0 7 14 20 1 4
1(F) Mean 207.0 230.7 260.2 319.2 229.7 243.8
S.D. 7.2 9.4 11.8 21.9 9.5 12.3
N 11 11 11 11 10 10
2(F) Mean 210.9 234.4 260.8 320.1 234.7 250.9
S.D. 16.4 17.2 18.9 26.1 17.8 18.9
N 12 12 12 12 12 12
3(F) Mean 206.7 233.8 262.5 318.8 242.7 248.4
S.D. 5.8 7.8 10.7 18.6 15.3 14.8
N 12 12 12 12 12 12
4(F) Mean 212.9 239.6 265.8 325.5 247.2* 257.7
S.D. 7.8 9.7 15.5 24.2 12.7 14.8
N 11 11 11 11 11 11

Dose Levels:

Group 1: 0 (Control), Group 2: 100 mg/kg bw/day, Group 3: 300 mg/kg bw/day, Group 4: 1000 mg/kg bw/day

M Male

F Female

S.D. Standard deviation

N Number of animals/litters

* Significantly different from control group p<0.05

Table 4. Group Mean Body Weight Gains

Increase in Body Weight (g)
Day Numbers Relative to Start Date
Abs %
Gain Gain
Group From: 1 8 15 22 29 36 1 1
(Sex) To: 8 15 22 29 36 43 43 43
1(M) Mean 12.6 20.2 8.4 16.7 13.7 10.6 82.1 26.5
S.D. 4.5 6.4 5.6 3.8 5.6 4.7 18.1 5.8
N 12 12 12 12 12 12 12 12
2(M) Mean 13.3 18.4 10.0 15.2 16.4 8.6 81.8 26.5
S.D. 3.6 6.8 9.9 4.3 5.4 3.5 17.3 5.6
N 12 12 12 12 12 12 12 12
3(M) Mean 14.8 19.0 10.6 19.9 17.2 7.8 89.3 28.8
S.D. 7.8 5.5 5.5 4.8 4.8 3.9 17.0 5.5
N 12 12 12 12 12 12 12 12
4(M) Mean 13.4 19.7 11.3 17.5 16.8 7.7 86.3 27.8
S.D. 8.8 4.7 5.9 4.9 3.5 4.3 15.9 4.9
N 12 12 12 12 12 12 12 12
Group From: 1 8 1 1
(Sex) To: 8 15 15 15
1(F) Mean 7.4 10.3 17.7 9.4
S.D. 4.7 4.2 5.0 2.7
N 12 12 12 12
2(F) Mean 6.6 11.5 18.1 9.6
S.D. 4.8 4.6 4.1 2.3
N 12 12 12 12
3(F) Mean 8.2 9.3 17.4 9.2
S.D. 4.3 5.3 6.4 3.3
N 12 12 12 12
4(F) Mean 9.9 11.0 20.9 11.0
S.D. 6.4 5.3 7.0 3.8
N 12 12 12 12
Increase in Body Weight (g) Cumulative Body Weight Change (g)
Days Days
Gestation Lactation Gestation
Group From: 0 7 14 1 0 0
(sex) To: 7 14 20 4 14 20
1(F) Mean 23.7 29.5 59.0 14.1 53.2 112.2
S.D. 5.8 6.6 14.1 8.4 10.0 21.8
N 11 11 11 10 11 11
2(F) Mean 23.5 26.4 59.3 16.3 49.9 109.2
S.D. 5.0 5.3 9.7 9.4 9.6 17.6
N 12 12 12 12 12 12
3(F) Mean 27.1 28.8 56.3 5.8 55.8 112.1
S.D. 3.3 6.3 13.3 8.0 7.2 16.4
N 12 12 12 12 12 12
4(F) Mean 26.7 26.2 59.6 10.5 52.9 112.5
S.D. 5.6 8.2 14.8 9.1 12.4 20.6
N 11 11 11 11 11 11

Dose Levels:

Group 1: 0 (Control), Group 2: 100 mg/kg bw/day, Group 3: 300 mg/kg bw/day, Group 4: 1000 mg/kg bw/day

M Male

F Female

S.D. Standard deviation

N Number of animals/litters

Table 5. Group Mean Hematological Values

Group (sex) Hb RBC Hct MCH MCV MCHC WBC Neut Lymph Mono Eos Bas CT PLT APTT
g/dl 10^12/l % pg fl g/dl 10^9/l 10^9/l 10^9/l 10^9/l 10^9/l 10^9/l Seconds 10^9/l Seconds
1(M) Mean 16.90 9.064 47.94 18.64 52.90 35.28 7.18 1.060 6.066 0.000n 0.054 0.000n 9.14 742.4 14.76
S.D. 0.63 0.339 1.33 0.65 1.19 1.02 1.82 0.287 1.608 0.000 0.078 0.000 0.19 158.5 0.93
N 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5
2(M) Mean 16.54 8.756 47.72 18.92 54.56 34.68 6.70 0.902 5.774 0.000n 0.026 0.000n 8.92 648.0 15.30
S.D. 0.35 0.314 0.55 0.87 2.03 0.54 1.19 0.438 1.005 0.000 0.037 0.000 0.15 67.5 0.39
N 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5
3(M) Mean 16.48 8.614 47.04 19.18 54.64 35.08 6.58 1.142 5.408 0.000n 0.030 0.000n 8.84 625.4 14.84
S.D. 0.47 0.188 0.83 0.73 1.44 0.81 1.08 0.542 1.296 0.000 0.041 0.000 0.17 51.8 1.75
N 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5
4(M) Mean 16.30 8.718 47.22 18.74 54.22 34.52 5.60 1.316 4.376 0.000n 0.024 0.000n 8.94 650.6 15.54
S.D. 0.39 0.390 1.21 0.86 2.01 0.33 0.90 0.346 0.942 0.000 0.033 0.000 0.27 25.0 0.48
N 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5
1(F) Mean 13.14 6.862 38.64 19.16 56.30 34.04 6.48 2.508 3.974 0.000n 0.000n 0.000n 8.12 994.0 14.60
S.D. 1.24 0.643 3.55 0.47 1.15 0.17 1.03 1.232 0.858 0.000 0.000 0.000 0.24 92.7 1.47
N 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5
2(F) Mean 13.48 6.952 39.98 19.42 57.56 33.74 6.12 2.232 3.888 0.000n 0.000n 0.000n 8.16 959.8 13.28
S.D. 0.66 0.326 1.43 0.61 1.77 0.43 1.62 1.247 0.503 0.000 0.000 0.000 0.11 94.0 1.15
N 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5
3(F) Mean 13.40 7.214 40.02 18.62 55.50 33.52 5.58 1.844 3.736 0.000n 0.000n 0.000n 8.16 1059.2 13.30
S.D. 0.50 0.409 1.70 0.61 1.08 0.63 1.23 0.845 1.002 0.000 0.000 0.000 0.27 83.1 1.52
N 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5
4(F) Mean 13.64 7.084 40.12 19.26 56.66 34.00 7.66 2.728 4.934 0.000n 0.000n 0.000n 8.14 876.0 13.44
S.D. 0.61 0.419 2.13 0.53 0.99 0.42 1.79 1.546 0.578 0.000 0.000 0.000 0.21 246.4 1.47
N 5 5 5 5 5 5 5 5 5 5 5 5 5 5

5

Dose Levels: Group 1 - 0 (Control), Group 2 - 100 mg/kg bw/day, Group 3 - 300 mg/kg bw/day, Group 4 - 1000 mg/kg bw/day

M Male

F Female

S.D. Standard deviation

N Number of animals/litters

n Data not appropriate for statistical analysis

Parameters measured on potassium EDTA - treated blood:

Hemoglobin (Hb)

Total erythrocyte count (RBC)

Hematocrit (Hct)

Erythrocyte indices - Mean Corpuscular Hemoglobin (MCH), Mean Corpuscular Volume (MCV), Mean Corpuscular Hemoglobin Concentration (MCHC)

Total leukocyte count (WBC)

Differential leukocyte count - Neutrophils (Neut), Lymphocytes (Lymph), Monocytes (Mono), Eosinophils (Eos), Basophils (Bas) Platelet count (PLT)

Parameters measured on citrate-treated blood:

Prothrombin time (CT)

Activated partial thromboplastin time (APTT)

Table 6. Group Mean Blood Chemical Values

Group (Sex) Urea Glucose Tot. Prot. Albumin A/G Na+ K+ Cl- Ca++ P ASAT ALAT AP Creat Chol Bili Bile Acid
mg/dl mg/dl g/dl g/dl Ratio mmol/l mmol/l mmol/l mmol/l mmol/l IU/l IU/l IU/l mg/dl mg/dl mg/dl μmol/l
1(M) Mean 47.0 157.0 7.642 3.96 1.076 146.6 4.346 100.2 2.788 2.62 73.2 57.8 158.4 0.686 106.8 0.186 8.78
S.D. 2.4 7.7 0.388 0.11 0.087 1.7 0.309 0.8 0.093 0.15 15.8 8.8 17.6 0.074 13.3 0.075 3.92
N 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5
2(M) Mean 49.4 172.6 7.332 3.90 1.140 147.2 4.264 101.8* 2.838 2.12** 74.8 54.0 170.8 0.784 101.0 0.112 8.16
S.D. 6.7 4.4 0.292 0.07 0.083 0.8 0.106 0.8 0.034 0.29 9.9 6.1 43.4 0.044 13.0 0.035 3.99
N 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5
3(M) Mean 52.2 162.8 7.208 3.90 1.178 147.0 4.632 102.2* 2.782 2.14** 84.0 56.4 166.2 0.836 98.8 0.154 8.12
S.D. 12.3 28.6 0.366 0.12 0.130 1.7 0.685 1.6 0.034 0.21 22.1 5.2 31.5 0.245 16.2 0.091 5.93
N 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5
4(M) Mean 42.6 181.0* 7.228 3.88 1.160 148.0 4.188 102.0* 2.800 2.16** 98.6 55.6 169.6 0.730 96.0 0.124 6.66
S.D. 3.9 18.5 0.283 0.11 0.065 2.8 0.338 1.0 0.031 0.27 25.0 6.7 16.3 0.056 8.0 0.028 2.47
N 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5
1(F) Mean 52.6 118.2 6.624 3.80 1.344 145.6 4.318 102.8 2.644 1.14 87.0 70.4 124.6 0.786 81.0 0.088 10.04
S.D. 7.5 15.1 0.238 0.20 0.067 1.5 0.257 2.0 0.093 0.45 15.1 15.7 49.1 0.150 14.6 0.008 3.07
N 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5
2(F) Mean 60.0 132.6 6.670 3.84 1.352 146.4 4.674 103.6 2.714 1.08 82.8 74.4 91.4 0.868 82.4 0.088 26.42
S.D. 13.0 20.5 0.305 0.17 0.039 1.5 0.732 1.5 0.181 0.37 11.8 17.5 8.3 0.148 6.7 0.019 14.78
N 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5
3(F) Mean 56.4 131.8 7.016 3.94 1.286 147.8 3.952 104.8 2.596 0.64 82.8 67.0 112.8 0.870 71.6 0.068* 7.38
S.D. 5.1 9.5 0.247 0.11 0.074 1.5 0.118 0.8 0.106 0.15 12.0 15.0 20.2 0.082 12.8 0.008 3.60
N 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5
4(F) Mean 47.0 155.0** 6.656 3.68 1.258* 145.4 3.948 103.4 2.502 0.84 99.6 76.2 135.2 0.844 83.8 0.072* 10.94
S.D. 9.2 23.1 0.418 0.22 0.026 1.1 0.343 0.5 0.115 0.26 18.3 13.8 75.3 0.056 12.8 0.004 9.67
N 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5

Dose Levels: Group 1 - 0 (Control), Group 2 - 100 mg/kg bw/day, Group 3 - 300 mg/kg bw/day, Group 4 - 1000 mg/kg bw/day

M Male

F Female

S.D. Standard deviation

N Number of animals/litters

n Data not appropriate for statistical analysis

* Significantly different from control group p<0.05

** Significantly different from control group p<0.01

Parameters measured on lithium heparin treated blood:

Urea

Calcium (Ca++)

Glucose

Inorganic phosphorus (P)

Total protein (Tot.Prot.)

Aspartate aminotransferase (ASAT)

Albumin

Alanine aminotransferase (ALAT)

Albumin/Globulin (A/G) ratio (by calculation)

Alkaline phosphatase (AP)

Sodium (Na+)

Creatinine (Creat)

Potassium (K+)

Total cholesterol (Chol)

Chloride (Cl-)

Total bilirubin (Bili)

Bile acids

Table 7. Group Mean Organ Weights with Corresponding Relative (% of Body Weight) Organ Weights

MALES FEMALES
0 100 300 1000 0 100 300 1000
Control mg/kg bw/day Control mg/kg bw/day
Terminal Bodyweight Mean (g) 392.9 392.2 399.2 398.3 248.6 254.8 251.3 261.7
S.D. 20.7 20.6 22.5 22.6 14.1 19.9 15.8 15.0
N 12 12 12 12 12 12 12 12
Adrenals Mean (g) 0.08528 0.08226 0.08246 0.08522 0.09580 0.09800 0.09698 0.10408
S.D. 0.00190 0.01406 0.01055 0.00888 0.01520 0.01361 0.01103 0.01774
N 5 5 5 5 5 5 5 5
Mean (%) 0.022 0.021 0.020 0.022 0.040 0.040 0.039 0.040
S.D. 0.001 0.003 0.003 0.003 0.005 0.006 0.005 0.006
N 5 5 5 5 5 5 5 5
Brain (Including Cerebrum,
Cerebellum And Pons)
Mean (g) 1.88670 1.92172 1.93068 1.91456 1.72716 1.71518 1.73446 1.81950
S.D. 0.03837 0.05397 0.06150 0.22249 0.08880 0.11007 0.05664 0.06049
N 5 5 5 5 5 5 5 5
Mean (%) 0.476 0.487 0.479 0.482 0.722 0.705 0.705 0.708
S.D. 0.024 0.018 0.024 0.053 0.041 0.026 0.009 0.048
N 5 5 5 5 5 5 5 5
Epididymides Mean (g) 1.75418 1.69644 1.81648 1.73282
S.D. 0.27530 0.17860 0.21857 0.14971
N 12 12 12 12
Mean (%) 0.446 0.435 0.456 0.437
S.D. 0.064 0.059 0.058 0.049
N 12 12 12 12
Heart Mean (g) 1.17996 1.10376 1.22666 1.25292 0.83040 0.91786 0.85436 0.88298
S.D. 0.07471 0.14288 0.12751 0.13954 0.07639 0.07389 0.08927 0.13537
N 5 5 5 5 5 5 5 5
Mean (%) 0.298 0.278 0.303 0.317 0.347 0.377 0.348 0.342
S.D. 0.027 0.024 0.024 0.048 0.034 0.021 0.041 0.04
N 5 5 5 5 5 5 5 5
Kidneys Mean (g) 2.43284 2.25590 2.51100 2.41284 1.57000 1.46824 1.46996 1.69592
S.D. 0.15977 0.19345 0.33264 0.10838 0.12636 0.16048 0.05858 0.08434
N 5 5 5 5 5 5 5 5
Mean (%) 0.614 0.571 0.622 0.608 0.655 0.605 0.598 0.660
S.D. 0.041 0.040 0.078 0.032 0.034 0.075 0.022 0.053
N 5 5 5 5 5 5 5 5
Liver Mean (g) 13.6384 13.1027 13.4021 13.6948 10.9844 11.0291 10.9512 11.9167
S.D. 1.01250 1.79533 1.56517 1.53317 1.40468 0.98740 0.99183 0.69743
N 5 5 5 5 5 5 5 5
Mean (%) 3.435 3.314 3.320 3.443 4.577 4.541 4.444 4.634
S.D. 0.139 0.416 0.374 0.270 0.402 0.446 0.212 0.314
N 5 5 5 5 5 5 5 5
Ovaries Mean (g) 0.11540 0.11941 0.11821 0.11930
S.D. 0.01275 0.01705 0.01858 0.01711
N 10 12 12 11
Mean (%) 0.047 0.047 0.047 0.045
S.D. 0.004 0.008 0.006 0.005
N 10 12 12 11
Pituitary Mean (g) 0.01177 0.01264 0.01392 0.01300 0.01340 0.01565* 0.01560* 0.01531*
S.D. 0.00211 0.00285 0.00390 0.00274 0.00194 0.00215 0.00236 0.00231
N 12 12 11 12 10 12 11 10
Mean (%) 0.003 0.003 0.004 0.003 0.005 0.006* 0.006* 0.006*
S.D. 0.001 0.001 0.001 0.001 0.001 0.001 0.001 0.001
N 12 12 11 12 10 12 11 10
Prostate Mean (g) 0.70769 0.67011 0.75388 0.67923
S.D. 0.14633 0.14353 0.22936 0.11531
N 12 12 12 12
Mean (%) 0.180 0.171 0.189 0.171
S.D. 0.037 0.037 0.055 0.030
N 12 12 12 12
Seminal Vesicles Mean (g) 2.07506 1.97583 1.95986 2.00458
S.D. 0.52402 0.27468 0.28542 0.47709
N 12 12 12 12
Mean (%) 0.528 0.506 0.491 0.503
S.D. 0.129 0.083 0.069 0.110
N 12 12 12 12
Spleen Mean (g) 0.76272 0.68878 0.68720 0.87458 0.58394 0.66942 0.54512 0.63062
S.D. 0.11332 0.10365 0.13722 0.12493 0.03934 0.10353 0.07306 0.09472
N 5 5 5 5 5 5 5 5
Mean (%) 0.192 0.174 0.171 0.221 0.244 0.275 0.221 0.245
S.D. 0.024 0.017 0.037 0.037 0.015 0.039 0.027 0.033
N 5 5 5 5 5 5 5 5
Testes Mean (g) 3.82673 3.86518 3.79398 3.79556
S.D. 0.26592 0.29204 0.22589 0.23812
N 12 12 12 12
Mean (%) 0.975 0.988 0.951 0.954
S.D. 0.067 0.083 0.035 0.061
N 12 12 12 12
Thymus Mean (g) 0.38442 0.40064 0.41422 0.43558 0.22768 0.23756 0.20504 0.22760
S.D. 0.08239 0.06301 0.03961 0.07949 0.02056 0.04589 0.01541 0.03992
N 5 5 5 5 5 5 5 5
Mean (%) 0.097 0.102 0.103 0.109 0.095 0.098 0.084 0.088
S.D. 0.023 0.021 0.013 0.018 0.007 0.022 0.009 0.015
N 5 5 5 5 5 5 5 5
Thyroid/Parathyroid Mean (g) 0.02164 0.02434 0.02448 0.02166 0.02440 0.01808 0.01884 0.02270
S.D. 0.00730 0.00359 0.00421 0.00277 0.00335 0.00323 0.00324 0.00771
N 5 5 5 5 5 5 5 5
Mean (%) 0.005 0.006 0.006 0.005 0.010 0.007 0.008 0.009
S.D. 0.002 0.001 0.001 0.001 0.001 0.002 0.001 0.003
N 5 5 5 5 5 5 5 5
Uterus & Cervix Mean (g) 0.73185 0.72131 0.69785 0.70894
S.D. 0.12800 0.19822 0.13247 0.10660
N 10 12 12 11
Mean (%) 0.296 0.282 0.278 0.272
S.D. 0.049 0.070 0.054 0.050
N 10 12 12 11

S.D. Standard deviation

N Number of animals/litters

* Significantly different from control group p<0.05

Table 8. Summary Incidence of Necropsy Findings

MALES
Removal Reason: SCHEDULED KILL 0 100 300 1000
(Control) mg/kg bw/day
Number of Animals Examined: 12 12 12 12
Group:  (1) (2) (3) (4)
Lungs (With Bronchi);
Submitted  (11#) (12) (12) (12)
No Visible Lesions  11 10 9 10
Discolouration 0 0 0 1
Discolouration; red  0 2 3 1
FEMALES
Removal Reason: SCHEDULED KILL 0 100 300 1000
(Control) mg/kg bw/day
Number of Animals Examined: 12 12 12 12
Group:  (1) (2) (3) (4)
Lungs (With Bronchi);
Submitted  (12) (11#) (12) (12)
No Visible Lesions  12 9 9 9
Discolouration; red  0 2 3 3
Uterus & Cervix;
Submitted  (12) (11#) (12) (12)
No Visible Lesions  12 10 12 11
Enlarged 0 0 0 1
Fluid Filled 0 0 0 1
Damaged On Removal 0 1 0 0

# = One animal not submitted in error

Table 9. Summary of Reproductive Performance - Groups Values

Dose Group (mg/kg bw/day)
0 (Control) 100 300 1000
Males
Initial group size 12 12 12 12
Paired 12 12 12 12
Failed to induce pregnancy in female partner 1 0 0 1
Induced pregnancy in female partner 11 12 12 11
Surviving to terminal necropsy 12 12 12 12
Females
Initial group size 12 12 12 12
Paired 12 12 12 12
Non-pregnant 1 0 0 1
Total Litter Loss 1 0 0 0
Reared young to Day 5 of age 10 12 12 11

Table 10. Tabular Summary Report of Effects on Reproduction/Development

Observations Dose Level (mg/kg bw/day)
0 (Control) 100 300 1000
Paired animals n 12 12 12 12
Females showing evidence of copulation n 12 12 12 12
Pregnant females n 11 12 12 11
Conception Days 1-5 n 12 10 12 12
Conception Days 12 n 0 1 0 0
Conception Days 14 n 0 1 0 0
Gestation = 22 Days n 1 2 3 2
Gestation = 22 ½ Days n 7 4 8 6
Gestation = 23 Days n 0 1 0 0
Gestation = 23 ½ Days n 2 5 1 2
Gestation = 24 Days n 0 0 0 1
Gestation = 24 ½ Days n 1 0 0 0
Dams with live young born n 11 12 12 11
Dams with live young at Day 4 post partum n 10 12 12 11
Corpora lutea/dam x 13.9 12.5 11.6 12.1
Implants/dam x 13.9 12.5 11.5 12.1
Live offspring/dam at Day 1 post partum x 12.8 11.5 10.3 11.1
Live offspring/dam at Day 4 post partum x 12.5 11.5 9.8 11.1
Sex ratio: % males at Day 1 post partum x 40.6 51.0 45.9 40.9
Sex ratio: % males at Day 4 post partum x 41.0 51.0 45.9 40.9
Litter weight (g) at Day 1 post partum x 72.04 68.58 58.91 66.20
Litter weight (g) at Day 4 post partum x 97.05 97.40 78.81 93.52
Male offspring weight (g) at Day 1 post partum x 5.93 6.29 6.00 6.32
Male offspring weight (g) at Day 4 post partum x 8.24 8.90 8.42 8.96
Female offspring weight (g) at Day 1 post partum x 5.59 5.82 5.67 6.06
Female offspring weight (g) at Day 4 post partum x 7.79 8.43 7.90 8.69
LOSS OF OFFSPRING/DAM
Pre-implantation (corpora lutea minus implantations)
0 n 11 12 11 11
1 n 0 0 1 0
2 n 0 0 0 0
3 n 0 0 0 0
5 n 0 0 0 0
6 n 0 0 0 0
Pre-natal (implantations minus live births)
0 n 4 5 5 7
1 n 3 3 3 1
2 n 2 3 2 2
3 n 1 1 1 0
4 n 1 0 1 0
6 n 0 0 0 1
Post natal (live births minus offspring alive on Day 4 post partum)
0 n 8 12 9 11
1 n 1 0 2 0
2 n 1 0 0 0
4 n 0 0 1 0

n = Number

x = Mean

Table 11. Group Mean Litter Size and Litter Weights and Survival Indices

Group Number of Corpora Lutea Number of Implantation Sites Total Number of Offspring Born Number of Live Offspring Litter Weight (g) Offspring Weight (g) Offspring Body Weight Change (g) Live Birth Index (%) Viability Index (%)
Day 1 Day 4 Days 1-4
Day 1 Day 4 Day 1 Day 4 Males Females Males Females Males Females
1 Mean 13.9 13.9 12.9 12.8 12.5 72.04 97.05 5.93 5.59 8.24 7.79 2.31 2.20 99.4 97.9
S.D. 1.7 1.7 2.6 2.4 2.3 10.29 12.92 0.77 0.76 1.54 1.52 0.82 0.84 2.0 4.8
N 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10
2 Mean 12.5 12.5 11.5 11.5 11.5 68.58 97.40 6.29 5.82 8.90 8.43 2.61 2.60 100.0 100.0
S.D. 2.4 2.4 2.3 2.3 2.3 10.03 14.42 0.60 0.58 1.28 1.27 0.73 0.78 0.0 0.0
N 12 12 12 12 12 12 12 12 12 12 12 12 12 12 12
3 Mean 11.6 11.5 10.4 10.3 9.8 58.91 78.81 6.00 5.67 8.42 7.90 2.41 2.24 95.8 96.5
S.D. 3.2 3.3 3.8 4.1 3.7 20.75 27.43 0.74 0.67 1.31 1.28 0.70 0.82 14.4 7.9
N 12 12 12 12 12 12 12 11 12 11 12 11 12 12 12
4 Mean 12.1 12.1 11.1 11.1 11.1 66.20 93.52 6.32 6.06 8.96 8.69 2.64 2.63 100.0 100.0
S.D. 4.6 4.6 4.1 4.1 4.1 19.66 26.63 0.72 0.72 1.53 1.52 0.95 0.91 0.0 0.0
N 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11

Group 1: 0 (Control), Group 2: 100 mg/kg bw/day, Group 3: 300 mg/kg bw/day, Group 4: 1000 mg/kg bw/day

S.D. Standard deviation

N Number of animals/litters

Conclusions:
The oral administration of the substance to rats by gavage, at dose levels of 100, 300 and 1000 mg/kg bw/day, was tolerated well. The ‘No Observed Effect Level’ (NOEL) for reproductive toxicity was considered to be 1000 mg/kg bw/day.

As there were no treatment-related adverse effects observed in the study and the test item was tolerated well, according to the CLP Regulation the substance does not need to be classified with regards to reproductive toxicity. The oral route was selected as the most appropriate route of exposure, based on the physical properties of the test item, and the results of the study are believed to be of value in predicting the likely toxicity of the test item to man.
Executive summary:

The study was designed to investigate the systemic toxicity and potential adverse effects of the test item on reproduction (including offspring development) and is designed to be compatible with the requirements of the OECD Guidelines for Testing of Chemicals No. 422 “Combined Repeated Dose Toxicity Study with the Reproduction/ Developmental Toxicity Screening Test” and with Commission Regulation (EC) No 440/2008 of 30 May 2008.

The test item was administered by gavage to three groups, each of twelve male and twelve female Wistar Han™:RccHan™:WIST strain rats, for up to eight weeks (including a two week pre-pairing phase, pairing, gestation and early lactation for females), at dose levels of 100, 300 and 1000 mg/kg bw/day. A control group of twelve males and twelve females was dosed with vehicle alone (Arachis oil BP).

Clinical signs, behavioral assessments, body weight change and food and water consumption were monitored during the study.

Pairing of animals within each dose group was undertaken on a one male: one female basis within each treatment group on Day 15 of the study, with females subsequently being allowed to litter and rear their offspring to Day 5 of lactation.

During the lactation phase, daily clinical observations were performed on all surviving offspring, together with litter size and offspring weights and assessment of surface righting reflex.

Extensive functional observations were performed on five selected males from each dose group after the completion of the pairing phase, and for five selected parental females from each dose group on Day 4 post partum. Hematology and blood chemistry were evaluated prior to termination on five selected males and females from each dose group.

Adult males were terminated on Day 43 or Day 44 followed by the termination of all females and offspring on Day 5 post partum. Any female which did not produce a pregnancy was terminated on or after Day 25 post coitum. All animals were subjected to a gross necropsy examination and histopathological evaluation of selected tissues was performed.

Adult Responses

Mortality - There were no unscheduled deaths on study.

Clinical Observations - There were no clinical signs of toxicity related to the test item.

Behavioral Assessment - There were no treatment-related effects in the behavioral parameters measured.

Functional Performance Tests - There were no treatment-related changes in the functional performance.

Sensory Reactivity Assessments - There were no treatment-related changes in sensory reactivity.

Body Weight - There was no adverse effect on body weight development for animals of either sex treated with 100, 300 or 1000 mg/kg bw/day.

Food Consumption - There were no effects detected in food consumption for animals of either sex treated with 100, 300 or 1000 mg/kg bw/day when compared with controls.

Water Consumption - Visual inspection of water residues did not indicate any effect of treatment on water intake throughout the study.

Reproductive Performance

Mating - No treatment-related effects were detected in mating performance.

Fertility - There were no treatment-related differences in fertility.

Gestation Lengths - There were no differences in gestation lengths. The distribution for treated females was comparable to controls as gestation lengths were between 22 and 24½ days.

Litter Responses

Offspring Litter Size, Sex Ratio and Viability - There was no effect of treatment on the mean number of corpora lutea, implantation sites, or pre- and post implantation losses. Of the litters born, no effect of treatment was evident in litter size at birth and subsequently on Days 1 and 4 post partum. Sex ratio was comparable with controls.

Offspring Growth and Development - Offspring body weights and litter weights at birth and on Days 1 and 4 post partum were

comparable to controls. There was no effect on surface righting reflex. There were no clinical signs of toxicity observed in offspring, or any macroscopic abnormalities detected at terminal kill.

Laboratory Investigations

Hematology - There were no treatment-related changes in the hematological parameters measured.

Blood Chemistry - No toxicologically significant changes were detected in blood chemical parameters measured.

Pathology

Necropsy -There were no treatment-related macroscopic abnormalities detected.

Organ Weights -There were no treatment-related effects in the organ weights measured.

Histopathology -No findings were noted which could be related to treatment with the test item. Findings observed were considered to be within the normal range of background alterations that are seen in untreated animals of this age and strain.

The oral administration of the substance to rats by gavage, at dose levels of 100, 300 and 1000 mg/kg bw/day, was tolerated well. The ‘No Observed Effect Level’ (NOEL) for reproductive toxicity was considered to be 1000 mg/kg bw/day. The substance is not classified as toxic to reproduction according to the CLP Regulation.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report date:
2016

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:
Adopted 22 March 1996
Deviations:
yes
Remarks:
No analysis was carried out to determine the stability of the test item formulation. Deviations from the target range for relative humidity. Necropsy details for 2 animals not recorded. For histopathology examinations some tissues were not processed.
GLP compliance:
yes (incl. QA statement)
Remarks:
Certificate is as an attachement to the study report
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Tetrasodium enneatitanium icosaoxide hydrate
EC Number:
601-472-6
Cas Number:
117314-20-2
Molecular formula:
Sodium form: Na4Ti9O20 × n H2O Sodium/hydrogen form: 50 % Na4Ti9O20 × n H2O, 50 % Na2H2Ti9O20 × n H2O
IUPAC Name:
Tetrasodium enneatitanium icosaoxide hydrate
Test material form:
solid: particulate/powder
Remarks:
powder
Details on test material:
- Substance type: commercial product (pure active substance)
- Physical state: Solid powder
- Storage condition of test material: Ambient temperature, humidity and pressure. Stored in sealed containers in darkness.
- Stability under test conditions: Stable
- Purity: ca 100 %
- Particle size distribution: 2.92% <100μm
- Crystal structure: TiO6-octaedra
- Density: 2.83 x 10^3 kg/m3
- pH value: the pH value of the substance in an aqueous solution is appr. 11

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
Wistar Han™:RccHan™:WIST strain rats. The rat was selected for this study as it is a readily available rodent species historically used in safety evaluation studies and is acceptable to appropriate regulatory authorities.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories UK Ltd., Oxon, UK.
- Age at study initiation: Approximately 12 weeks
- Weight at study initiation: males 288-328 g, females 173-203 g
- Fasting period before study: No fasting
- Housing: Initially, all animals were housed in groups of three in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding. During the pairing phase, animals were transferred to polypropylene grid floor cages suspended over trays lined with absorbent paper on a one male: one female basis within each dose group. Following evidence of successful mating, the males were returned to their original cages. Mated females were housed individually during gestation and lactation in solid floor polypropylene cages with stainless steel mesh lids and softwood flakes.
- Diet (e.g. ad libitum): Pelleted diet (Rodent 2018C Teklad Global Certified Diet, Harlan Laboratories U.K Ltd., Oxon, UK.), free access to food
- Water (e.g. ad libitum): Tap water, free access to water
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25 °C
- Humidity (%): 30 to 70% with deviations from the target range up to 85.36%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
The test item and control substance (Arachis oil BP) were administered daily by gavage using a stainless steel cannula attached to a disposable plastic syringe.
Vehicle:
arachis oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was prepared at the appropriate concentrations as a suspension in Arachis oil BP. Due to the complex nature of the test item and its limited solubility in organic and aqueous media, a substance specific quantitative method of analysis could not be developed and no stability determined. The test item was formulated within two hours of it being applied to the test system; it is assumed that the formulation was stable for this duration. The concentration of test item in the formulations was determined using a gravimetric technique. Formulations were prepared daily.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Arachis oil BP was used because the test item did not dissolve/suspend in distilled water.
- Concentration in vehicle: 0, 25, 75 and 250 mg/mL
- Amount of vehicle (if gavage): 4 mL/kg treatment volume. The volume of test and control item administered to each animal was based on the most recent scheduled body weight and was adjusted at weekly intervals.

Analytical verification of doses or concentrations:
yes
Remarks:
Samples of the test item formulation were analyzed gravimetrically for the concentration of the test item.
Details on analytical verification of doses or concentrations:
Due to the complex nature of the test item and its limited solubility in organic and aqueous media, a substance specific quantitative method of analysis could not be developed. The concentration of test item in the formulations was determined using a gravimetric technique. Therefore formulations were prepared daily.

Samples of the test item formulation were taken and analyzed for concentration of SrTreat. The results indicate the accurate use of the test item and Arachis Oil BP as vehicle during the study, and that the prepared formulations were within acceptable ranges for the purpose of this study. The formulations were found to be homogeneously prepared. The analytical procedure had acceptable recoveries of test item in the vehicle. The method of analysis was validated and proven to be suitable for use.
Duration of treatment / exposure:
The test item was administered by gavage to groups of rats for up to eight weeks (including a two week pre-pairing phase, pairing, gestation and early lactation for females).
Frequency of treatment:
The test item was administered daily.
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
m/f
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
m/f
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Remarks:
m/f
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
m/f
No. of animals per sex per dose:
Control group (vehicle alone): 12 males, 12 females
Dose level 100 mg/kg bw/day: 12 males, 12 females
Dose level 300 mg/kg bw/day: 12 males, 12 females
Dose level 1000 mg/kg bw/day: 12 males, 12 females
For further details, see Table 1 in 'Any other information on results incl. tables'.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were chosen based on the results of previous toxicity work (SrTreat fourteen day repeated dose oral (gavage) range-finding toxicity study in the rat, see Attached background material).

In the range-finding toxicity study, the test item was administered by gavage to three groups, each of three male and three female Wistar Han™:RccHan™:WIST strain rats, for fourteen consecutive days, at dose levels of 250, 500 and 1000 mg/kg bw/day. A control group of three males and three females was dosed with vehicle alone (Arachis oil BP). Clinical signs, body weight change, dietary intake and water consumption were monitored during the study. All animals were subjected to gross necropsy examination.

Results:
Mortality -There were no unscheduled deaths on the study.
Clinical Observations - There were no clinical signs apparent during the study.
Body Weight- There was no adverse effects on body weight development for either sex at 1000, 500 or 250 mg/kg bw/day.
Food Consumption - There was no obvious effect on food consumption or food conversion efficiency for either sex at 1000, 500 or 250 mg/kg bw/day.
Water Consumption - There was no consistent effect on water consumption for either sex at 1000, 500 or 250 mg/kg bw/day.
Necropsy - There were no macroscopic abnormalities were detected.

The oral administration of the test item to rats by gavage, at dose levels of 250, 500 and 1000 mg/kg bw/day was well tolerated. Based on the results from this study a dose level of 1000 mg/kg bw/day was considered to be suitable as a high dose level for the OECD 422 study together with 100 and 300 mg/kg bw/day as the low and intermediate dose levels, respectively.

- Rationale for animal assignment: The animals were randomly allocated to treatment groups using a stratified body weight randomization procedure and the group mean body weights were then determined to ensure similarity between the treatment groups. Animals were paired on a 1 male: 1 female basis within each dose group.

- Section schedule rationale (if not random): The tissues from five selected control and 1000 mg/kg bw/day dose group animals, any animals dying during the study, and any animals which failed to mate or did not achieve a pregnancy were prepared as paraffin blocks, sectioned and stained for subsequent microscopic examination. Adult males were killed on Day 43 or Day 44. Adult females were killed on Day 5 post partum. Any females which failed to achieve pregnancy or produce a litter were killed on or after Day 25 post coitum.



Positive control:
No positive control

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: No

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily clinical observations. All animals were examined for overt signs of toxicity, ill-health and behavioral change immediately before dosing, soon after dosing, and one hour after dosing during the working week, at weekends and public holidays (except for females during parturition where applicable).

During the lactation phase, daily clinical observations were performed on all surviving offspring. Pregnant females were allowed to give birth and maintain their offspring until Day 5 post partum. Clinical signs were also recorded during this period.

On Day 43, the one hour post dose clinical observations were not performed for female 37 in error. Female 37 immediately before and after dosing on Day 43 and Day 44 did not show any clinical signs of toxicity, therefore, the omitted observations were considered not to affect the purpose or integrity of the study.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were recorded on Day 1 (prior to dosing) and then weekly for males until termination and weekly for females until pairing. During pairing phase females were weighed daily until mating was confirmed. Body weights were then recorded for females on Days 0, 7, 14 and 20 post coitum, and on Days 1 and 4 post partum. Body weights were also recorded at terminal kill.

FOOD CONSUMPTION:
- During the pre-pairing period, weekly food consumption was recorded for each cage of adults. This was continued for males after the mating phase. For females showing evidence of mating, food consumption was recorded for the periods covering post coitum Days 0-7, 7-14 and 14-20. For females with live litters, food consumption was recorded on Days 1 and 4 post partum.

FOOD EFFICIENCY:
- Food efficiency (the ratio of body weight change/dietary intake) was calculated retrospectively for males throughout the study period (with the exception of the mating phase) and for females during the pre-pairing phase. Due to offspring growth and milk production, food efficiency could not be accurately calculated during gestation and lactation.

WATER CONSUMPTION: Yes
- Time schedule for examinations: Water intake was observed daily by visual inspection of water bottles for any overt changes.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Hematological investigations were performed on five males and five females selected from each test and control group prior to termination (Day 42 for males and Day 4 post partum for females).
- Anaesthetic used for blood collection: No data
- Animals fasted: No
- How many animals: 10
- Parameters examined: Hemoglobin (Hb), Erythrocyte count (RBC), Hematocrit (Hct), Erythrocyte indices (mean corpuscular hemoglobin (MCH), mean corpuscular volume (MCV), mean corpuscular hemoglobin concentration (MCHC)), Total leukocyte count (WBC), Differential leukocyte count (neutrophils (Neut), lymphocytes (Lymph), monocytes (Mono), eosinophils (Eos), basophils (Bas)), Platelet count (PLT), Reticulocyte count (Retic) (Methylene blue stained slides were prepared but reticulocytes were not assessed). Prothrombin time (CT) was assessed by ‘Innovin’ and Activated partial thromboplastin time (APTT) was assessed by ‘Actin FS’.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood chemical investigations were performed on five males and five females selected from each test and control group prior to termination (Day 42 for males and Day 4 post partum for females).
- Animals fasted: No
- How many animals: 10
- Parameters examined: Urea, Calcium (Ca++), Glucose, Inorganic phosphorus (P), Total protein (Tot.Prot.), Aspartate aminotransferase (ASAT), Albumin, Alanine aminotransferase (ALAT), Albumin/Globulin (A/G) ratio (by calculation), Alkaline phosphatase (AP), Sodium (Na+), Creatinine (Creat), Potassium (K+), Total cholesterol (Chol), Chloride (Cl-), Total bilirubin (Bili), Bile acids

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Prior to the start of treatment and at weekly intervals thereafter, all animals were observed for signs of functional/behavioral toxicity. Functional performance tests were also performed on five selected males (during Week 6,on completion of the pairing phase) and females (at Day 4 post partum) from each dose level, prior to termination, together with an assessment of sensory reactivity to various stimuli.
- Battery of functions tested: Forelimb/Hindlimb grip strength, motor activity and sensory reactivity to auditory, visual and
proprioceptive stimuli (Grasp response, Touch escape, Vocalization, Pupil reflex, Toe pinch, Blink reflex, Tail pinch, Startle reflex, Finger approach)

-Behavioral Assessments (Parameters observed: Gait, Hyper/Hypothermia, Tremors, Skin color, Twitches, Respiration, Convulsions , Palpebral closure, Bizarre/Abnormal/Stereotypic behavior, Urination, Salivation , Defecation, Pilo-erection, Transfer arousal, Exophthalmia, Tail elevation, Lachrymation)
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
All adult animals and offspring, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

The following organs were dissected free from fat and weighed before fixation from five selected males and five selected females from each dose group: Adrenals, Prostate, Brain, Seminal vesicles, Epididymides, Spleen, Heart, Testes, Kidneys, Thymus, Liver, Thyroid (weighed post-fixation with Parathyroid), Ovaries, Uterus (weighed with Cervix), Pituitary (post fixation)

The following tissues were weighed from all remaining animals: Prostate, Seminal vesicles, Epididymides, Testes, Ovaries, Uterus (weighed with Cervix), Pituitary (post fixation)

On the day of necropsy, necropsy details for Male 11 from the Control dose group and Female 42 from the 100 mg/kg bw/day dose group were not recorded on the data capture system, in error. The required organ weights for both animals were recorded. As there were sufficient data available for adequate interpretation of results, this deviation from the Study Plan was considered not to have affected the scientific integrity of the study or the
results obtained.

HISTOPATHOLOGY: Yes
Samples of the following tissues were removed from five selected males and five selected females from each dose group:
Adrenals, Muscle (skeletal), Aorta (thoracic), Ovaries, Bone & bone marrow (femur including stifle joint), Pancreas, Bone & bone marrow (sternum), Pituitary, Brain (including cerebrum, cerebellum and pons), Prostate, Caecum, Rectum, Coagulating gland, Salivary glands (submaxillary), Colon, Sciatic nerve, Duodenum, Seminal vesicles, Epididymides, Skin, Esophagus, Spinal cord (cervical, mid-thoracic and lumbar), Eyes, Gross lesions, Spleen, Heart, Stomach, Ileum (including peyer’s patches), Thyroid/parathyroid, Jejunum, Trachea, Kidneys, Testes, Liver, Thymus, Lungs (with bronchi), Urinary bladder, Lymph nodes (mandibular and mesenteric), Uterus/Cervix, Mammary gland, Vagina.

The tissues from five selected control and 1000 mg/kg bw/day dose group animals, any animals dying during the study, and any animals which failed to mate or did not achieve a pregnancy were prepared as paraffin blocks, sectioned and stained for subsequent microscopic examination.

Samples of the following tissues were preserved from all remaining animals: Ovaries, Pituitary, Prostate, Coagulating gland, Seminal vesicles, Epididymides, Gross lesions, Testes, Uterus/Cervix, Mammary gland, Vagina

Of these tissues from the remaining control and 1000 mg/kg bw/day animals and animals which did not achieve a pregnancy were also processed.

In addition, sections of testes from all control and 1000 mg/kg bw/day males were also stained and examined. Detailed qualitative examination of the testes was undertaken.

The following tissues were not processed due to technician error at necropsy and/or at post fix: The lungs with bronchi from animal numbers 32, 41, 56, 58, 72, 82 and 91 as gross lesions, and the colon (animal 1), vagina (animal 89) and pituitary (animal 91). The additional missing tissues recorded in the histopathology examination data can be due to the actual processing and trimming of the tissues. While it is accepted that this deviation was less than ideal, overall it is considered that this deviation had no adverse impact on the scientific purpose of the study as the remaining
examined tissues showed no treatment related findings.
Other examinations:
Reproductive performance, offspring viability, clinical signs of offspring, offspring body weight and gross pathology of offspring were also examined to determine toxicity to reproduction and development: reported in Endpoint study record 7.8.1 Toxicity to reproduction.001 (see field "Cross-reference").

Statistics:
Where considered appropriate, quantitative data was subjected to statistical analysis to detect the significance of intergroup differences from control; statistical significance was achieved at a level of p<0.05. Statistical analysis was performed on the following parameters:

Grip Strength, Motor Activity, Body Weight, Body Weight Change, Food Consumption during gestation and lactation, Pre-Coital Interval, Gestation Length, Litter Size, Litter Weight, Sex Ratio, Corpora Lutea, Implantation Sites, Implantation Losses, Viability Indices, Offspring Body Weight, Offspring Body Weight Change, Offspring Surface Righting, Hematology, Blood Chemistry, Absolute Organ Weights, Body Weight-Relative Organ Weights.

Data were analyzed using the decision tree from the ProvantisTM Tables and Statistics Module. Data not analyzed by the Provantis data capture system were assessed separately using the R Environment for Statistical Computing. For a more detailed description of the statistical analysis performed, see the field "Any other information on materials and methods incl. tables".

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
There were no clinical signs of toxicity related to the test item. One 1000 mg/kg bw/day male showed signs of increased salivation on Day 1 and one 300 mg/kg bw/day female showed signs of generalised fur loss on Days 35 to 42. These observations were considered to be of no toxicological importance.

For results in tabular form, see Table 2 in 'Any other information on results incl. tables'.
Mortality:
no mortality observed
Description (incidence):
There were no unscheduled deaths on study (see Table 2 in 'Any other information on results incl. tables').
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There were no adverse effects detected at 100, 300 or 1000 mg/kg bw/day for males throughout the study and for females during pre-mating, gestation or lactation phase. At 300 or 1000 mg/kg bw/day actual body weight values for females on Day 1 of lactation were slightly higher than controls achieving statistical significance for the later. This was considered to be due to the slightly lower litter size for these females and as the actual body weight was higher than controls it was deemed not to be an adverse effect.

For results in tabular form, see Tables 3 and 4 in 'Any other information on results incl. tables'.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Not a feeding study. However, there were no effects detected in food consumption for animals of either sex treated with 100, 300 or 1000 mg/kg bw/day when compared with controls.
Food efficiency:
no effects observed
Description (incidence and severity):
There were no effects detected in food efficiencies for animals of either sex treated with 100, 300 or 1000 mg/kg bw/day when compared with controls.
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Not a drinking water study. However, visual inspection of water residues did not indicate any effect of treatment on water intake throughout the study.
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
There were no treatment-related effects detected in the hematological parameters measured (see Table 5 in 'Any other information on results incl. tables').
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
No toxicologically significant changes were detected in blood chemical parameters. Treated males from all dose groups showed statistically significant reductions in inorganic phosphorus and a statistically significant increase in chloride concentrations. All affected parameters showed no dose relationship and all individual values were within the background control ranges. As ther were no histopathology findings these observations were considered to be of no toxicological significance.

Males and females treated with 1000 mg/kg bw/day showed statistically significant increases in glucose levels with these females also showing a statistically significant reduction in Albumin/Globulin ratio. At 300 or 1000 mg/kg bw/day, females also showed a statistically significant reduction in bilirubin, but without any dose relationship. All individual values were within background control ranges and in the absence of histopathology correlates these findings were deemed to be of no toxicological significance.

For results in tabular form, see Table 6 in 'Any other information on results incl. tables'.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
No treatment-related effects were detected in behavioral assessments. One female treated with 1000 mg/kg bw/day showed signs of hunched posture and pilo erection on Day 4 post partum. This was considered incidental and of no toxicological importance.

There were no treatment-related changes in sensory reactivity.

There were no treatment-related changes in the functional performance. Males treated with 1000 mg/kg bw/day showed a statistically significant reduction in the hind limb grip strength during test 1 and an increase in grip strength during hind limb test 3. Due to the lack of any consistency, these findings were considered to be incidental. Females treated with 1000 or 300 mg/kg bw/day showed a statistically significant reductions during the hind limb strength test 3. These females showed comparable values to control during the preceding two grip strength tests and in the absence of any signs of neurotoxicity, this finding was deemed likely to be incidental.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No treatment-related changes were evident in the organ weights measured. Females from all treatment groups showed a statistically significant increase in pituitary weights in both absolute and relative to terminal body weight when comparerd with controls. There was no dose relationship present and no histopathological correlation, therefore this was considered to be unrelated to treatment with the test item.

For results in tabular form, see Table 7 in 'Any other information on results incl. tables'.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no treatment-related findings detected at necropsy. Reddened lungs were observed in some animals of either sex treated with the test item. There was, however, no dose-dependence and although some of these were not histopathologically processed and examined, there were no treatment related histopathological observations on the tissues examined. Additionally such observations are common in this type of study and this finding was considered unrelated to treatment with the test item. For offspring, there were no macroscopic abnormalities detected at terminal kill.

For results in tabular form, see Table 8 in 'Any other information on results incl. tables'.
Neuropathological findings:
no effects observed
Description (incidence and severity):
Histopathological findings, including examination of the brain (incl. cerebrum, cerebellum and pons): no findings were noted which could be related to treatment with the test item (see attached Illustration in section "Overall remarks, attachments").

Organ weight findings, including the brains: there were no treatment-related effects in the organ weights measured (see Table 7 in 'Any other information on results incl. tables').
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
No findings were noted which could be related to treatment with the test item (see attached Illustration in section "Overall remarks, attachments"). Findings observed were considered to be within the normal range of background findings that are seen in untreated animals of this age and strain.
Histopathological findings: neoplastic:
not specified
Other effects:
no effects observed
Description (incidence and severity):
See field "Cross-reference": results on reproductive performance, offspring viability, clinical signs of offspring, offspring body weight and gross pathology of offspring reported in endpoint study record 7.8.1 Toxicity to reproduction.001. No adverse effects observed.
Details on results:
No treatment related adverse effects observed.

Effect levels

Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: NOEL = highest dose tested. No treatment related adverse effects observed.

Target system / organ toxicity

Critical effects observed:
no

Any other information on results incl. tables

Table 1. Allocation of animals to treatment groups

Treatment Group Dose Level Treatment Volume Concentration Animal Numbers
(mg/kg bw/day) (mL/kg) (mg/mL) Male Female
Control 0 4 0 12 (1-12) 12 (13-24)
Low 100 4 25 12 (25-36) 12 (37-48)
Intermediate 300 4 75 12 (49-60) 12 (61-72)
High 1000 4 250 12 (73-84) 12 (85-96)

The numbers in parentheses ( ) show the individual animal numbers allocated to each treatment

group.

Table 2. Summary Incidence of Daily Clinical Observations

Day Numbers Relative to Start Date
Group 1 Group 2 Group 3 Group 4
0(Control) 100 mg/kg bw/day 300 mg/kg bw/day 1000 mg/kg bw/day
Sex: Male
Scheduled kill
Number of Animals 12 12 12 12
Days from - to  43 44 43 44 43 44 43 44
Increased salivation
Number of Animals . . . 1
Days from - to  . . . 1 1
Sex: Female 
Littering
Number of Animals . 1 . 1
Days from - to  . 41 41 . 41 41
Not Observed Due to Littering
Number of Animals . 1 . 1
Days from - to  . 41 41 . 41 41
Scheduled kill
Number of Animals 12 12 12 12
Days from - to  42 46 42 55 42 46 42 46
Generalised fur loss
Number of Animals . . 1 .
Days from - to  . . 35 42 .

. No data available

Table 3. Group Mean Body Weight Values

Body Weights (g)
Day Numbers Relative to Start Date
Group (Sex) 1 8 15 22 29 36 43
1(M) Mean 309.9 322.5 342.7 351.1 367.8 381.4 392.0
S.D. 6.9 9.9 14.4 14.5 15.4 19.5 20.9
N 12 12 12 12 12 12 12
2(M) Mean 309.2 322.4 340.8 350.8 366.0 382.4 391.0
S.D. 7.0 7.8 9.6 15.1 17.2 19.8 19.8
N 12 12 12 12 12 12 12
3(M) Mean 309.7 324.5 343.5 354.1 374.0 391.2 398.9
S.D. 12.6 17.4 19.6 21.1 22.0 22.7 23.0
N 12 12 12 12 12 12 12
4(M) Mean 310.8 324.3 343.9 355.3 372.8 389.5 397.2
S.D. 10.6 13.0 15.8 18.5 21.5 21.0 20.8
N 12 12 12 12 12 12 12
1(F) Mean 188.6 196.0 206.3
S.D. 5.9 7.4 6.5
N 12 12 12
2(F) Mean 188.1 194.7 206.2
S.D. 7.3 9.7 7.8
N 12 12 12
3(F) Mean 188.7 196.8 206.1
S.D. 4.7 7.6 8.8
N 12 12 12
4(F) Mean 190.7 200.6 211.6
S.D. 7.7 9.1 10.5
N 12 12 12
Day Numbers
Gestation Lactation
0 7 14 20 1 4
1(F) Mean 207.0 230.7 260.2 319.2 229.7 243.8
S.D. 7.2 9.4 11.8 21.9 9.5 12.3
N 11 11 11 11 10 10
2(F) Mean 210.9 234.4 260.8 320.1 234.7 250.9
S.D. 16.4 17.2 18.9 26.1 17.8 18.9
N 12 12 12 12 12 12
3(F) Mean 206.7 233.8 262.5 318.8 242.7 248.4
S.D. 5.8 7.8 10.7 18.6 15.3 14.8
N 12 12 12 12 12 12
4(F) Mean 212.9 239.6 265.8 325.5 247.2* 257.7
S.D. 7.8 9.7 15.5 24.2 12.7 14.8
N 11 11 11 11 11 11

Dose Levels:

Group 1: 0 (Control), Group 2: 100 mg/kg bw/day, Group 3: 300 mg/kg bw/day, Group 4: 1000 mg/kg bw/day

M Male

F Female

S.D. Standard deviation

N Number of animals/litters

* Significantly different from control group p<0.05

Table 4. Group Mean Body Weight Gains

Increase in Body Weight (g)
Day Numbers Relative to Start Date
Abs %
Gain Gain
Group From: 1 8 15 22 29 36 1 1
(Sex) To: 8 15 22 29 36 43 43 43
1(M) Mean 12.6 20.2 8.4 16.7 13.7 10.6 82.1 26.5
S.D. 4.5 6.4 5.6 3.8 5.6 4.7 18.1 5.8
N 12 12 12 12 12 12 12 12
2(M) Mean 13.3 18.4 10.0 15.2 16.4 8.6 81.8 26.5
S.D. 3.6 6.8 9.9 4.3 5.4 3.5 17.3 5.6
N 12 12 12 12 12 12 12 12
3(M) Mean 14.8 19.0 10.6 19.9 17.2 7.8 89.3 28.8
S.D. 7.8 5.5 5.5 4.8 4.8 3.9 17.0 5.5
N 12 12 12 12 12 12 12 12
4(M) Mean 13.4 19.7 11.3 17.5 16.8 7.7 86.3 27.8
S.D. 8.8 4.7 5.9 4.9 3.5 4.3 15.9 4.9
N 12 12 12 12 12 12 12 12
Group From: 1 8 1 1
(Sex) To: 8 15 15 15
1(F) Mean 7.4 10.3 17.7 9.4
S.D. 4.7 4.2 5.0 2.7
N 12 12 12 12
2(F) Mean 6.6 11.5 18.1 9.6
S.D. 4.8 4.6 4.1 2.3
N 12 12 12 12
3(F) Mean 8.2 9.3 17.4 9.2
S.D. 4.3 5.3 6.4 3.3
N 12 12 12 12
4(F) Mean 9.9 11.0 20.9 11.0
S.D. 6.4 5.3 7.0 3.8
N 12 12 12 12
Increase in Body Weight (g) Cumulative Body Weight Change (g)
Days Days
Gestation Lactation Gestation
Group From: 0 7 14 1 0 0
(sex) To: 7 14 20 4 14 20
1(F) Mean 23.7 29.5 59.0 14.1 53.2 112.2
S.D. 5.8 6.6 14.1 8.4 10.0 21.8
N 11 11 11 10 11 11
2(F) Mean 23.5 26.4 59.3 16.3 49.9 109.2
S.D. 5.0 5.3 9.7 9.4 9.6 17.6
N 12 12 12 12 12 12
3(F) Mean 27.1 28.8 56.3 5.8 55.8 112.1
S.D. 3.3 6.3 13.3 8.0 7.2 16.4
N 12 12 12 12 12 12
4(F) Mean 26.7 26.2 59.6 10.5 52.9 112.5
S.D. 5.6 8.2 14.8 9.1 12.4 20.6
N 11 11 11 11 11 11

Dose Levels:

Group 1: 0 (Control), Group 2: 100 mg/kg bw/day, Group 3: 300 mg/kg bw/day, Group 4: 1000 mg/kg bw/day

M Male

F Female

S.D. Standard deviation

N Number of animals/litters

Table 5. Group Mean Hematological Values

Group (sex) Hb RBC Hct MCH MCV MCHC WBC Neut Lymph Mono Eos Bas CT PLT APTT
g/dl 10^12/l % pg fl g/dl 10^9/l 10^9/l 10^9/l 10^9/l 10^9/l 10^9/l Seconds 10^9/l Seconds
1(M) Mean 16.90 9.064 47.94 18.64 52.90 35.28 7.18 1.060 6.066 0.000n 0.054 0.000n 9.14 742.4 14.76
S.D. 0.63 0.339 1.33 0.65 1.19 1.02 1.82 0.287 1.608 0.000 0.078 0.000 0.19 158.5 0.93
N 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5
2(M) Mean 16.54 8.756 47.72 18.92 54.56 34.68 6.70 0.902 5.774 0.000n 0.026 0.000n 8.92 648.0 15.30
S.D. 0.35 0.314 0.55 0.87 2.03 0.54 1.19 0.438 1.005 0.000 0.037 0.000 0.15 67.5 0.39
N 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5
3(M) Mean 16.48 8.614 47.04 19.18 54.64 35.08 6.58 1.142 5.408 0.000n 0.030 0.000n 8.84 625.4 14.84
S.D. 0.47 0.188 0.83 0.73 1.44 0.81 1.08 0.542 1.296 0.000 0.041 0.000 0.17 51.8 1.75
N 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5
4(M) Mean 16.30 8.718 47.22 18.74 54.22 34.52 5.60 1.316 4.376 0.000n 0.024 0.000n 8.94 650.6 15.54
S.D. 0.39 0.390 1.21 0.86 2.01 0.33 0.90 0.346 0.942 0.000 0.033 0.000 0.27 25.0 0.48
N 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5
1(F) Mean 13.14 6.862 38.64 19.16 56.30 34.04 6.48 2.508 3.974 0.000n 0.000n 0.000n 8.12 994.0 14.60
S.D. 1.24 0.643 3.55 0.47 1.15 0.17 1.03 1.232 0.858 0.000 0.000 0.000 0.24 92.7 1.47
N 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5
2(F) Mean 13.48 6.952 39.98 19.42 57.56 33.74 6.12 2.232 3.888 0.000n 0.000n 0.000n 8.16 959.8 13.28
S.D. 0.66 0.326 1.43 0.61 1.77 0.43 1.62 1.247 0.503 0.000 0.000 0.000 0.11 94.0 1.15
N 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5
3(F) Mean 13.40 7.214 40.02 18.62 55.50 33.52 5.58 1.844 3.736 0.000n 0.000n 0.000n 8.16 1059.2 13.30
S.D. 0.50 0.409 1.70 0.61 1.08 0.63 1.23 0.845 1.002 0.000 0.000 0.000 0.27 83.1 1.52
N 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5
4(F) Mean 13.64 7.084 40.12 19.26 56.66 34.00 7.66 2.728 4.934 0.000n 0.000n 0.000n 8.14 876.0 13.44
S.D. 0.61 0.419 2.13 0.53 0.99 0.42 1.79 1.546 0.578 0.000 0.000 0.000 0.21 246.4 1.47
N 5 5 5 5 5 5 5 5 5 5 5 5 5 5

5

Dose Levels: Group 1 - 0 (Control), Group 2 - 100 mg/kg bw/day, Group 3 - 300 mg/kg bw/day, Group 4 - 1000 mg/kg bw/day

M Male

F Female

S.D. Standard deviation

N Number of animals/litters

n Data not appropriate for statistical analysis

Parameters measured on potassium EDTA - treated blood:

Hemoglobin (Hb)

Total erythrocyte count (RBC)

Hematocrit (Hct)

Erythrocyte indices - Mean Corpuscular Hemoglobin (MCH), Mean Corpuscular Volume (MCV), Mean Corpuscular Hemoglobin Concentration (MCHC)

Total leukocyte count (WBC)

Differential leukocyte count - Neutrophils (Neut), Lymphocytes (Lymph), Monocytes (Mono), Eosinophils (Eos), Basophils (Bas) Platelet count (PLT)

Parameters measured on citrate-treated blood:

Prothrombin time (CT)

Activated partial thromboplastin time (APTT)

Table 6. Group Mean Blood Chemical Values

Group (Sex) Urea Glucose Tot. Prot. Albumin A/G Na+ K+ Cl- Ca++ P ASAT ALAT AP Creat Chol Bili Bile Acid
mg/dl mg/dl g/dl g/dl Ratio mmol/l mmol/l mmol/l mmol/l mmol/l IU/l IU/l IU/l mg/dl mg/dl mg/dl μmol/l
1(M) Mean 47.0 157.0 7.642 3.96 1.076 146.6 4.346 100.2 2.788 2.62 73.2 57.8 158.4 0.686 106.8 0.186 8.78
S.D. 2.4 7.7 0.388 0.11 0.087 1.7 0.309 0.8 0.093 0.15 15.8 8.8 17.6 0.074 13.3 0.075 3.92
N 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5
2(M) Mean 49.4 172.6 7.332 3.90 1.140 147.2 4.264 101.8* 2.838 2.12** 74.8 54.0 170.8 0.784 101.0 0.112 8.16
S.D. 6.7 4.4 0.292 0.07 0.083 0.8 0.106 0.8 0.034 0.29 9.9 6.1 43.4 0.044 13.0 0.035 3.99
N 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5
3(M) Mean 52.2 162.8 7.208 3.90 1.178 147.0 4.632 102.2* 2.782 2.14** 84.0 56.4 166.2 0.836 98.8 0.154 8.12
S.D. 12.3 28.6 0.366 0.12 0.130 1.7 0.685 1.6 0.034 0.21 22.1 5.2 31.5 0.245 16.2 0.091 5.93
N 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5
4(M) Mean 42.6 181.0* 7.228 3.88 1.160 148.0 4.188 102.0* 2.800 2.16** 98.6 55.6 169.6 0.730 96.0 0.124 6.66
S.D. 3.9 18.5 0.283 0.11 0.065 2.8 0.338 1.0 0.031 0.27 25.0 6.7 16.3 0.056 8.0 0.028 2.47
N 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5
1(F) Mean 52.6 118.2 6.624 3.80 1.344 145.6 4.318 102.8 2.644 1.14 87.0 70.4 124.6 0.786 81.0 0.088 10.04
S.D. 7.5 15.1 0.238 0.20 0.067 1.5 0.257 2.0 0.093 0.45 15.1 15.7 49.1 0.150 14.6 0.008 3.07
N 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5
2(F) Mean 60.0 132.6 6.670 3.84 1.352 146.4 4.674 103.6 2.714 1.08 82.8 74.4 91.4 0.868 82.4 0.088 26.42
S.D. 13.0 20.5 0.305 0.17 0.039 1.5 0.732 1.5 0.181 0.37 11.8 17.5 8.3 0.148 6.7 0.019 14.78
N 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5
3(F) Mean 56.4 131.8 7.016 3.94 1.286 147.8 3.952 104.8 2.596 0.64 82.8 67.0 112.8 0.870 71.6 0.068* 7.38
S.D. 5.1 9.5 0.247 0.11 0.074 1.5 0.118 0.8 0.106 0.15 12.0 15.0 20.2 0.082 12.8 0.008 3.60
N 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5
4(F) Mean 47.0 155.0** 6.656 3.68 1.258* 145.4 3.948 103.4 2.502 0.84 99.6 76.2 135.2 0.844 83.8 0.072* 10.94
S.D. 9.2 23.1 0.418 0.22 0.026 1.1 0.343 0.5 0.115 0.26 18.3 13.8 75.3 0.056 12.8 0.004 9.67
N 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5

Dose Levels: Group 1 - 0 (Control), Group 2 - 100 mg/kg bw/day, Group 3 - 300 mg/kg bw/day, Group 4 - 1000 mg/kg bw/day

M Male

F Female

S.D. Standard deviation

N Number of animals/litters

n Data not appropriate for statistical analysis

* Significantly different from control group p<0.05

** Significantly different from control group p<0.01

Parameters measured on lithium heparin treated blood:

Urea

Calcium (Ca++)

Glucose

Inorganic phosphorus (P)

Total protein (Tot.Prot.)

Aspartate aminotransferase (ASAT)

Albumin

Alanine aminotransferase (ALAT)

Albumin/Globulin (A/G) ratio (by calculation)

Alkaline phosphatase (AP)

Sodium (Na+)

Creatinine (Creat)

Potassium (K+)

Total cholesterol (Chol)

Chloride (Cl-)

Total bilirubin (Bili)

Bile acids

Table 7. Group Mean Organ Weights with Corresponding Relative (% of Body Weight) Organ Weights

MALES FEMALES
0 100 300 1000 0 100 300 1000
Control mg/kg bw/day Control mg/kg bw/day
Terminal Bodyweight Mean (g) 392.9 392.2 399.2 398.3 248.6 254.8 251.3 261.7
S.D. 20.7 20.6 22.5 22.6 14.1 19.9 15.8 15.0
N 12 12 12 12 12 12 12 12
Adrenals Mean (g) 0.08528 0.08226 0.08246 0.08522 0.09580 0.09800 0.09698 0.10408
S.D. 0.00190 0.01406 0.01055 0.00888 0.01520 0.01361 0.01103 0.01774
N 5 5 5 5 5 5 5 5
Mean (%) 0.022 0.021 0.020 0.022 0.040 0.040 0.039 0.040
S.D. 0.001 0.003 0.003 0.003 0.005 0.006 0.005 0.006
N 5 5 5 5 5 5 5 5
Brain (Including Cerebrum,
Cerebellum And Pons)
Mean (g) 1.88670 1.92172 1.93068 1.91456 1.72716 1.71518 1.73446 1.81950
S.D. 0.03837 0.05397 0.06150 0.22249 0.08880 0.11007 0.05664 0.06049
N 5 5 5 5 5 5 5 5
Mean (%) 0.476 0.487 0.479 0.482 0.722 0.705 0.705 0.708
S.D. 0.024 0.018 0.024 0.053 0.041 0.026 0.009 0.048
N 5 5 5 5 5 5 5 5
Epididymides Mean (g) 1.75418 1.69644 1.81648 1.73282
S.D. 0.27530 0.17860 0.21857 0.14971
N 12 12 12 12
Mean (%) 0.446 0.435 0.456 0.437
S.D. 0.064 0.059 0.058 0.049
N 12 12 12 12
Heart Mean (g) 1.17996 1.10376 1.22666 1.25292 0.83040 0.91786 0.85436 0.88298
S.D. 0.07471 0.14288 0.12751 0.13954 0.07639 0.07389 0.08927 0.13537
N 5 5 5 5 5 5 5 5
Mean (%) 0.298 0.278 0.303 0.317 0.347 0.377 0.348 0.342
S.D. 0.027 0.024 0.024 0.048 0.034 0.021 0.041 0.04
N 5 5 5 5 5 5 5 5
Kidneys Mean (g) 2.43284 2.25590 2.51100 2.41284 1.57000 1.46824 1.46996 1.69592
S.D. 0.15977 0.19345 0.33264 0.10838 0.12636 0.16048 0.05858 0.08434
N 5 5 5 5 5 5 5 5
Mean (%) 0.614 0.571 0.622 0.608 0.655 0.605 0.598 0.660
S.D. 0.041 0.040 0.078 0.032 0.034 0.075 0.022 0.053
N 5 5 5 5 5 5 5 5
Liver Mean (g) 13.6384 13.1027 13.4021 13.6948 10.9844 11.0291 10.9512 11.9167
S.D. 1.01250 1.79533 1.56517 1.53317 1.40468 0.98740 0.99183 0.69743
N 5 5 5 5 5 5 5 5
Mean (%) 3.435 3.314 3.320 3.443 4.577 4.541 4.444 4.634
S.D. 0.139 0.416 0.374 0.270 0.402 0.446 0.212 0.314
N 5 5 5 5 5 5 5 5
Ovaries Mean (g) 0.11540 0.11941 0.11821 0.11930
S.D. 0.01275 0.01705 0.01858 0.01711
N 10 12 12 11
Mean (%) 0.047 0.047 0.047 0.045
S.D. 0.004 0.008 0.006 0.005
N 10 12 12 11
Pituitary Mean (g) 0.01177 0.01264 0.01392 0.01300 0.01340 0.01565* 0.01560* 0.01531*
S.D. 0.00211 0.00285 0.00390 0.00274 0.00194 0.00215 0.00236 0.00231
N 12 12 11 12 10 12 11 10
Mean (%) 0.003 0.003 0.004 0.003 0.005 0.006* 0.006* 0.006*
S.D. 0.001 0.001 0.001 0.001 0.001 0.001 0.001 0.001
N 12 12 11 12 10 12 11 10
Prostate Mean (g) 0.70769 0.67011 0.75388 0.67923
S.D. 0.14633 0.14353 0.22936 0.11531
N 12 12 12 12
Mean (%) 0.180 0.171 0.189 0.171
S.D. 0.037 0.037 0.055 0.030
N 12 12 12 12
Seminal Vesicles Mean (g) 2.07506 1.97583 1.95986 2.00458
S.D. 0.52402 0.27468 0.28542 0.47709
N 12 12 12 12
Mean (%) 0.528 0.506 0.491 0.503
S.D. 0.129 0.083 0.069 0.110
N 12 12 12 12
Spleen Mean (g) 0.76272 0.68878 0.68720 0.87458 0.58394 0.66942 0.54512 0.63062
S.D. 0.11332 0.10365 0.13722 0.12493 0.03934 0.10353 0.07306 0.09472
N 5 5 5 5 5 5 5 5
Mean (%) 0.192 0.174 0.171 0.221 0.244 0.275 0.221 0.245
S.D. 0.024 0.017 0.037 0.037 0.015 0.039 0.027 0.033
N 5 5 5 5 5 5 5 5
Testes Mean (g) 3.82673 3.86518 3.79398 3.79556
S.D. 0.26592 0.29204 0.22589 0.23812
N 12 12 12 12
Mean (%) 0.975 0.988 0.951 0.954
S.D. 0.067 0.083 0.035 0.061
N 12 12 12 12
Thymus Mean (g) 0.38442 0.40064 0.41422 0.43558 0.22768 0.23756 0.20504 0.22760
S.D. 0.08239 0.06301 0.03961 0.07949 0.02056 0.04589 0.01541 0.03992
N 5 5 5 5 5 5 5 5
Mean (%) 0.097 0.102 0.103 0.109 0.095 0.098 0.084 0.088
S.D. 0.023 0.021 0.013 0.018 0.007 0.022 0.009 0.015
N 5 5 5 5 5 5 5 5
Thyroid/Parathyroid Mean (g) 0.02164 0.02434 0.02448 0.02166 0.02440 0.01808 0.01884 0.02270
S.D. 0.00730 0.00359 0.00421 0.00277 0.00335 0.00323 0.00324 0.00771
N 5 5 5 5 5 5 5 5
Mean (%) 0.005 0.006 0.006 0.005 0.010 0.007 0.008 0.009
S.D. 0.002 0.001 0.001 0.001 0.001 0.002 0.001 0.003
N 5 5 5 5 5 5 5 5
Uterus & Cervix Mean (g) 0.73185 0.72131 0.69785 0.70894
S.D. 0.12800 0.19822 0.13247 0.10660
N 10 12 12 11
Mean (%) 0.296 0.282 0.278 0.272
S.D. 0.049 0.070 0.054 0.050
N 10 12 12 11

S.D. Standard deviation

N Number of animals/litters

* Significantly different from control group p<0.05

Table 8. Summary Incidence of Necropsy Findings

MALES
Removal Reason: SCHEDULED KILL 0 100 300 1000
(Control) mg/kg bw/day
Number of Animals Examined: 12 12 12 12
Group:  (1) (2) (3) (4)
Lungs (With Bronchi);
Submitted  (11#) (12) (12) (12)
No Visible Lesions  11 10 9 10
Discolouration 0 0 0 1
Discolouration; red  0 2 3 1
FEMALES
Removal Reason: SCHEDULED KILL 0 100 300 1000
(Control) mg/kg bw/day
Number of Animals Examined: 12 12 12 12
Group:  (1) (2) (3) (4)
Lungs (With Bronchi);
Submitted  (12) (11#) (12) (12)
No Visible Lesions  12 9 9 9
Discolouration; red  0 2 3 3
Uterus & Cervix;
Submitted  (12) (11#) (12) (12)
No Visible Lesions  12 10 12 11
Enlarged 0 0 0 1
Fluid Filled 0 0 0 1
Damaged On Removal 0 1 0 0

# = One animal not submitted in error

Applicant's summary and conclusion

Conclusions:
The oral administration of the test item to rats by gavage, at dose levels of 100, 300 and 1000 mg/kg bw/day, was tolerated well. The ‘No Observed Effect Level’ (NOEL) for systemic toxicity was therefore considered to be 1000 mg/kg bw/day.

As there were no treatment-related adverse effects observed in the study and the test item was tolerated well, according to the CLP Regulation the substance does not need to be classified with regards to systemic toxicity, as Specific Target Organ Toxicant. The oral route was selected as the most appropriate route of exposure, based on the physical properties of the test item. The results of the study are believed to be of value in predicting the likely toxicity of the test item to man.
Executive summary:

The study was designed to investigate the systemic toxicity and potential adverse effects of the test item on reproduction and is designed to be compatible with the requirements of the OECD Guidelines for Testing of Chemicals No. 422 “Combined Repeated Dose Toxicity Study with the Reproduction/ Developmental Toxicity Screening Test” and with Commission Regulation (EC) No 440/2008 of 30 May 2008.

The test item was administered by gavage to three groups, each of twelve male and twelve female Wistar Han™:RccHan™:WIST strain rats, for up to eight weeks (including a two week pre-pairing phase, pairing, gestation and early lactation for females), at dose levels of 100, 300 and 1000 mg/kg bw/day. A control group of twelve males and twelve females was dosed with vehicle alone (Arachis oil BP).

Clinical signs, behavioral assessments, body weight change and food and water consumption were monitored during the study. Extensive functional observations were performed on five selected males from each dose group after the completion of the pairing phase, and for five selected parental females from each dose group on Day 4 post partum. Hematology and blood chemistry were evaluated prior to termination on five selected males and females from each dose group.

Adult males were terminated on Day 43 or Day 44 followed by the termination of all females and offspring on Day 5 post partum. Any female which did not produce a pregnancy was terminated on or after Day 25 post coitum. All animals were subjected to a gross necropsy examination and histopathological evaluation of selected tissues was performed.

Adult Responses

Mortality - There were no unscheduled deaths on study.

Clinical Observations - There were no clinical signs of toxicity related to the test item.

Behavioral Assessment - There were no treatment-related effects in the behavioral parameters measured.

Functional Performance Tests - There were no treatment-related changes in the functional performance.

Sensory Reactivity Assessments - There were no treatment-related changes in sensory reactivity.

Body Weight - There was no adverse effect on body weight development for animals of either sex treated with 100, 300 or 1000 mg/kg bw/day.

Food Consumption - There were no effects detected in food consumption for animals of either sex treated with 100, 300 or 1000 mg/kg bw/day when compared with controls.

Water Consumption - Visual inspection of water residues did not indicate any effect of treatment on water intake throughout the study.

Laboratory Investigations

Hematology - There were no treatment-related changes in the hematological parameters measured.

Blood Chemistry - No toxicologically significant changes were detected in blood chemical parameters measured.

Pathology

Necropsy -There were no treatment-related macroscopic abnormalities detected.

Organ Weights -There were no treatment-related effects in the organ weights measured.

Histopathology -No findings were noted which could be related to treatment with the test item. Findings observed were considered to be within the normal range of background alterations that are seen in untreated animals of this age and strain.

The oral administration of the test item to rats by gavage, at dose levels of 100, 300 and 1000 mg/kg bw/day, was tolerated well. The ‘No Observed Effect Level’ (NOEL) for systemic toxicity was therefore considered to be 1000 mg/kg bw/day. The substance is not classified for systemic toxicity according to the CLP Regulation, as no adverse effects were observed.

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