Dipotassium hexacyanocobalt(II)-ferrate(II)

Toxicity to reproduction

Administrative data

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:

9.6.2015-29.2.2016

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:

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:

09.6.2015-29.2.2016

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 integrity or validity 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. The concentration of test item in the formulations was determined using a gravimetric technique. Deviations from the target range for relative humidity.

GLP compliance:

yes (incl. QA statement)

Remarks:

Certificate is as an attachement 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 314-345 g, females 168-216 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 94.06%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

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. Formulations were prepared daily. The concentration of test item in the formulations was determined using a gravimetric technique.

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, 6, 20 and 60 mg/mL
- Amount of vehicle (if gavage): 5 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 and dosed within two hours of preparation.

Samples of the test item formulation were taken and analyzed for concentration of the test item. The results indicate that the prepared formulations were within 99% to 109% of the nominal concentration. Thus the required content limit of +/- 10% with reference to the nominal content was met. The results indicate the accurate use of the test item and Arachis Oil BP as vehicle during the 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.

Dose / conc.:

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

Remarks:

m/f

Dose / conc.:

30 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

No. of animals per sex per dose:

Control group (vehicle alone): 12 males, 12 females
Dose level 30 mg/kg bw/day: 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
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 (CsTreat 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 development, food consumption and water intake were monitored during the study. All animals were subjected to gross necropsy examination at termination.

Results:
Mortality -There were no unscheduled deaths on the study.

Clinical Observations - At 1000 mg/kg bw/day, one male showed clinical signs of dehydration, decreased respiratory rate, emaciation, hunched posture and tiptoe gait on Day 15 of the study. This appeared to be an isolated incident as no other animal from this dosage or from other dosage groups.

Body Weight - Reduction in body weight gains was evident in animals of either sex treated with 1000 and 500 mg/kg bw/day when compared to controls. Actual body weight losses were evident in these animals of either sex between Days 1 and 4 and continued in 1000 mg/kg bw/day males during the second week of treatment. Recovery in body weight gain was evident in females from Day 4 onwards. Males treated with 250 mg/kg bw/day, showed no obvious effects on body weight gain, and females at this level appear to be unaffected by treatment.

Food Consumption - At 500 and 1000 mg/kg bw/day, mean food consumption and food conversion efficiency for males was lower than control throughout the study. For males at 250 mg/kg bw/day, there were no consistent differences from control in food intake that indicated an effect of treatment. For females, food consumption did not indicate any obvious effect of treatment at 250, 500 or 1000 mg/kg bw/day throughout the study. However food conversion efficiency at 1000 mg/kg bw/day was lower between Days 1-4. Recovery was evident thereafter an no such effect was evident at 250 mg/kg bw/day.

Water Consumption - There was no obvious effect on water consumption in animals of either sex treated at 1000, 500 or 250 mg/kg bw/day. There were incidences where males treated with 1000 mg/kg bw/day showed an increase in water consumption during the treatment period.

Necropsy - At 1000 mg/kg bw/day, all males showed reddened discolouration in the glandular region in the stomach and one male showed blue coloured stomach contents. No similar findings were found in females treated with 1000 mg/kg bw/day or animals of either sex at 500 or 250 mg/kg bw/day.

The oral administration of the test itemt to rats by gavage, at dose levels of 250, 500 and 1000 mg/kg bw/day, resulted in a reduction in body weight gain and food consumption at 1000 and 500 mg/kg bw/day. Macroscopic abnormalities were also evident in 1000 mg/kg bw/day. Based on the results from this study a dose level of 300 mg/kg bw/day was considered to be suitable as a high dose level for the OECD 422 study together with 30 and 100 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 300 mg/kg bw/day dose group animals and any animals dying during the study were prepared as paraffin blocks, sectioned and stained for subsequent microscopic examination. Adult males were killed on Day 43 or Day 44. Surviving 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

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 (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.

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 surviving test and control groups 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 surviving test and control groups 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 from each dose level, and five females from groups 1 to 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)

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 300 mg/kg bw/day dose group animals and any animals dying during the study 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 300 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 300 mg/kg bw/day males were also stained and examined. Detailed qualitative examination of the testes was undertaken.

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 to same study").

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".

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

There were no adverse clinical signs in females treated with 300 mg/kg bw/day up to Day 38 (around the time of parturition), or in the remaining animals of either sex throughout the study (see Table 2 in 'Any other information on results incl. tables').

On Day 38, clinical signs of hypothermia, decreased respiratory rate, diarrhoea, hunched posture, and/or pilo-erection were apparent in some females treated with 300 mg/kg bw/day and were considered to be due to difficulty in parturition. The surviving females from this group were terminated prematurely.

There were sporadic instances of staining of fur by test item in some of the females receiving 300 mg/kg bw/day during Weeks 5 and 6. One male each treated with 100 or 30 mg/kg bw/day showed signs of noisy respiration on sporadic days. One male treated with 100 mg/kg bw/day showed staining around the eye on Days 1 to 4. These observations were considered to be isolated incidents and of no toxicological significance.

Mortality:

mortality observed, treatment-related

Description (incidence):

On Day 38 of the study when the first set of females was due to litter, two females treated with 300 mg/kg bw/day and one female treated with 100 mg/kg bw/day were found dead; there were no prior adverse clinical signs for these early decedents. A further female treated with 300 mg/kg bw/day showed signs of difficulty during parturition including hypothermia, hunched posture, pilo-erection and was later terminated on the same day. At necropsy the two females found dead from the 300 mg/kg bw/day dose group had dead pups inside the uterus with one of the females showing enlarged adrenal glands. The remaining female from this dose group that was killed in extremis had eleven live pups within the uterus. The macroscopic findings for this female consisted of yellow colored contents in caecum, colon, duodenum, ileum and jejunum and enlarged adrenal glands.

The early decedent from the 100 mg/kg bw/day dose gr up had completed parturition but 6/14 pups were found dead. There were, however no macroscopic findings at necropsy for the offspring. Some of the remaining pregnant females treated with 300 mg/kg bw/day also showed signs of deterioration and difficulty during parturition and it was decided to terminate all surviving females from this dose group on Day 38; of these, one was found to be non-pregnant, two hadsuccessfully delivered live offspring and six had dead fetuses in the uterus.

There were no further unscheduled deaths on the study. For results in tabular form, see Table 2 in 'Any other information on results incl. tables'.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Males treated with 300 mg/kg bw/day, showed statistically significant reductions in group mean body weight gains throughout the study excluding the final week of dosing. Males treated with 100 mg/kg bw/day also showed instances of reduced body weight gains achieving statistical significance in Weeks 2 and 5. Overall body weight gain in both groups was reduced when compared to controls (approximately 29% and 42 % at 100 or 300 mg/kg bw/day, respectively). Group mean body weight gains in males given 30 mg/kg bw/day were generally similar to controls.

During the pre-pairing phase, females treated with 300 or 100 mg/kg bw/day showed slightly lower group mean body weight gains than controls although not attaining statistical significance. The group mean body weight gains for these females during gestation Days 14 to 20 were also lower than controls in a dose related manner achieving statistical significance. This resulted in reduced cumulative body weight gains over Days 0 to 20 gestation and reduced body weights on Day 20 of gestation; statistical significance was achieved at 300 mg/kg bw/day. A reduction in body weight gain was also evident during lactation in females treated with 100 mg/kg bw/day but without attaining statistical significance; there was a high degree of inter-individual variation and this observation was considered likely to be incidental. There was no effect on body weight gains during pre-pairing, gestation or lactation phases of the study for females treated with 30 mg/kg bw/day.

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):

effects observed, treatment-related

Description (incidence and severity):

Not a feeding study. However, during the pre-pairing phase, food consumption for both sexes was comparable to controls. Any differences from controls were generally not dose related and were considered to be incidental. For males treated with 300 or 100 mg/kg bw/day, the last two weeks of treatment showed a slight reduction in food intake with the corresponding females also showing a dose related reduction in food intake during gestation Days 14 to 20. Food consumptions for males and females treated with 30 mg/kg bw/day was comparable to controls.

For results in tabular form, see Table 5 in 'Any other information on results incl. tables'.

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

Males treated with 300 or 100 mg/kg bw/day, generally showed lower food conversion efficiency throughout the study when compared to controls. Food conversion efficiency was lower than controls for the corresponding females during Week 1 of pre-pairing phase and remained low for the 300 mg/kg bw/day females during Week 2. There was no effect of treatment at 30 mg/kg bw/day on food conversion efficiency for animals of either sex.

For results in tabular form, see Table 6 in 'Any other information on results incl. tables'.

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

no effects observed

Description (incidence and severity):

Not a drinking 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:

effects observed, treatment-related

Description (incidence and severity):

Due to the early termination of females treated with 300 mg/kg bw/day hematology parameters were not assessed for these females.

All treated animals showed a dose related increase in levels of hemoglobin, hematocrit and erythrocyte count which was statistically significant. The majority of individual values for males treated with 300 or 100 mg/kg bw/day were above the background control ranges, whereas most animals treated with 30 mg/kg bw/day had individual values within the background control ranges. At all dose levels males also showed statistically significantly lower mean corpuscular hemoglobin concentrations in relation to controls. There was no dose relationship and all individual values were within the background control ranges with the corresponding values in females also being similar to controls.

Reticulocytes in females treated with 30 and 100 mg/kg bw/day were statistically significantly higher when compared to controls. Some females treated with 100 mg/kg bw/day showed individual values that were above the background control ranges.

Treated males from 100 and 300 mg/kg bw/day dose groups showed a dose related reduction in platelet count which was statistically significant. Individual values for males treated with 300 mg/kg bw/day were above the background control ranges, whereas males treated with the 100 mg/kg bw/day showed individual values within the background control ranges. The corresponding females also showed a lower platelet count when compared to controls but
without any dose relationship or attaining statistical significance.

Females treated with 100 mg/kg bw/day showed a statistically significant reduction in lymphocytes count. All individual values were within background control ranges; and the corresponding values in males were comparable to controls.

For results in tabular form, see Table 7 in 'Any other information on results incl. tables'.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

There were no changes of toxicological significance up to a dose level of 300 mg/kg bw/day in males and up to a dose level of 100 mg/kg bw/day in females (see Table 8 in 'Any other information on results incl. tables').

At 300 and 100 mg/kg bw/day, males showed a statistically significant increase in levels of bile acid and aspartate aminotransferase and a statistically significant reduction in cholesterol and bilirubin when compared to controls. Although these differences were dose related, the majority of individual values were within the background control ranges.

Males treated with 300 mg/kg bw/day showed a statistically significant reduction in total protein, albumin and calcium concentrations when compared to controls. There was also a statistically significant increase in levels of chloride concentration. There was no dose relationship for any of these parameters and with the exception of chloride concentration most individual values for the remaining parameters were within the background control ranges. Females treated with 30 or 100 mg/kg bw/day also showed a non dose related increase in chloride levels with 2/5 of the latter females showing individual values that exceeded the background control ranges.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

There were no treatment-related effects detected in the behavioral parameters measured in males up to a dose level of 300 mg/kg bw/day and in females up to a dose level of 100 mg/kg bw/day.

There were no treatment-related changes in the functional performance in males up to a dose level of 300 mg/kg bw/day and in females up to a dose level of 100 mg/kg bw/day.

There were no treatment-related changes in sensory reactivity in males up to a dose level of 300 mg/kg bw/day and in females up to a dose level of 100 mg/kg bw/day.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Adrenals: Females treated with 30 or 100 mg/kg bw/day showed a dose-related statistically significant increase in adrenal weights relative to terminal body weight. The relationship for absolute weights was not clear with females treated with 30 mg/kg bw/day showing a marginal but statistically significant reduction in absolute weight but females receiving 100 mg/kg bw/day showing an increase. The majority of the individual values were within the background control ranges.

Liver: Females treated with 30 mg/kg bw/day, showed a dose-related statistically significant increase in liver weight in relation to body weight. The relationship for absolute weights was less clear with females treated with 100 mg/kg bw/day showing a statistically significant reduction in absolute weight whilst an increase was evident at 30 mg/kg bw/day. The majority of the individual values were within the background control ranges.

Kidneys: Males treated with 300 or 100 mg/kg bw/day showed a dose-related statistically significant reduction in kidney weight both absolute and relative to terminal body weight. The majority of the individual values were within the background control ranges.

Prostate: Males treated with 300 mg/kg bw/day showed a statistically significant reduction in prostate weight both absolute and relative to terminal body weights. The majority of the individual values were within the background control ranges.

Seminal Vesicles: Males treated with 300 or 100 mg/kg bw/day showed a statistically significant reduction in seminal vesicle weight both absolute and relative to terminal body weight. A dose relationship was apparent for absolute weights. The majority of individual values were within the background control ranges.

For results in tabular form, see Table 9 in 'Any other information on results incl. tables'.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Premature Decedents
At 300 mg/kg bw/day, two females were found dead and one was killed in extremis around the time of parturition (day 38 of dosing). Of these, Female 95 (found dead) showed enlarged adrenals whilst Female 91 (killed in extremis) had enlarged adrenals and yellow colored contents in the caecum, colon, duodenum, ileum and jejunum.

The remaining females treated with 300 mg/kg bw/day were also terminated prematurely on Day 38. All these females had black colored contents in the stomach with some animals also showing yellow discoloration or sloughing of the glandular or non glandular region of the stomach. The presence of black and/or yellow colored contents in other parts of the mottled appearing liver.

There were no macroscopic findings for the female treated with 100 mg/kg bw/day that was found dead on Day 38.

Terminal Sacrifice
There were no treatment-related macroscopic findings at terminal necropsy for males up to a dose level of 300 mg/kg bw/day and females for females up to a dose level of 100 mg/kg bw/day.

At scheduled termination, one male treated with 300 and 30 mg/kg bw/day and one female treated with 100 and 30 mg/kg bw/day was shown to have reddened lungs at necropsy. At 30 mg/kg bw/day, one male showed small epididymides and small testes. One female showed one malformed kidney being smaller in size, misshaped and pale. These findings were considered incidental.

For results in tabular form, see Table 10 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 relating to the brains of the test animals which could be related to treatment with the test item (see attached Illustration in section "Overall remarks, attachments").

Organ weight findings, including the brains: no findings were noted relating to the brains of the test animals which could be related to treatment with the test item (see Table 9 in 'Any other information on results incl. tables').

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

There were several findings that were considered to be of toxicological significance (see attached Illustration in section "Overall remarks, attachments").

The following treatment related microscopic abnormalities were detected:

Premature Decedents
Gastrointestinal tract: There was minimal to moderate ulceration present in the glandular area of the stomach in 10/12 females treated with 300 mg/kg bw/day. There was inflammatory change present in one or more areas of the intestines, most pronounced in the caecum in 7/12 of these females.

Thymus, Spleen and Lymph Nodes: Lymphoid depletion or lymphocytolysis was present in the thymus, spleen and lymph nodes of up to 10/12 females. There was an increase in hematopoiesis in the spleen of some females.

Bone marrow (sternum): Decreased cellularity was present in the bone marrow (sternum) of 5/12 animals.

Adrenal glands: 5/12 females treated with 300 mg/kg bw/day showed cortical hypertrophy in the adrenal gland.

Liver: Hepatocellular atrophy (presenting as a diffuse change with homogenous, deeply staining cells without normal lobular pattern often concentrated in the centrilobular area occasionally giving the appearance of periportal enlargement) was present in 10/12 animals. Periportal fat-type vacuolation was also present in one female, hematopoiesis present in 3/12 females.

A further one female treated with 100 mg/kg bw/day had given birth but was found dead. This animal also showed similar changes in the liver, stomach, intestines, bone marrow and lymphoid system.

Terminal Sacrifice
Spleen: An increase in the amount of hematopoiesis in the spleen of all treated males and the change in males treated with 30 mg/kg bw/day was equivocal.

Thymus: Thymic lymphoid depletion was present in one female treated with 100 mg/kg bw/day but was not present in females treated with 30 mg/kg bw/day.

Bone marrow (sternum): An increased cellularity in the bone marrow (sternum) of all treated males and the change in males treated with 30 mg/kg bw/day were equivocal.

Adrenal glands: Minimal cortical hypertrophy in the adrenal gland was present in one female treated with 30 mg/kg bw/day and two females treated with 100 mg/kg bw/day.

Liver: Hepatocellular atrophy was present three females treated with 100 mg/kg bw/day; these findings were low grade changes and did not occur in females treated with 30 mg/kg bw/day.

There were no changes in the stomach, intestines or mesenteric lymph node of females treated with 30 or 100 mg/kg bw/day.

There were no test item-related microscopic findings in the testes, including following the qualitative examination of the stages of spermatogenesis in the testes (no test item-related abnormalities in the integrity of the various cell types present within the different stages of the sperm cycle) or following the evaluation of the uterus or evaluation of follicles and corpora lutea in the ovaries.

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 obvious adverse effects observed that would lead to the classification of the substance with regard to reproductive toxicity.

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: overall effects

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

haematopoietic

Organ:

other: haematopoietic tissues

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

not specified

Relevant for humans:

yes

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

gastrointestinal tract

Organ:

other: gastrointestinal tract

Treatment related:

yes

Dose response relationship:

not specified

Relevant for humans:

yes

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	5	0	12 (1-12)	12 (13-24)
Low	30	5	6	12 (25-36)	12 (37-48)
Intermediate	100	5	20	12 (49-60)	12 (61-72)
High	300	5	60	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)		30 mg/kg bw/day		100 mg/kg bw/day		300 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
Staining around the eyes
	Number of Animals	.		.		1		.
	Days from - to	.		.		1	4	.
Noisy respiration
	Number of Animals	.		1		1		.
	Days from - to	.		35	39	34	35	.
Sex: Female
Littering
	Number of Animals	1		1		.		.
	Days from - to	40	40	39	39	.		.
Not Observed Due to Littering
	Number of Animals	1		1		.		1
	Days from - to	40	40	39	39	.		37	37
Killed (SD/NACWO Request)
	Number of Animals	.		.		.		9
	Days from - to	.		.		.		38	38
Killed `in extremis'
	Number of Animals	.		.		.		1
	Days from - to	.		.		.		38	38
Scheduled kill
	Number of Animals	12		12		11		.
	Days from - to	42	45	42	45	42	54	.
Found dead
	Number of Animals	.		.		1		2
	Days from - to	.		.		38	38	38	38
Decreased respiratory rate
	Number of Animals	.		.		.		3
	Days from - to	.		.		.		38	38
Pilo-erection
	Number of Animals	.		.		.		4
	Days from - to	.		.		.		38	38
Hypothermia
	Number of Animals	.		.		.		1
	Days from - to	.		.		.		38	38
Hunched posture
	Number of Animals	.		.		.		4
	Days from - to	.		.		.		38	38
Fur stained by test material
	Number of Animals	.		.		.		4
	Days from - to	.		.		.		30	37
Diarrhoea
	Number of Animals	.		.		.		4
	Days from - to	.		.		.		38	38

. 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	329.8	339.6	352.2	361.3	374.3	388.4	395.3
	S.D.	5.0	9.4	12.7	16.6	19.2	21.5	24.7
	N	12	12	12	12	12	12	12
2(M)	Mean	330.8	339.6	349.1	357.9	371.6	383.8	393.3
	S.D.	6.6	9.0	12.3	12.8	16.4	18.2	19.2
	N	12	12	12	12	12	12	12
3(M)	Mean	326.1	333.6	341.4	347.7	361.1	368.8	372.4
	S.D.	9.1	13.0	14.5	18.0	19.9	22.5	23.1
	N	12	12	12	12	12	12	12
4(M)	Mean	327.4	329.3	335.8	337.3	346.0	349.6	354.7
	S.D.	6.1	7.0	10.3	12.9	14.5	17.8	19.1
	N	12	12	12	12	12	12	12
1(F)	Mean	199.3	208.1	217.2
	S.D.	9.2	11.4	12.8
	N	12	12	12
2(F)	Mean	192.3	200.5	210.3
	S.D.	9.0	10.6	9.2
	N	12	12	12
3(F)	Mean	192.2	199.3	206.1
	S.D.	8.9	9.7	12.1
	N	12	12	12
4(F)	Mean	191.6	197.7	204.3
	S.D.	12.8	11.7	10.2
	N	12	12	12
		Day Numbers
		Gestation					Lactation
		0	7	14	20		1	4
1(F)	Mean	219.3	241.3	271.2	332.1		249.0	259.7
	S.D.	13.0	14.8	18.4	26.0		18.3	15.7
	N	12	12	12	12		11	11
2(F)	Mean	213.1	236.9	264.6	324.0		240.6	253.0
	S.D.	13.0	13.6	19.7	32.6		11.4	16.2
	N	11	11	11	11		10	10
3(F)	Mean	210.2	232.8	260.8	310.5		235.7	242.4
	S.D.	12.9	12.8	12.9	17.2		18.0	16.8
	N	11	11	11	11		10	10
4(F)	Mean	204.5	226.9	256.0	292.2**		#	#
	S.D.	13.5	17.2	17.4	23.4
	N	11	11	11	9

Dose Levels:

Group 1: 0 (Control), Group 2: 30 mg/kg bw/day, Group 3: 100 mg/kg bw/day, Group 4: 300 mg/kg bw/day

# = females terminated on Day 38 of the study

M Male

F Female

S.D. Standard deviation

N Number of animals/litters

* Significantly different from control group p<0.05

** Significantly different from control group p<0.01

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	9.8	12.6	9.1	13.0	14.2	6.8	65.5	19.8
	S.D.	6.0	5.4	7.9	3.5	3.8	5.2	21.7	6.4
	N	12	12	12	12	12	12	12	12
2(M)	Mean	8.8	9.5	8.8	13.7	12.2	9.6	62.6	18.9
	S.D.	4.4	7.9	5.5	5.6	3.2	4.7	16.0	4.7
	N	12	12	12	12	12	12	12	12
3(M)	Mean	7.5	7.8*	6.3	13.4	7.7**	3.7	46.3	14.2
	S.D.	4.8	5.1	6.6	4.4	3.8	5.6	17.5	5.2
	N	12	12	12	12	12	12	12	12
4(M)	Mean	1.9**	6.4*	1.5**	8.8*	3.6**	5.1	27.3	8.3
	S.D.	4.4	4.7	5.7	3.7	5.3	4.6	14.7	4.4
	N	12	12	12	12	12	12	12	12
Group	From:	1	8					1	1
(Sex)	To:	8	15					15	15
1(F)	Mean	8.8	9.1					17.8	8.9
	S.D.	4.6	6.3					5.3	2.4
	N	12	12					12	12
2(F)	Mean	8.3	9.8					18.1	9.5
	S.D.	5.6	5.7					5.6	3.0
	N	12	12					12	12
3(F)	Mean	7.2	6.8					13.9	7.2
	S.D.	4.1	6.2					6.1	3.1
	N	12	12					12	12
4(F)	Mean	6.1	6.7					12.8	6.8
	S.D.	4.4	4.5					6.4	3.6
	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	22.0	29.8	60.9	10.7	51.8	112.8
	S.D.	5.0	6.6	9.4	8.1	8.7	16.4
	N	12	12	12	11	12	12
2(F)	Mean	23.8	27.7	59.4	12.4	51.5	110.9
	S.D.	5.5	7.6	16.7	12.8	11.5	26.3
	N	11	11	11	10	11	11
3(F)	Mean	22.6	28.0	49.7*	6.7	50.6	100.4
	S.D.	4.5	3.3	10.9	7.0	5.7	10.4
	N	11	11	11	10	11	11
4(F)	Mean	22.4	29.1	37.6**	#	51.5	88.9*
	S.D.	6.6	4.5	11.4		5.0	14.0
	N	11	11	9		11	11

Dose Levels:

Group 1: 0 (Control), Group 2: 30 mg/kg bw/day, Group 3: 100 mg/kg bw/day, Group 4: 300 mg/kg bw/day

# = females terminated on Day 38 of the study

M Male

F Female

S.D. Standard deviation

N Number of animals/litters

* Significantly different from control group p<0.05

** Significantly different from control group p<0.01

Table 5. Group Mean Food Consumptions

	Day Numbers Relative to Start Date
Group	From:	1	8	29	36
(Sex)	To:	8	15	36	43
1(M)	Mean	17.9	18.0	19.6	19.5
	N	12	12	12	12
2(M)	Mean	18.1	18.1	19.7	19.2
	N	12	12	12	12
3(M)	Mean	16.7	16.2	17.6	16.4
	N	12	12	12	12
4(M)	Mean	16.9	17.7	16.9	16.2
	N	12	12	12	12
Group	From:	1	8#
(Sex)	To:	8	15
1(F)	Mean	15.2	15.4
	N	12	12
2(F)	Mean	16.1	13.9
	N	12	9
3(F)	Mean	17.2	14.7
	N	12	6
4(F)	Mean	13.8	14.2
	N	12	12
	Day Numbers
		Gestation			Lactation
Group	From:	0	7	14	1
(Sex)	To:	7	14	20	4
1(F)	Mean	18.8	22.0	23.6	23.0
	S.D.	1.1	2.3	2.8	6.0
	N	12	12	12	9
2(F)	Mean	19.0	22.1	22.7	24.7
	S.D.	2.1	3.2	2.6	5.9
	N	11	11	11	10
3(F)	Mean	18.2	21.4	21.8	17.4
	S.D.	1.4	1.7	2.4	4.1
	N	11	11	11	9
4(F)	Mean	18.4	21.1	17.9***	¤
	S.D.	2.4	2.7	2.5
	N	11	10	8

Food Consumption = g/animal/day.

Dose Levels:

Group 1: 0 (Control), Group 2: 30 mg/kg bw/day, Group 3: 100 mg/kg bw/day, Group 4: 300 mg/kg bw/day

# = due to technician error cages 16, 21 and 24 have been excluded from group means

¤ = data unavailable due to early termination of level

M Male

F Female

S.D. Standard deviation

N Number of animals/litters

*** Significantly different from control group p<0.001

Table 6. Food Efficiency

	Day Numbers Relative to Start Date
Group	From:	1	8	29	36
(Sex)	To:	8	15	36	43
1(M)	Mean	7.8	9.9	10.3	4.9
	N	12	12	12	12
2(M)	Mean	7.0	7.4	8.8	7.1
	N	12	12	12	12
3(M)	Mean	6.5	6.8	6.2	3.1
	N	12	12	12	12
4(M)	Mean	1.5	5.2	2.9	4.4
	N	12	12	12	12
Group	From:	1	8
(Sex)	To:	8	15
1(F)	Mean	8.3	8.2
	N	12	12
2(F)	Mean	7.7	10.1
	N	12	9
3(F)	Mean	6.0	8.2
	N	12	6
4(F)	Mean	6.3	6.7
	N	12	12

Dose Levels:

Group 1: 0 (Control), Group 2: 30 mg/kg bw/day, Group 3: 100 mg/kg bw/day, Group 4: 300 mg/kg bw/day

M Male

F Female

S.D. Standard deviation

N Number of animals/litters

Table 7. Group Mean Hematological Values

Group (sex)		Hb	RBC	Hct	MCH	MCV	MCHC	WBC	Neut	Lymph	Mono	Eos	Bas	CT	PLT	APTT	Retics
		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	16.50	8.562	46.62	19.28	54.44	35.42	6.84	0.970	5.814	0.000n	0.056	0.000n	8.76	545.4	13.92	4.04
	S.D.	0.62	0.251	1.96	0.40	1.32	0.38	1.34	0.288	1.419	0.000	0.077	0.000	0.27	80.8	1.51	0.62
	N	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5
2(M)	Mean	18.14*	9.548**	52.60**	19.00	55.10	34.46*	7.58	1.562	5.976	0.000n	0.044	0.000n	8.92	525.2	15.16	4.86
	S.D.	1.20	0.563	3.56	0.87	2.03	0.45	2.65	0.956	1.770	0.000	0.060	0.000	0.69	61.7	2.50	0.88
	N	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5
3(M)	Mean	21.68**	11.254**	63.18**	19.28	56.18	34.34*	6.14	0.970	5.110	0.000n	0.038	0.000n	9.66	457.2*	15.66	4.32
	S.D.	1.02	0.675	3.25	0.48	1.05	0.38	0.80	0.370	0.778	0.000	0.036	0.000	0.82	22.2	1.69	0.41
	N	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5
4(M)	Mean	22.84**	11.944**	65.94**	19.14	55.22	34.62*	7.86	0.892	6.934	0.000n	0.034	0.000n	9.70	376.4**	13.94	4.74
	S.D.	1.17	0.586	3.17	0.60	1.44	0.40	0.84	0.380	1.162	0.000	0.076	0.000	0.91	102.7	1.15	0.58
	N	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5
1(F)	Mean	13.32	6.976	39.26	19.12	56.34	33.94	6.58	1.488	5.092	0.000n	0.000n	0.000n	8.48	908.2	12.86	6.58
	S.D.	0.75	0.470	2.45	0.75	2.35	0.26	1.01	0.744	0.306	0.000	0.000	0.000	0.50	170.3	1.63	0.84
	N	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5
2(F)	Mean	14.84*	7.668*	43.92*	19.36	57.26	33.80	6.32	2.218	4.104	0.000n	0.000n	0.000n	8.24	797.8	13.08	8.24**
	S.D.	1.25	0.707	4.07	0.24	0.15	0.34	3.44	2.110	1.359	0.000	0.000	0.000	0.50	124.0	1.13	0.54
	N	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5
3(F)	Mean	16.44**	8.268**	48.80**	19.86	59.02	33.66	5.36	1.882	3.478*	0.000n	0.000n	0.000n	8.16	757.0	14.12	9.74**
	S.D.	0.76	0.111	2.56	0.79	2.60	0.48	2.17	0.896	1.295	0.000	0.000	0.000	0.24	91.8	0.44	0.89
	N	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5

Dose Levels: Group 1 - 0 (Control), Group 2 - 30 mg/kg bw/day, Group 3 - 100 mg/kg bw/day, Group 4 - 300 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 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)

Reticulocyte count (Retics)

Parameters measured on citrate-treated blood:

Prothrombin time (CT)

Activated partial thromboplastin time (APTT)

Table 8. 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	160.6	7.024	3.74	1.158	148.0	4.216	102.6	2.730	2.20	72.2	48.6	138.0	0.742	96.0	0.136	5.70
	S.D.	3.0	31.3	0.553	0.11	0.122	2.0	0.248	1.5	0.097	0.31	7.8	6.0	26.5	0.056	26.3	0.040	1.01
	N	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5
2(M)	Mean	43.6	155.8	6.932	3.76	1.182	148.4	4.372	103.8	2.666	1.84	84.8	51.0	147.4	0.776	87.8	0.110	8.36
	S.D.	8.8	18.6	0.400	0.11	0.077	0.9	0.547	1.1	0.079	0.21	13.6	6.1	23.3	0.070	13.0	0.038	2.70
	N	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5
3(M)	Mean	43.4	120.0*	7.418	3.90	1.112	148.4	4.396	103.0	2.734	2.06	96.4*	53.2	172.4	0.768	71.6**	0.084*	11.30**
	S.D.	4.3	15.3	0.328	0.16	0.070	3.0	0.353	1.2	0.112	0.63	16.2	6.6	50.4	0.042	1.5	0.046	2.60
	N	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5
4(M)	Mean	41.8	160.0	5.840**	3.20**	1.234	148.8	4.688	106.8**	2.520**	1.66	112.8**	57.2	136.2	0.726	56.2**	0.016**	13.40**
	S.D.	4.4	23.8	0.395	0.16	0.052	0.4	0.638	1.3	0.040	0.27	39.4	8.9	27.3	0.076	6.5	0.011	4.71
	N	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5
1(F)	Mean	44.8	133.6	6.532	3.74	1.324	146.4	4.170	103.0	2.710	1.06	86.4	62.2	143.0	0.948	91.6	0.064	23.16
	S.D.	7.1	20.3	0.648	0.37	0.040	1.5	0.116	1.9	0.154	0.57	20.2	24.7	109.0	0.236	11.1	0.015	18.00
	N	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5
2(F)	Mean	45.4	137.0	6.256	3.48	1.252	149.2	4.338	106.8*	2.614	0.52	92.0	56.8	112.2	0.818	76.8	0.062	12.26
	S.D.	11.2	12.2	0.772	0.60	0.175	3.2	0.596	3.3	0.130	0.04	34.6	13.5	25.8	0.138	19.8	0.008	11.84
	N	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5
3(F)	Mean	52.2	132.2	6.516	3.76	1.386	147.6	4.184	106.6*	2.652	0.66	79.4	56.8	137.4	0.854	74.4	0.062	9.36
	S.D.	3.6	12.4	0.365	0.18	0.057	1.5	0.445	1.7	0.171	0.42	8.3	7.8	46.3	0.068	11.1	0.008	3.47
	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 - 30 mg/kg bw/day, Group 3 - 100 mg/kg bw/day, Group 4 - 300 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 9. Group Mean Organ Weights with Corresponding Relative (% of Body Weight) Organ Weights

		MALES				FEMALES
		0	30	100	300	0	30	100	300
		Control	mg/kg bw/day			Control	mg/kg bw/day
Terminal Bodyweight	Mean (g)	395.8	393.8	374.5	355.3	259.7	252.9	241.1
	S.D.	25.3	19.7	23.3	18.9	16.5	17.1	16.4
	N	12	12	12	12	12	12	11
Adrenals	Mean (g)	0.07022	0.07550	0.07516	0.07210	0.09590	0.09510*	0.10672**
	S.D.	0.01231	0.00859	0.01068	0.01696	0.01396	0.01500	0.02918
	N	5	5	5	5	5	5	5
	Mean (%)	0.018	0.019	0.020	0.021	0.035	0.038*	0.045**
	S.D.	0.004	0.001	0.002	0.004	0.004	0.005	0.009
	N	5	5	5	5	5	5	5
Brain (Including Cerebrum, Cerebellum And Pons)	Mean (g)	1.97200	1.92486	1.99846	1.92282	1.85840	1.72084	1.73108
	S.D.	0.10215	0.15308	0.10167	0.13286	0.06758	0.07946	0.05395
	N	5	5	5	5	5	5	5
	Mean (%)	0.511	0.481	0.536	0.550	0.682	0.700	0.742
	S.D.	0.023	0.019	0.045	0.037	0.031	0.046	0.044
	N	5	5	5	5	5	5	5
Epididymides	Mean (g)	1.48781	1.48413	1.45452	1.41631
	S.D.	0.14906	0.30587	0.10444	0.19569
	N	12	12	12	12
	Mean (%)	0.376	0.376	0.390	0.398
	S.D.	0.037	0.075	0.040	0.044
	N	12	12	12	12
Heart	Mean (g)	1.24044	1.30996	0.96188	1.06524	0.94754	0.90450	0.89762
	S.D.	0.14610	0.20342	0.10404	0.21239	0.17241	0.17023	0.06659
	N	5	5	5	5	5	5	5
	Mean (%)	0.320	0.328	0.258	0.304	0.347	0.365	0.384
	S.D.	0.025	0.045	0.034	0.052	0.058	0.053	0.026
	N	5	5	5	5	5	5	5
Kidneys	Mean (g)	2.60834	2.25078	2.12342*	1.88502*	1.69866	1.56786	1.61864
	S.D.	0.61941	0.19761	0.08122	0.23142	0.18732	0.15490	0.25860
	N	5	5	5	5	5	5	5
	Mean (%)	0.676	0.565	0.570*	0.540*	0.624	0.636	0.690
	S.D.	0.161	0.066	0.052	0.067	0.079	0.058	0.075
	N	5	5	5	5	5	5	5
Liver	Mean (g)	12.9290	11.9379	11.9103	11.8279	11.2310	11.3161*	11.1483*
	S.D.	1.26107	0.84340	1.00069	1.17351	0.58563	1.67927	2.00872
	N	5	5	5	5	5	5	5
	Mean (%)	3.352	2.989	3.184	3.383	4.121	4.579*	4.738*
	S.D.	0.366	0.144	0.162	0.300	0.233	0.524	0.539
	N	5	5	5	5	5	5	5
Ovaries	Mean (g)					0.11010	0.11668	0.11234
	S.D.					0.01937	0.02418	0.02442
	N					11	10	10
	Mean (%)					0.042	0.046	0.047
	S.D.					0.007	0.008	0.011
	N					11	10	10
Pituitary	Mean (g)	0.01174	0.01297	0.01155	0.01067	0.01667	0.01669	0.01461
	S.D.	0.00248	0.00234	0.00202	0.00197	0.00228	0.00512	0.00174
	N	12	12	12	11	11	9	10
	Mean (%)	0.003	0.003	0.003	0.003	0.006	0.006	0.006
	S.D.	0.001	0.001	0.001	0.001	0.001	0.002	0.001
	N	12	12	12	11	11	9	10
Prostate	Mean (g)	0.68968	0.63371	0.60710	0.51978**
	S.D.	0.11748	0.16226	0.14203	0.08947
	N	12	12	12	12
	Mean (%)	0.174	0.161	0.162	0.147**
	S.D.	0.024	0.039	0.035	0.027
	N	12	12	12	12
Seminal Vesicles	Mean (g)	1.94050	1.84904	1.59451*	1.50923*
	S.D.	0.55025	0.33359	0.55241	0.30269
	N	12	12	12	12
	Mean (%)	0.494	0.471	0.426*	0.426*
	S.D.	0.149	0.089	0.141	0.087
	N	12	12	12	12
Spleen	Mean (g)	0.70462	0.78266	0.76826	0.77962	0.68320	0.73588	0.64726
	S.D.	0.08802	0.16750	0.09232	0.03436	0.08846	0.09501	0.06136
	N	5	5	5	5	5	5	5
	Mean (%)	0.182	0.196	0.205	0.223	0.251	0.298	0.277
	S.D.	0.020	0.044	0.017	0.012	0.036	0.031	0.025
	N	5	5	5	5	5	5	5
Testes	Mean (g)	3.56021	3.50226	3.54632	3.54373
	S.D.	0.30975	0.83156	0.56755	0.27723
	N	12	12	12	12
	Mean (%)	0.901	0.887	0.950	0.998
	S.D.	0.075	0.204	0.164	0.062
	N	12	12	12	12
Thymus	Mean (g)	0.34894	0.31898	0.39868	0.39736	0.31312	0.22936	0.24802
	S.D.	0.11172	0.11676	0.11450	0.07675	0.06041	0.06198	0.06618
	N	5	5	5	5	5	5	5
	Mean (%)	0.089	0.079	0.108	0.114	0.115	0.094	0.107
	S.D.	0.025	0.027	0.035	0.023	0.024	0.028	0.031
	N	5	5	5	5	5	5	5
Thyroid/Parathyroid	Mean (g)	0.02074	0.02100	0.01874	0.01930	0.02228	0.02204	0.02000
	S.D.	0.00456	0.00604	0.00646	0.00523	0.00562	0.00428	0.00317
	N	5	5	5	4	5	5	5
	Mean (%)	0.005	0.005	0.005	0.005	0.008	0.009	0.009
	S.D.	0.001	0.002	0.002	0.001	0.002	0.002	0.001
	N	5	5	5	4	5	5	5
Uterus & Cervix	Mean (g)					0.69819	0.62455	0.71666
	S.D.					0.13221	0.12797	0.30585
	N					11	10	10
	Mean (%)					0.266	0.246	0.295
	S.D.					0.042	0.050	0.121
	N					11	10	10

S.D. Standard deviation

N Number of animals/litters

* Significantly different from control group p<0.05

** Significantly different from control group p<0.01

Table 10. Summary Incidence of Necropsy Findings

		MALES
Removal Reason: SCHEDULED KILL		0	30	100	300
		(Control)	mg/kg bw/day
	Number of Animals Examined:	12	12	12	12
	Group:	(1)	(2)	(3)	(4)
Epididymides;
Submitted		(12)	(12)	(12)	(12)
No Visible Lesions		12	11	12	12
Small		0	1	0	0
Lungs (With Bronchi);
Submitted		(12)	(12)	(12)	(12)
No Visible Lesions		12	11	12	11
Reddened		0	1	0	1
Testes;
Submitted		(12)	(12)	(12)	(12)
No Visible Lesions		12	11	12	12
Small		0	1	0	0
		FEMALES
Removal Reason: SCHEDULED KILL		0	30	100
		(Control)	mg/kg bw/day
	Number of Animals Examined:	12	12	11
	Group:	(1)	(2)	(3)
Kidneys;
Submitted		(12)	(12)	(11)
No Visible Lesions		12	11	11
Malformed		0	1	0
Lungs (With Bronchi);
Submitted		(12)	(12)	(11)
No Visible Lesions		12	11	10
Reddened		0	1	1
Uterus & Cervix;
Submitted		(12)	(12)	(11)
No Visible Lesions		12	11	11
Damaged On Removal		0	1	0
		FEMALES
Removal Reason: FOUND DEAD				100	300
				mg/kg bw/day
	Number of Animals Examined:			1	2
	Group:			(3)	(4)
Adrenals;
Submitted				(1)	(2)
No Visible Lesions				1	1
Enlarged				0	1
		FEMALES
Removal Reason: KILLED (AT SD/NACWO REQUEST)					300
					mg/kg bw/day
	Number of Animals Examined:				9
	Group:				(4)
Adrenals;
Submitted					(9)
No Visible Lesions					9
Caecum;
Submitted					(9)
No Visible Lesions					7
Coloured Contents; black					2
Duodenum;
Submitted					(9)
No Visible Lesions					8
Coloured Contents; yellow					1
Ileum (Including Peyers Patches);
Submitted					(9)
No Visible Lesions					7
Coloured Contents; black					1
Coloured Contents; yellow					1
Jejunum;
Submitted					(9)
No Visible Lesions					8
Coloured Contents; yellow					1
Liver;
Submitted					(9)
No Visible Lesions					8
Mottled Appearance					1
Lungs (With Bronchi);
Submitted					(9)
No Visible Lesions					8
Reddened					1
Spleen;
Submitted					(9)
No Visible Lesions					8
Small					1
Stomach;
Submitted					(9)
No Visible Lesions					0
Coloured Contents; black					9
Discolouration; yellow; glandular region					1
Discolouration; yellow; non-glandular region					1
Sloughing; glandular region					1
		FEMALES
Removal Reason: KILLED IN EXTREMIS					300
					mg/kg bw/day
	Number of Animals Examined:				1
	Group:				(4)
Adrenals;
Submitted					(1)
No Visible Lesions					0
Enlarged					1
Caecum;
Submitted					(1)
No Visible Lesions					0
Coloured Contents; yellow					1
Colon;
Submitted					(1)
No Visible Lesions					0
Coloured Contents; yellow					1
Duodenum;
Submitted					(1)
No Visible Lesions					0
Coloured Contents; yellow					1
Ileum (Including Peyers Patches);
Submitted					(1)
No Visible Lesions					0
Coloured Contents; yellow					1
Jejunum;
Submitted					(1)
No Visible Lesions					0
Coloured Contents; yellow					1

Conclusions:

The oral administration of the substance to rats by gavage, at dose levels of 30, 100 and 300 mg/kg bw/day resulted in the premature termination or early death of all females around the time of parturition at 300 mg/kg bw/day. One female given 100 mg/kg bw/day was also found dead shortly after completing parturition. These deaths were considered to be treatment-related with histopathological changes in the gastrointestinal tract considered to be the primary cause along with secondary changes to the liver, adrenals and lympho-reticular system at the point of parturition when the intrinsic stress on these females was notably increased. The surviving animals of either sex treated with 100 mg/kg bw/day and males given 300 mg/kg bw/day showed adverse changes in the hematology profile which were associated with toxicologically significant alterations in the spleen and sternal bone marrow.

The dose level of 30 mg/kg bw/day was considered to be the ‘No Observed Adverse Effect Level’ (NOAEL) for systemic toxicity in animals of either sex.

Based on a toxicological interpretation of all the results, the substance is classified with regard to specific target organ toxicity - repeated exposure, with the classification STOT-RE Cat 2: H373 (May cause damage to organs through prolonged or repeated exposure). Classification of the substance is appropriate based on the dose levels 100 and 300 mg/kg bw/day, where mortality at the time of parturition associated with gastrointestinal toxicity and secondary stress-related responses as well as dose-related changes in the haematopoietic system and the liver were observed. The haematopoietic tissues and the liver are suggested as the targets rather than a single organ. At higher doses the gastrointestinal tract is also considered a target.

Executive summary:

Introduction

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.

Methods

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 30, 100 and 300 mg/kg bw/day. A control group of twelve males and twelve females was dosed with vehicle alone (Arachis oil BP).

On Day 38, two females and one female treated with 300 and 100 mg/kg bw/day respectively, were found dead and an additional female treated with 300 mg/kg bw/day was killed in extremis after showing clinical signs during parturition. The remaining females treated with 300 mg/kg bw/day showed further signs of deterioration and difficulty during parturition, and were terminated early on welfare grounds.

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.

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 surviving dose group on Day 4 post partum. Hematology and blood chemistry were evaluated prior to termination on five selected males and females from each surviving dose group.

Adult males were terminated on Day 43 or 44, followed by the termination of all surviving females and offspring on Day 5 post partum. Any surviving 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.

Results

Adult Responses

Mortality - Two females treated with 300 mg/kg bw/day were found dead around the time of parturition whilst another female was killed in extremis due to clinical signs associated with difficulty in parturition. Another female from 100 mg/kg bw/day dose group was found dead shortly after completing parturition.

Some of the remaining females treated with 300 mg/kg bw/day also showed signs of deterioration and difficulty during parturition and therefore all the females from this dose group were terminated earlier than scheduled (Day 38 of dosing). These deaths were considered to be treatment-related. There were no further unscheduled deaths on the study.

Clinical Observations - There were no clinical signs of toxicity related to the test item in females treated with 300 mg/kg bw/day up to Day 38 (around the time of parturition) or in the remaining animals of either sex throughout the study. On Day 38, clinical signs were evident in females treated with 300 mg/kg bw/day. These included hypothermia, decreased respiratory rate, diarrhoea, hunched posture, pilo-erection and fur staining and were deemed to be due to difficulty in parturition.

Behavioral Assessment - There were no treatment-related effects at any dose level.

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 - Males treated with 300 mg/kg bw/day, showed a statistically significant reduction in group mean body weight gains throughout the study excluding the final week. During Weeks 2 and 5 of treatment, males treated with 100 mg/kg bw/day also showed a statistically significant reduction in group mean body weight gain when compared to controls. Overall body weight

gain in both groups was reduced when compared to controls and this finding was considered to be adverse.

During the pre-pairing phase, females treated with 300 or 100 mg/kg bw/day showed lower group mean body weight gains although not attaining statistical significance. The group mean body weight gains during gestation Days 14 to 20 for these females were lower than controls. The overall effects on body weight development for females treated with 300 mg/kg bw/day were deemed to be adverse. There was no adverse effect on body weight gains during lactation for females treated with 30 mg/kg bw/day.

Food Consumption - During the pre-pairing phase food consumption for both sexes were comparable to controls. For males treated with 300 or 100 mg/kg bw/day, the last two weeks of treatment showed a slight reduction in food intake. Food intake for females treated with 300 or 100 mg/kg bw/day during gestation Days 14 to 20 also showed achieving statistical significance for the former.

Males treated with 300 or 100 mg/kg bw/day generally showed lower food conversion efficiency throughout the study when compared to controls. Food conversion efficiency for the corresponding females was lower than controls during Week 1 of pre-pairing phase and remained low for the 300 mg/kg bw/day females during Week 2. For 30 mg/kg bw/day animals food conversion efficiency was comparable with controls, where measured.

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

Laboratory Investigations

Due to the early termination of females treated with 300 mg/kg bw/day the hematology and blood chemical parameters were not assessed for these females.

Hematology - At all dose levels, hematology evaluations identified increases in hemoglobin, erythrocyte counts hematocrit, reticulocyte counts (females only) and reduced mean corpuscular hemoglobin concentration (males only). Animals of either sex treated with 100 mg/kg bw/day and males receiving 300 mg/kg bw/day showed an increase in lymphocytes count. Taking into consideration the associated histopathology alterations, the hematology changes at 100 mg/kg bw/day were considered to be of toxicological significance.

Blood Chemistry - There were no changes of toxicological significance up to a dose level of 300 mg/kg bw/day in males and up to a dose level of 100 mg/kg bw/day in females.

Pathology

Necropsy - Early decedents: At 300 mg/kg bw/day, some females that were found dead or killed prematurely showed findings of enlarged adrenals, black color contents in the stomach, caecum and ileum and/or yellow color contents in the duodenum, ileum and jejunum. One female had discolored yellow glandular region in the stomach and sloughing of the glandular region. One other female had a mottled appearance of the liver, reddened lungs, a small spleen, and the stomach was discolored yellow on the non glandular region. These findings were considered to be related to treatment.

Terminal Sacrifice: At scheduled termination, one male treated with 300 and 30 mg/kg bw/day and one female each treated with 100 and 30 mg/kg bw/day was shown to have reddened lungs at necropsy. At 30 mg/kg bw/day, one male shown to have small epididymides and testes. One female was shown to have one malformed kidney, being smaller in size, misshapen and pale. These

findings were considered to be incidental and not related to treatment.

Organ Weights - Females treated with 30 or 100 mg/kg bw/day showed a dose-related statistically significant increase in adrenal weights relative to terminal body weight. The relationship for absolute weights was not clear with females treated with 30 mg/kg bw/day showing a marginal but statistically significant reduction in absolute weight but females receiving 100 mg/kg bw/day

showing an increase. The majority of the individual values were within the background control ranges.

There was no histological correlation with the reduced weights in the prostate or seminal vesicles at 300 or 100 mg/kg bw/day and, as the weights were within background ranges this may be linked to the overall body weight reduction. There were no toxicological significant effects were detected in males or females treated with 30 mg/kg bw/day.

Histopathology - Premature Decedents: Minimal to moderate ulceration was present in the glandular area of the stomach in 10/12

animals. Inflammatory change was present in one or more areas of the intestines, most pronounced in the caecum in 7/12 animals. Of these animals 5/12 showed cortical hypertrophy in the adrenal gland.

Hepatocellular atrophy; (presenting as a diffuse change with homogenous, deeply staining cells without normal lobular pattern often concentrated in the centrilobular area occasionally giving the appearance of periportal enlargement) was present in 10/12 animals. Periportal fattype vacuolation was present in one animal.

Hematopoiesis was present in 3/12 animals. Lymphoid depletion or lymphocytolysis was present in the thymus, spleen and lymph nodes of up to 10/12 animals. There was an increase in hematopoiesis in the spleen of some animals. Decreased cellularity was present in the bone marrow (sternum) of 5/12 animals. A further one 100 mg/kg bw/day female had given birth but was found dead. This animal also showed similar changes in the liver, stomach, intestines, bone marrow and lymphoid system. These findings were considered to be of toxicological significance.

Terminal Sacrifice: There was an increase in the amount of hematopoiesis in the spleen and an increased cellularity in the bone marrow (sternum) of all groups treated with the test item when compared to control animals. The change in males treated with 30 mg/kg bw/day was equivocal.

Minimal cortical hypertrophy in the adrenal gland was present in 1/5 and 2/5 females treated with 30 or 100 mg/kg bw/day; respectively. There was hepatocellular atrophy in the liver in 3/5 females treated with 100 mg/kg bw/day. These were low grade changes and did not occur in females treated with 30 mg/kg bw/day.

Thymic lymphoid depletion was present in one female treated with 100 mg/kg bw/day but was not present in females treated with 30 mg/kg bw/day. There were no changes in the stomach, intestines or mesenteric lymph node of females treated with 100 or 30 mg/kg bw/day.

There were no test item-related microscopic findings in the testes, including following the qualitative examination of the stages of spermatogenesis in the testes (no test item-related abnormalities in the integrity of the various cell types present within the different stages of the sperm cycle) or following the evaluation of the uterus or evaluation of follicles and corpora lutea in the ovaries.

Conclusion

The oral administration of CsTreat to rats by gavage, at dose levels of 30, 100 and 300 mg/kg bw/day resulted in the premature termination or early death of all females around the time of parturition at 300 mg/kg bw/day. One female given 100 mg/kg bw/day was also found dead shortly after completing parturition. These deaths were considered to be treatment-related with histopathological changes in the gastrointestinal tract considered to be the primary cause along with secondary changes to the liver, adrenals and lympho-reticular system at the point of parturition when the intrinsic stress on these females was notably increased. The surviving animals of either sex treated with 100 mg/kg bw/day and males given 300 mg/kg bw/day showed adverse changes in hematology profile which were associated with toxicologically significant alterations in the spleen and sternal bone marrow.

A dose level of 30 mg/kg bw/day was therefore considered to be the ‘No Observed Adverse Effect Level’ (NOAEL) for systemic toxicity in animals of either sex. Based on toxicological interpretation of all the results, the substance is classified with regard to specific target organ toxicity - repeated exposure, with the classification STOT-RE Cat 2: H373 (May cause damage to organs through prolonged or repeated exposure). The haematopoietic tissues and the liver are suggested as the targets rather than a single organ. At higher doses the gastrointestinal tract is also considered a target.

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. The concentration of test item in the formulations was determined using a gravimetric technique. Deviations from the target range for relative humidity.

GLP compliance:

yes

Remarks:

Certificate is as an attachement to the study report

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

λ²-cobalt(2+) potassium hexacyanoirondiuide

EC Number:

603-073-2

Cas Number:

12549-23-4

Molecular formula:

K2[CoFe(CN)6]

IUPAC Name:

λ²-cobalt(2+) potassium hexacyanoirondiuide

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: 1.05% <100μm
- Crystal structure: Face-centered cubic structure
- Density: 2.21 x 10^3 kg/m3
- pH value: the substance has a neutral pH value in an aqueous solution

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 314-345 g, females 168-216 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 94.06%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

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. Formulations were prepared daily. The concentration of test item in the formulations was determined using a gravimetric technique.

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, 6, 20 and 60 mg/mL
- Amount of vehicle (if gavage): 5 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 and/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 (how): 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 and dosed within two hours of preparation.

Samples of the test item formulation were taken and analyzed for concentration of the test item. The results indicate that the prepared formulations were within 99% to 109% of the nominal concentration. Thus the required content limit of +/- 10% with reference to the nominal content was met. The results indicate the accurate use of the test item and Arachis Oil BP as vehicle during the 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

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Remarks:

P, m/f

Doses / concentrations 2

Dose / conc.:

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

Remarks:

P, m/f

Doses / concentrations 3

Dose / conc.:

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

Remarks:

P, m/f

Doses / concentrations 4

Dose / conc.:

300 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 30 mg/kg bw/day: 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
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 (CsTreat 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 development, food consumption and water intake were monitored during the study. All animals were subjected to gross necropsy examination at termination.

Results:
Mortality -There were no unscheduled deaths on the study.

Clinical Observations - At 1000 mg/kg bw/day, one male showed clinical signs of dehydration, decreased respiratory rate, emaciation, hunched posture and tiptoe gait on Day 15 of the study. This appeared to be an isolated incident as no other animal from this dosage or from other dosage groups.

Body Weight - Reduction in body weight gains was evident in animals of either sex treated with 1000 and 500 mg/kg bw/day when compared to controls. Actual body weight losses were evident in these animals of either sex between Days 1 and 4 and continued in 1000 mg/kg bw/day males during the second week of treatment. Recovery in body weight gain was evident in females from Day 4 onwards. Males treated with 250 mg/kg bw/day, showed no obvious effects on body weight gain, and females at this level appear to be unaffected by treatment.

Food Consumption - At 500 and 1000 mg/kg bw/day, mean food consumption and food conversion efficiency for males was lower than control throughout the study. For males at 250 mg/kg bw/day, there were no consistent differences from control in food intake that indicated an effect of treatment. For females, food consumption did not indicate any obvious effect of treatment at 250, 500 or 1000 mg/kg bw/day throughout the study. However food conversion efficiency at 1000 mg/kg bw/day was lower between Days 1-4. Recovery was evident thereafter an no such effect was evident at 250 mg/kg bw/day.

Water Consumption - There was no obvious effect on water consumption in animals of either sex treated at 1000, 500 or 250 mg/kg bw/day. There were incidences where males treated with 1000 mg/kg bw/day showed an increase in water consumption during the treatment period.

Necropsy - At 1000 mg/kg bw/day, all males showed reddened discolouration in the glandular region in the stomach and one male showed blue coloured stomach contents. No similar findings were found in females treated with 1000 mg/kg bw/day or animals of either sex at 500 or 250 mg/kg bw/day.

The oral administration of the test item to rats by gavage, at dose levels of 250, 500 and 1000 mg/kg bw/day, resulted in a reduction in body weight gain and food consumption at 1000 and 500 mg/kg bw/day. Macroscopic abnormalities were also evident in 1000 mg/kg bw/day. Based on the results from this study a dose level of 300 mg/kg bw/day was considered to be suitable as a high dose level for the OECD 422 study together with 30 and 100 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

Examinations

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 (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 surviving test and control groups 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 surviving test and control groups 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 from each dose level, and five females from groups 1 to 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 300 mg/kg bw/day males and any animals dying during the study 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 300 mg/kg bw/day dose group animals and any animals dying during the study 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 from groups 1, 2 and 3 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
- 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.

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.

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 surviving 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 300 mg/kg bw/day dose group animals and any animals dying during the study 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 300 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 300 mg/kg bw/day males were also stained and examined. Detailed qualitative examination of the testes was undertaken.

Since there were indications of treatment-related changes, examination was subsequently extended to include similarly prepared sections of liver, mesenteric lymph nodes, thymus, stomach and intestines, adrenal glands (females only) and sternum with bone marrow and spleen (both sexes) from animals in the low and intermediate groups (30 and 100 mg/kg bw/day, respectively).

Postmortem examinations (offspring):

SACRIFICE
- At Day 5 post partum, all surviving offspring (F1) 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

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

There were no clinical signs of toxicity related to the test item in females treated with 300 mg/kg bw/day up to Day 38 (around the time of parturition) or in the remaining animals of either sex throughout the study (see Table 2 in 'Any other information on results incl. tables').

On Day 38, clinical signs were evident in females treated with 300 mg/kg bw/day. These included hypothermia, decreased respiratory rate, diarrhoea, hunched posture, pilo-erection and fur staining and were deemed to be due to difficulty in parturition. The surviving females from this group were terminated prematurely.

Mortality:

mortality observed, treatment-related

Description (incidence):

Two females treated with 300 mg/kg bw/day were found dead around the time of parturition whilst another female was killed in extremis due to clinical signs associated with difficulty in parturition. Another female from 100 mg/kg bw/day dose group was found dead shortly after completing parturition. Some of the remaining females treated with 300 mg/kg bw/day also showed signs of deterioration and difficulty during parturition and therefore all the females from this dose group were terminated earlier than scheduled (Day 38 of dosing). These deaths were considered to be treatment-related. There were no further unscheduled deaths on the study (see Table 2 in 'Any other information on results incl. tables').

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Males treated with 300 mg/kg bw/day showed a statistically significant reduction in group mean body weight gains throughout the study excluding the final week. During Weeks 2 and 5 of treatment, males treated with 100 mg/kg bw/day also showed a statistically significant reduction in group mean body weight gain when compared to controls. Overall body weight gain in both groups was reduced when compared to controls and this finding was considered to be adverse.

During the pre-pairing phase, females treated with 300 or 100 mg/kg bw/day showed lower group mean body weight gains although not attaining statistical significance. The group mean body weight gains during gestation Days 14 to 20 for these females were lower than controls. The overall effects on body weight development for females treated with 300 mg/kg bw/day were deemed to be adverse. There was no adverse effect on body weight gains during lactation for females treated with 30 mg/kg bw/day.

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):

effects observed, treatment-related

Description (incidence and severity):

Not a feeding study. However, during the pre-pairing phase food consumption for both sexes were comparable to controls. For males treated with 300 or 100 mg/kg bw/day, the last two weeks of treatment showed a slight reduction in food intake. Food intake for females treated with 300 or 100 mg/kg bw/day during gestation Days 14 to 20 also showed achieving statistical significance for the former. Food consumptions for males and females treated with 30 mg/kg bw/day was comparable to controls.

For results in tabular form, see Table 5 in 'Any other information on results incl. tables'.

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

Males treated with 300 or 100 mg/kg bw/day generally showed lower food conversion efficiency throughout the study when compared to controls. Food conversion efficiency for the corresponding females was lower than controls during Week 1 of pre-pairing phase and remained low for the 300 mg/kg bw/day females during Week 2. There was no effect of treatment at 30 mg/kg bw/day on food conversion efficiency for animals of either sex.

For results in tabular form, see Table 6 in 'Any other information on results incl. tables'.

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:

effects observed, treatment-related

Description (incidence and severity):

Due to the early termination of females treated with 300 mg/kg bw/day the hematology parameters were not assessed for these females.

At all dose levels, hematology evaluations identified increases in hemoglobin, erythrocyte counts hematocrit, reticulocyte counts (females only) and reduced mean corpuscular hemoglobin concentration (males only). Most animals treated with 30 mg/kg bw/day had individual values within the background control ranges. Animals of either sex treated with 100 mg/kg bw/day and males receiving 300 mg/kg bw/day showed an increase in lymphocytes count. Taking into consideration the associated histopathology alterations, the hematology changes at 100 mg/kg bw/day were considered to be of toxicological significance.

For results in tabular form, see Table 7 in 'Any other information on results incl. tables'.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Due to the early termination of females treated with 300 mg/kg bw/day the blood chemical parameters were not assessed for these females.
There were no changes of toxicological significance up to a dose level of 300 mg/kg bw/day in males and up to a dose level of 100 mg/kg bw/day in females.

For results in tabular form, see Table 8 in 'Any other information on results incl. tables'.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Behavioral Assessment - There were no treatment-related effects at any dose level.
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.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Histopathological findings, including examination of the brain (incl. cerebrum, cerebellum and pons): no findings were noted relating to the brains of the test animals which could be related to treatment with the test item (see attached Illustration in section "Overall remarks, attachments").

Organ weight findings, including the brains: no findings were noted relating to the brains of the test animals which could be related to treatment with the test item (see Table 9 in 'Any other information on results incl. tables').

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

There were several findings that were considered to be of toxicological significance (see attached Illustration in section "Overall remarks, attachments").

Premature Decedents
Minimal to moderate ulceration was present in the glandular area of the stomach in 10/12 animals. Inflammatory change was present in one or more areas of the intestines, most pronounced in the caecum in 7/12 animals. Of these animals 5/12 showed cortical hypertrophy in the adrenal gland.

Hepatocellular atrophy; (presenting as a diffuse change with homogenous, deeply staining cells without normal lobular pattern often concentrated in the centrilobular area occasionally giving the appearance of periportal enlargement) was present in 10/12 animals. Periportal fattype vacuolation was present in one animal. Hematopoiesis was present in 3/12 animals.

Lymphoid depletion or lymphocytolysis was present in the thymus, spleen and lymph nodes of up to 10/12 animals. There was an increase in hematopoiesis in the spleen of some animals. Decreased cellularity was present in the bone marrow (sternum) of 5/12 animals.

A further one 100 mg/kg bw/day female had given birth but was found dead. This animal also showed similar changes in the liver, stomach, intestines, bone marrow and lymphoid system. These findings were considered to be of toxicological significance.

Terminal Sacrifice
There was an increase in the amount of hematopoiesis in the spleen and an increased cellularity in the bone marrow (sternum) of all groups treated with the test item when compared to control animals. The change in males treated with 30 mg/kg bw/day was equivocal.

Minimal cortical hypertrophy in the adrenal gland was present in 1/5 and 2/5 females treated with 30 or 100 mg/kg bw/day; respectively. There was hepatocellular atrophy in the liver in 3/5 females treated with 100 mg/kg bw/day.These were low grade changes and did not occur in females treated with 30 mg/kg bw/day. Thymic lymphoid depletion was present in one female treated with 100 mg/kg bw/day butwas not present in females treated with 30 mg/kg bw/day.

There were no changes in the stomach, intestines or mesenteric lymph node of females treated with 100 or 30 mg/kg bw/day. There were no test item-related microscopic findings in the testes, including following the qualitative examination of the stages of spermatogenesis in the testes (no test item-related abnormalities in the integrity of the various cell types present within the different stages of the sperm cycle) or following the evaluation of the uterus or evaluation of follicles and corpora lutea in the ovaries.

Histopathological findings: neoplastic:

not specified

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not specified

Description (incidence and severity):

There were no treatment-related differences in fertility.

There were no test item-related microscopic findings in the testes, including following the qualitative examination of the stages of spermatogenesis in the testes (no test item-related abnormalities in the integrity of the various cell types present within the different stages of the sperm cycle) or following the evaluation of the uterus or evaluation of follicles and corpora lutea in the ovaries.

Reproductive function: sperm measures:

not specified

Description (incidence and severity):

There were no treatment-related differences in fertility.

There were no test item-related microscopic findings in the testes, including following the qualitative examination of the stages of spermatogenesis in the testes (no test item-related abnormalities in the integrity of the various cell types present within the different stages of the sperm cycle) or following the evaluation of the uterus or evaluation of follicles and corpora lutea in the ovaries.

Reproductive performance:

no effects observed

Description (incidence and severity):

No treatment-related effects were detected in reproductive performance.

Mating - No treatment-related effects were detected in mating performance.
The majority of females mated within four days of pairing with one female treated with 100 mg/kg bw/day mating after thirteen days of pairing.

Fertility - There were no treatment-related differences in fertility.
One low (30 mg/kg bw/day), one intermediate (100 mg/kg bw/day) and one high (300 mg/kg bw/day) dose female (No’s 44, 66 and 88; respectively) were found to be non-pregnant following positive evidence of mating. All three females appeared to have been cycling normally. Whilst marked tubular atrophy in the testes and lack of sperm in the epididymis for male 32 (paired with female 44) is likely to have been the cause of the infertility for this pair, there was no obvious reason for the lack of pregnancy for the remaining two females.

Gestation Lengths - There were no treatment related differences in gestation lengths.
There were no differences in gestation lengths up to a dose level of 100 mg/kg bw/day. The distribution for treated females was comparable to controls as gestation lengths were between 22 and 23½ days.

For results in tabular form, see Tables 11 and 12 in 'Any other information on results incl. tables'.

Effect levels (P0)

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: overall effects

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

There were no adverse clinical signs of toxicity observed in offspring up to a dose level of 100 mg/kg bw/day, or any macroscopic abnormalities detected at terminal kill. There was no effect on surface righting reflex values as they were similar to controls.

Mortality / viability:

no mortality observed

Description (incidence and severity):

There was no obvious adverse effect of treatment on the mean number of corpora lutea, implantation sites, pre- and post implantation losses, litter size, offspring viability or sex ratio (see table 13 in 'Any other information on results incl. tables').

In total, 11, 10 and 10 females from control, 30 and 100 mg/kg bw/day dose groups respectively, gave birth to a live litter and successfully reared young to Day 5 of age.

Slightly lower corpora lutea values were evident in females treated with 100 mg/kg bw/day. Consequently litter size at birth, Day 1 and 4 post partum was slightly lower than controls. Statistical significance was not achieved for these parameters therefore this was considered most likely to be due to biological variation. One control and one female treated with 30 mg/kg bw/day showed a total litter loss on Day 2 and 3 post partum, respectively. Following the early termination of females treated with 300 mg/kg bw/day these females could not be assessed.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

At 100 mg/kg bw/day, offspring (F1) weights on Day 1 post partum were similar to controls whilst the corresponding litter weights were lower than controls which deemed to be due to a slightly lower litter size at this dose level. Offspring body weight gains at this dose level over Days 1 to 4 post partum were slightly lower than controls resulting in lower offspring and litter weights on Day 4 post partum. Although statistical significance was not achieved for any of these differences other than the litter weight on Day 4 post partum, taking into consideration the slightly lower litter size at 100 mg/kg bw/day, these offspring normally would have been expected to gain more or comparable body weights to control. There were neither any adverse clinical signs for the offspring at this dose level nor any evidence of the lack of care by the dams during the lactation period and toxicological significance of this finding remains unclear.

At 30 mg/kg bw/day, offspring body weight gains over Day 1 to 4 post partum the corresponding body weights on Day 4 post partum were also slightly lower than controls. Statistical significance was not achieved and taking into consideration the slightly larger litter size at this dose level and slightly higher litter weights on Day 1 and 4 post partum in relation to controls, this observation was deemed to be due to normal biological variation.

For results in tabular form, see table 13 in 'Any other information on results incl. tables'.

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 treatment-related macroscopic abnormalities detected at terminal kill up to a dose level of 100 mg/kg bw/day.

Histopathological findings:

not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

Dose descriptor:

other: No dose descriptors determined for the F1 generation

Generation:

F1

Based on:

other: No dose descriptors determined for the F1 generation

Sex:

not specified

Remarks on result:

not measured/tested

Remarks:

No dose descriptors determined for the F1 generation

Overall reproductive toxicity

Reproductive 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	5	0	12 (1-12)	12 (13-24)
Low	30	5	6	12 (25-36)	12 (37-48)
Intermediate	100	5	20	12 (49-60)	12 (61-72)
High	300	5	60	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)		30 mg/kg bw/day		100 mg/kg bw/day		300 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
Staining around the eyes
	Number of Animals	.		.		1		.
	Days from - to	.		.		1	4	.
Noisy respiration
	Number of Animals	.		1		1		.
	Days from - to	.		35	39	34	35	.
Sex: Female
Littering
	Number of Animals	1		1		.		.
	Days from - to	40	40	39	39	.		.
Not Observed Due to Littering
	Number of Animals	1		1		.		1
	Days from - to	40	40	39	39	.		37	37
Killed (SD/NACWO Request)
	Number of Animals	.		.		.		9
	Days from - to	.		.		.		38	38
Killed `in extremis'
	Number of Animals	.		.		.		1
	Days from - to	.		.		.		38	38
Scheduled kill
	Number of Animals	12		12		11		.
	Days from - to	42	45	42	45	42	54	.
Found dead
	Number of Animals	.		.		1		2
	Days from - to	.		.		38	38	38	38
Decreased respiratory rate
	Number of Animals	.		.		.		3
	Days from - to	.		.		.		38	38
Pilo-erection
	Number of Animals	.		.		.		4
	Days from - to	.		.		.		38	38
Hypothermia
	Number of Animals	.		.		.		1
	Days from - to	.		.		.		38	38
Hunched posture
	Number of Animals	.		.		.		4
	Days from - to	.		.		.		38	38
Fur stained by test material
	Number of Animals	.		.		.		4
	Days from - to	.		.		.		30	37
Diarrhoea
	Number of Animals	.		.		.		4
	Days from - to	.		.		.		38	38

. 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	329.8	339.6	352.2	361.3	374.3	388.4	395.3
	S.D.	5.0	9.4	12.7	16.6	19.2	21.5	24.7
	N	12	12	12	12	12	12	12
2(M)	Mean	330.8	339.6	349.1	357.9	371.6	383.8	393.3
	S.D.	6.6	9.0	12.3	12.8	16.4	18.2	19.2
	N	12	12	12	12	12	12	12
3(M)	Mean	326.1	333.6	341.4	347.7	361.1	368.8	372.4
	S.D.	9.1	13.0	14.5	18.0	19.9	22.5	23.1
	N	12	12	12	12	12	12	12
4(M)	Mean	327.4	329.3	335.8	337.3	346.0	349.6	354.7
	S.D.	6.1	7.0	10.3	12.9	14.5	17.8	19.1
	N	12	12	12	12	12	12	12
1(F)	Mean	199.3	208.1	217.2
	S.D.	9.2	11.4	12.8
	N	12	12	12
2(F)	Mean	192.3	200.5	210.3
	S.D.	9.0	10.6	9.2
	N	12	12	12
3(F)	Mean	192.2	199.3	206.1
	S.D.	8.9	9.7	12.1
	N	12	12	12
4(F)	Mean	191.6	197.7	204.3
	S.D.	12.8	11.7	10.2
	N	12	12	12
		Day Numbers
		Gestation					Lactation
		0	7	14	20		1	4
1(F)	Mean	219.3	241.3	271.2	332.1		249.0	259.7
	S.D.	13.0	14.8	18.4	26.0		18.3	15.7
	N	12	12	12	12		11	11
2(F)	Mean	213.1	236.9	264.6	324.0		240.6	253.0
	S.D.	13.0	13.6	19.7	32.6		11.4	16.2
	N	11	11	11	11		10	10
3(F)	Mean	210.2	232.8	260.8	310.5		235.7	242.4
	S.D.	12.9	12.8	12.9	17.2		18.0	16.8
	N	11	11	11	11		10	10
4(F)	Mean	204.5	226.9	256.0	292.2**		#	#
	S.D.	13.5	17.2	17.4	23.4
	N	11	11	11	9

Dose Levels:

Group 1: 0 (Control), Group 2: 30 mg/kg bw/day, Group 3: 100 mg/kg bw/day, Group 4: 300 mg/kg bw/day

M Male

F Female

S.D. Standard deviation

N Number of animals/litters

# = females terminated on Day 38 of the study

* Significantly different from control group p<0.05

** Significantly different from control group p<0.01

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	9.8	12.6	9.1	13.0	14.2	6.8	65.5	19.8
	S.D.	6.0	5.4	7.9	3.5	3.8	5.2	21.7	6.4
	N	12	12	12	12	12	12	12	12
2(M)	Mean	8.8	9.5	8.8	13.7	12.2	9.6	62.6	18.9
	S.D.	4.4	7.9	5.5	5.6	3.2	4.7	16.0	4.7
	N	12	12	12	12	12	12	12	12
3(M)	Mean	7.5	7.8*	6.3	13.4	7.7**	3.7	46.3	14.2
	S.D.	4.8	5.1	6.6	4.4	3.8	5.6	17.5	5.2
	N	12	12	12	12	12	12	12	12
4(M)	Mean	1.9**	6.4*	1.5**	8.8*	3.6**	5.1	27.3	8.3
	S.D.	4.4	4.7	5.7	3.7	5.3	4.6	14.7	4.4
	N	12	12	12	12	12	12	12	12
Group	From:	1	8					1	1
(Sex)	To:	8	15					15	15
1(F)	Mean	8.8	9.1					17.8	8.9
	S.D.	4.6	6.3					5.3	2.4
	N	12	12					12	12
2(F)	Mean	8.3	9.8					18.1	9.5
	S.D.	5.6	5.7					5.6	3.0
	N	12	12					12	12
3(F)	Mean	7.2	6.8					13.9	7.2
	S.D.	4.1	6.2					6.1	3.1
	N	12	12					12	12
4(F)	Mean	6.1	6.7					12.8	6.8
	S.D.	4.4	4.5					6.4	3.6
	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	22.0	29.8	60.9	10.7	51.8	112.8
	S.D.	5.0	6.6	9.4	8.1	8.7	16.4
	N	12	12	12	11	12	12
2(F)	Mean	23.8	27.7	59.4	12.4	51.5	110.9
	S.D.	5.5	7.6	16.7	12.8	11.5	26.3
	N	11	11	11	10	11	11
3(F)	Mean	22.6	28.0	49.7*	6.7	50.6	100.4
	S.D.	4.5	3.3	10.9	7.0	5.7	10.4
	N	11	11	11	10	11	11
4(F)	Mean	22.4	29.1	37.6**	#	51.5	88.9*
	S.D.	6.6	4.5	11.4		5.0	14.0
	N	11	11	9		11	11

Dose Levels:

Group 1: 0 (Control), Group 2: 30 mg/kg bw/day, Group 3: 100 mg/kg bw/day, Group 4: 300 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

** Significantly different from control group p<0.01

Table 5. Group Mean Food Consumptions

	Day Numbers Relative to Start Date
Group	From:	1	8	29	36
(Sex)	To:	8	15	36	43
1(M)	Mean	17.9	18.0	19.6	19.5
	N	12	12	12	12
2(M)	Mean	18.1	18.1	19.7	19.2
	N	12	12	12	12
3(M)	Mean	16.7	16.2	17.6	16.4
	N	12	12	12	12
4(M)	Mean	16.9	17.7	16.9	16.2
	N	12	12	12	12
Group	From:	1	8#
(Sex)	To:	8	15
1(F)	Mean	15.2	15.4
	N	12	12
2(F)	Mean	16.1	13.9
	N	12	9
3(F)	Mean	17.2	14.7
	N	12	6
4(F)	Mean	13.8	14.2
	N	12	12
	Day Numbers
		Gestation			Lactation
Group	From:	0	7	14	1
(Sex)	To:	7	14	20	4
1(F)	Mean	18.8	22.0	23.6	23.0
	S.D.	1.1	2.3	2.8	6.0
	N	12	12	12	9
2(F)	Mean	19.0	22.1	22.7	24.7
	S.D.	2.1	3.2	2.6	5.9
	N	11	11	11	10
3(F)	Mean	18.2	21.4	21.8	17.4
	S.D.	1.4	1.7	2.4	4.1
	N	11	11	11	9
4(F)	Mean	18.4	21.1	17.9***	¤
	S.D.	2.4	2.7	2.5
	N	11	10	8

Food Consumption = g/animal/day.

Dose Levels:

Group 1: 0 (Control), Group 2: 30 mg/kg bw/day, Group 3: 100 mg/kg bw/day, Group 4: 300 mg/kg bw/day

# = due to technician error cages 16, 21 and 24 have been excluded from group means

¤ = data unavailable due to early termination of level

M Male

F Female

S.D. Standard deviation

N Number of animals/litters

*** Significantly different from control group p<0.001

Table 6. Food Efficiency

	Day Numbers Relative to Start Date
Group	From:	1	8	29	36
(Sex)	To:	8	15	36	43
1(M)	Mean	7.8	9.9	10.3	4.9
	N	12	12	12	12
2(M)	Mean	7.0	7.4	8.8	7.1
	N	12	12	12	12
3(M)	Mean	6.5	6.8	6.2	3.1
	N	12	12	12	12
4(M)	Mean	1.5	5.2	2.9	4.4
	N	12	12	12	12
Group	From:	1	8
(Sex)	To:	8	15
1(F)	Mean	8.3	8.2
	N	12	12
2(F)	Mean	7.7	10.1
	N	12	9
3(F)	Mean	6.0	8.2
	N	12	6
4(F)	Mean	6.3	6.7
	N	12	12

Dose Levels:

Group 1: 0 (Control), Group 2: 30 mg/kg bw/day, Group 3: 100 mg/kg bw/day, Group 4: 300 mg/kg bw/day

M Male

F Female

S.D. Standard deviation

N Number of animals/litters

Table 7. Group Mean Hematological Values

Group (sex)		Hb	RBC	Hct	MCH	MCV	MCHC	WBC	Neut	Lymph	Mono	Eos	Bas	CT	PLT	APTT	Retics
		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	16.50	8.562	46.62	19.28	54.44	35.42	6.84	0.970	5.814	0.000n	0.056	0.000n	8.76	545.4	13.92	4.04
	S.D.	0.62	0.251	1.96	0.40	1.32	0.38	1.34	0.288	1.419	0.000	0.077	0.000	0.27	80.8	1.51	0.62
	N	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5
2(M)	Mean	18.14*	9.548**	52.60**	19.00	55.10	34.46*	7.58	1.562	5.976	0.000n	0.044	0.000n	8.92	525.2	15.16	4.86
	S.D.	1.20	0.563	3.56	0.87	2.03	0.45	2.65	0.956	1.770	0.000	0.060	0.000	0.69	61.7	2.50	0.88
	N	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5
3(M)	Mean	21.68**	11.254**	63.18**	19.28	56.18	34.34*	6.14	0.970	5.110	0.000n	0.038	0.000n	9.66	457.2*	15.66	4.32
	S.D.	1.02	0.675	3.25	0.48	1.05	0.38	0.80	0.370	0.778	0.000	0.036	0.000	0.82	22.2	1.69	0.41
	N	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5
4(M)	Mean	22.84**	11.944**	65.94**	19.14	55.22	34.62*	7.86	0.892	6.934	0.000n	0.034	0.000n	9.70	376.4**	13.94	4.74
	S.D.	1.17	0.586	3.17	0.60	1.44	0.40	0.84	0.380	1.162	0.000	0.076	0.000	0.91	102.7	1.15	0.58
	N	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5
1(F)	Mean	13.32	6.976	39.26	19.12	56.34	33.94	6.58	1.488	5.092	0.000n	0.000n	0.000n	8.48	908.2	12.86	6.58
	S.D.	0.75	0.470	2.45	0.75	2.35	0.26	1.01	0.744	0.306	0.000	0.000	0.000	0.50	170.3	1.63	0.84
	N	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5
2(F)	Mean	14.84*	7.668*	43.92*	19.36	57.26	33.80	6.32	2.218	4.104	0.000n	0.000n	0.000n	8.24	797.8	13.08	8.24**
	S.D.	1.25	0.707	4.07	0.24	0.15	0.34	3.44	2.110	1.359	0.000	0.000	0.000	0.50	124.0	1.13	0.54
	N	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5
3(F)	Mean	16.44**	8.268**	48.80**	19.86	59.02	33.66	5.36	1.882	3.478*	0.000n	0.000n	0.000n	8.16	757.0	14.12	9.74**
	S.D.	0.76	0.111	2.56	0.79	2.60	0.48	2.17	0.896	1.295	0.000	0.000	0.000	0.24	91.8	0.44	0.89
	N	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5

Dose Levels: Group 1 - 0 (Control), Group 2 - 30 mg/kg bw/day, Group 3 - 100 mg/kg bw/day, Group 4 - 300 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 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)

Reticulocyte count (Retics)

Parameters measured on citrate-treated blood:

Prothrombin time (CT)

Activated partial thromboplastin time (APTT)

Table 8. 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	160.6	7.024	3.74	1.158	148.0	4.216	102.6	2.730	2.20	72.2	48.6	138.0	0.742	96.0	0.136	5.70
	S.D.	3.0	31.3	0.553	0.11	0.122	2.0	0.248	1.5	0.097	0.31	7.8	6.0	26.5	0.056	26.3	0.040	1.01
	N	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5
2(M)	Mean	43.6	155.8	6.932	3.76	1.182	148.4	4.372	103.8	2.666	1.84	84.8	51.0	147.4	0.776	87.8	0.110	8.36
	S.D.	8.8	18.6	0.400	0.11	0.077	0.9	0.547	1.1	0.079	0.21	13.6	6.1	23.3	0.070	13.0	0.038	2.70
	N	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5
3(M)	Mean	43.4	120.0*	7.418	3.90	1.112	148.4	4.396	103.0	2.734	2.06	96.4*	53.2	172.4	0.768	71.6**	0.084*	11.30**
	S.D.	4.3	15.3	0.328	0.16	0.070	3.0	0.353	1.2	0.112	0.63	16.2	6.6	50.4	0.042	1.5	0.046	2.60
	N	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5
4(M)	Mean	41.8	160.0	5.840**	3.20**	1.234	148.8	4.688	106.8**	2.520**	1.66	112.8**	57.2	136.2	0.726	56.2**	0.016**	13.40**
	S.D.	4.4	23.8	0.395	0.16	0.052	0.4	0.638	1.3	0.040	0.27	39.4	8.9	27.3	0.076	6.5	0.011	4.71
	N	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5
1(F)	Mean	44.8	133.6	6.532	3.74	1.324	146.4	4.170	103.0	2.710	1.06	86.4	62.2	143.0	0.948	91.6	0.064	23.16
	S.D.	7.1	20.3	0.648	0.37	0.040	1.5	0.116	1.9	0.154	0.57	20.2	24.7	109.0	0.236	11.1	0.015	18.00
	N	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5
2(F)	Mean	45.4	137.0	6.256	3.48	1.252	149.2	4.338	106.8*	2.614	0.52	92.0	56.8	112.2	0.818	76.8	0.062	12.26
	S.D.	11.2	12.2	0.772	0.60	0.175	3.2	0.596	3.3	0.130	0.04	34.6	13.5	25.8	0.138	19.8	0.008	11.84
	N	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5
3(F)	Mean	52.2	132.2	6.516	3.76	1.386	147.6	4.184	106.6*	2.652	0.66	79.4	56.8	137.4	0.854	74.4	0.062	9.36
	S.D.	3.6	12.4	0.365	0.18	0.057	1.5	0.445	1.7	0.171	0.42	8.3	7.8	46.3	0.068	11.1	0.008	3.47
	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 - 30 mg/kg bw/day, Group 3 - 100 mg/kg bw/day, Group 4 - 300 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 9. Group Mean Organ Weights with Corresponding Relative (% of Body Weight) Organ Weights

		MALES				FEMALES
		0	30	100	300	0	30	100	300
		Control	mg/kg bw/day			Control	mg/kg bw/day
Terminal Bodyweight	Mean (g)	395.8	393.8	374.5	355.3	259.7	252.9	241.1
	S.D.	25.3	19.7	23.3	18.9	16.5	17.1	16.4
	N	12	12	12	12	12	12	11
Adrenals	Mean (g)	0.07022	0.07550	0.07516	0.07210	0.09590	0.09510*	0.10672**
	S.D.	0.01231	0.00859	0.01068	0.01696	0.01396	0.01500	0.02918
	N	5	5	5	5	5	5	5
	Mean (%)	0.018	0.019	0.020	0.021	0.035	0.038*	0.045**
	S.D.	0.004	0.001	0.002	0.004	0.004	0.005	0.009
	N	5	5	5	5	5	5	5
Brain (Including Cerebrum, Cerebellum And Pons)	Mean (g)	1.97200	1.92486	1.99846	1.92282	1.85840	1.72084	1.73108
	S.D.	0.10215	0.15308	0.10167	0.13286	0.06758	0.07946	0.05395
	N	5	5	5	5	5	5	5
	Mean (%)	0.511	0.481	0.536	0.550	0.682	0.700	0.742
	S.D.	0.023	0.019	0.045	0.037	0.031	0.046	0.044
	N	5	5	5	5	5	5	5
Epididymides	Mean (g)	1.48781	1.48413	1.45452	1.41631
	S.D.	0.14906	0.30587	0.10444	0.19569
	N	12	12	12	12
	Mean (%)	0.376	0.376	0.390	0.398
	S.D.	0.037	0.075	0.040	0.044
	N	12	12	12	12
Heart	Mean (g)	1.24044	1.30996	0.96188	1.06524	0.94754	0.90450	0.89762
	S.D.	0.14610	0.20342	0.10404	0.21239	0.17241	0.17023	0.06659
	N	5	5	5	5	5	5	5
	Mean (%)	0.320	0.328	0.258	0.304	0.347	0.365	0.384
	S.D.	0.025	0.045	0.034	0.052	0.058	0.053	0.026
	N	5	5	5	5	5	5	5
Kidneys	Mean (g)	2.60834	2.25078	2.12342*	1.88502*	1.69866	1.56786	1.61864
	S.D.	0.61941	0.19761	0.08122	0.23142	0.18732	0.15490	0.25860
	N	5	5	5	5	5	5	5
	Mean (%)	0.676	0.565	0.570*	0.540*	0.624	0.636	0.690
	S.D.	0.161	0.066	0.052	0.067	0.079	0.058	0.075
	N	5	5	5	5	5	5	5
Liver	Mean (g)	12.9290	11.9379	11.9103	11.8279	11.2310	11.3161*	11.1483*
	S.D.	1.26107	0.84340	1.00069	1.17351	0.58563	1.67927	2.00872
	N	5	5	5	5	5	5	5
	Mean (%)	3.352	2.989	3.184	3.383	4.121	4.579*	4.738*
	S.D.	0.366	0.144	0.162	0.300	0.233	0.524	0.539
	N	5	5	5	5	5	5	5
Ovaries	Mean (g)					0.11010	0.11668	0.11234
	S.D.					0.01937	0.02418	0.02442
	N					11	10	10
	Mean (%)					0.042	0.046	0.047
	S.D.					0.007	0.008	0.011
	N					11	10	10
Pituitary	Mean (g)	0.01174	0.01297	0.01155	0.01067	0.01667	0.01669	0.01461
	S.D.	0.00248	0.00234	0.00202	0.00197	0.00228	0.00512	0.00174
	N	12	12	12	11	11	9	10
	Mean (%)	0.003	0.003	0.003	0.003	0.006	0.006	0.006
	S.D.	0.001	0.001	0.001	0.001	0.001	0.002	0.001
	N	12	12	12	11	11	9	10
Prostate	Mean (g)	0.68968	0.63371	0.60710	0.51978**
	S.D.	0.11748	0.16226	0.14203	0.08947
	N	12	12	12	12
	Mean (%)	0.174	0.161	0.162	0.147**
	S.D.	0.024	0.039	0.035	0.027
	N	12	12	12	12
Seminal Vesicles	Mean (g)	1.94050	1.84904	1.59451*	1.50923*
	S.D.	0.55025	0.33359	0.55241	0.30269
	N	12	12	12	12
	Mean (%)	0.494	0.471	0.426*	0.426*
	S.D.	0.149	0.089	0.141	0.087
	N	12	12	12	12
Spleen	Mean (g)	0.70462	0.78266	0.76826	0.77962	0.68320	0.73588	0.64726
	S.D.	0.08802	0.16750	0.09232	0.03436	0.08846	0.09501	0.06136
	N	5	5	5	5	5	5	5
	Mean (%)	0.182	0.196	0.205	0.223	0.251	0.298	0.277
	S.D.	0.020	0.044	0.017	0.012	0.036	0.031	0.025
	N	5	5	5	5	5	5	5
Testes	Mean (g)	3.56021	3.50226	3.54632	3.54373
	S.D.	0.30975	0.83156	0.56755	0.27723
	N	12	12	12	12
	Mean (%)	0.901	0.887	0.950	0.998
	S.D.	0.075	0.204	0.164	0.062
	N	12	12	12	12
Thymus	Mean (g)	0.34894	0.31898	0.39868	0.39736	0.31312	0.22936	0.24802
	S.D.	0.11172	0.11676	0.11450	0.07675	0.06041	0.06198	0.06618
	N	5	5	5	5	5	5	5
	Mean (%)	0.089	0.079	0.108	0.114	0.115	0.094	0.107
	S.D.	0.025	0.027	0.035	0.023	0.024	0.028	0.031
	N	5	5	5	5	5	5	5
Thyroid/Parathyroid	Mean (g)	0.02074	0.02100	0.01874	0.01930	0.02228	0.02204	0.02000
	S.D.	0.00456	0.00604	0.00646	0.00523	0.00562	0.00428	0.00317
	N	5	5	5	4	5	5	5
	Mean (%)	0.005	0.005	0.005	0.005	0.008	0.009	0.009
	S.D.	0.001	0.002	0.002	0.001	0.002	0.002	0.001
	N	5	5	5	4	5	5	5
Uterus & Cervix	Mean (g)					0.69819	0.62455	0.71666
	S.D.					0.13221	0.12797	0.30585
	N					11	10	10
	Mean (%)					0.266	0.246	0.295
	S.D.					0.042	0.050	0.121
	N					11	10	10

S.D. Standard deviation

N Number of animals/litters

* Significantly different from control group p<0.05

** Significantly different from control group p<0.01

Table 10. Summary Incidence of Necropsy Findings

		MALES
Removal Reason: SCHEDULED KILL		0	30	100	300
		(Control)	mg/kg bw/day
	Number of Animals Examined:	12	12	12	12
	Group:	(1)	(2)	(3)	(4)
Epididymides;
Submitted		(12)	(12)	(12)	(12)
No Visible Lesions		12	11	12	12
Small		0	1	0	0
Lungs (With Bronchi);
Submitted		(12)	(12)	(12)	(12)
No Visible Lesions		12	11	12	11
Reddened		0	1	0	1
Testes;
Submitted		(12)	(12)	(12)	(12)
No Visible Lesions		12	11	12	12
Small		0	1	0	0
		FEMALES
Removal Reason: SCHEDULED KILL		0	30	100
		(Control)	mg/kg bw/day
	Number of Animals Examined:	12	12	11
	Group:	(1)	(2)	(3)
Kidneys;
Submitted		(12)	(12)	(11)
No Visible Lesions		12	11	11
Malformed		0	1	0
Lungs (With Bronchi);
Submitted		(12)	(12)	(11)
No Visible Lesions		12	11	10
Reddened		0	1	1
Uterus & Cervix;
Submitted		(12)	(12)	(11)
No Visible Lesions		12	11	11
Damaged On Removal		0	1	0
		FEMALES
Removal Reason: FOUND DEAD				100	300
				mg/kg bw/day
	Number of Animals Examined:			1	2
	Group:			(3)	(4)
Adrenals;
Submitted				(1)	(2)
No Visible Lesions				1	1
Enlarged				0	1
		FEMALES
Removal Reason: KILLED (AT SD/NACWO REQUEST)					300
					mg/kg bw/day
	Number of Animals Examined:				9
	Group:				(4)
Adrenals;
Submitted					(9)
No Visible Lesions					9
Caecum;
Submitted					(9)
No Visible Lesions					7
Coloured Contents; black					2
Duodenum;
Submitted					(9)
No Visible Lesions					8
Coloured Contents; yellow					1
Ileum (Including Peyers Patches);
Submitted					(9)
No Visible Lesions					7
Coloured Contents; black					1
Coloured Contents; yellow					1
Jejunum;
Submitted					(9)
No Visible Lesions					8
Coloured Contents; yellow					1
Liver;
Submitted					(9)
No Visible Lesions					8
Mottled Appearance					1
Lungs (With Bronchi);
Submitted					(9)
No Visible Lesions					8
Reddened					1
Spleen;
Submitted					(9)
No Visible Lesions					8
Small					1
Stomach;
Submitted					(9)
No Visible Lesions					0
Coloured Contents; black					9
Discolouration; yellow; glandular region					1
Discolouration; yellow; non-glandular region					1
Sloughing; glandular region					1
		FEMALES
Removal Reason: KILLED IN EXTREMIS					300
					mg/kg bw/day
	Number of Animals Examined:				1
	Group:				(4)
Adrenals;
Submitted					(1)
No Visible Lesions					0
Enlarged					1
Caecum;
Submitted					(1)
No Visible Lesions					0
Coloured Contents; yellow					1
Colon;
Submitted					(1)
No Visible Lesions					0
Coloured Contents; yellow					1
Duodenum;
Submitted					(1)
No Visible Lesions					0
Coloured Contents; yellow					1
Ileum (Including Peyers Patches);
Submitted					(1)
No Visible Lesions					0
Coloured Contents; yellow					1
Jejunum;
Submitted					(1)
No Visible Lesions					0
Coloured Contents; yellow					1

Table 11. Summary of Reproductive Performance - Groups Values

	Dose Group (mg/kg bw/day)
	0 (Control)	30	100	300
Males
Initial group size	12	12	12	12
Paired	12	12	12	12
Failed to induce pregnancy in female partner	0	1	1	1
Induced pregnancy in female partner	12	11	11	11
Surviving to terminal necropsy	12	12	12	12
Females
Initial group size	12	12	12	12
Found dead	0	0	1	2
Killed in extremis	0	0	0	1
Killed at SD/NACWO request	0	0	0	9
Paired	12	12	12	12
Non-pregnant	0	1	1	1
Total Litter Loss	1	1	0	1#
Reared young to Day 5 of age	11	10	10	0
Litter killed in extremis	0	0	1*	1@

# = One female had total litter loss prior to early termination.

@ = One litter killed in extremis due to early termination of dose level.

* = One litter killed in extremis due to dam found dead.

Table 12. Tabular Summary Report of Effects on Reproduction/Development

Observations		Dose Level (mg/kg bw/day)
		0 (Control)	30	100	300+
Paired animals	n	12	12	12	12
Females showing evidence of copulation	n	12	12	12	12
Pregnant females	n	12	11	11	11
Conception Days 1-4	n	12	11	10	11
Conception Days 13	n	0	0	1	0
Gestation = 22 Days	n	1	4	6	2+
Gestation = 22 ½ Days	n	8	5	5
Gestation = 23 Days	n	1	1	0
Gestation = 23 ½ Days	n	2	1	0
Dams with live young born	n	12	11	11	2+
Dams with live young at Day 4 post partum	n	11	10	10
Corpora lutea/dam	x	13.1#	13.7	11.7	14.0
Implants/dam	x	13.1#	13.3	11.1	13.0
Live offspring/dam at Day 1 post partum	x	11.8	12.4	9.6
Live offspring/dam at Day 4 post partum	x	10.7	12.3	9.2
Sex ratio: % males at Day 1 post partum	x	55.6	46.1	46.6
Sex ratio: % males at Day 4 post partum	x	50.1	46.4	47.4
Litter weight (g) at Day 1 post partum	x	67.69	72.02	53.77
Litter weight (g) at Day 4 post partum	x	89.29	97.94	68.08
Male offspring weight (g) at Day 1 post partum	x	5.97	5.83	5.85
Male offspring weight (g) at Day 4 post partum	x	8.85	8.12	7.92
Female offspring weight (g) at Day 1 post partum	x	5.77	5.81	5.61
Female offspring weight (g) at Day 4 post partum	x	8.54	7.92	7.47
LOSS OF OFFSPRING/DAM
Pre-implantation (corpora lutea minus implantations)#
0	n	11	7	6
1	n	0	3	2
2	n	0	1	2
Pre-natal (implantations minus live births)#
0	n	3	7	5
1	n	2	1	3
2	n	3	2	1
3	n	2	0	0
4	n	1	0	0
6	n	0	1	0
10	n	0	0	1
Post natal (live births minus offspring alive on Day 4 post partum)
0	n	10	9	9
1	n	0	2	0
4	n	0	0	1
12	n	1	0	0

n = Number

x = Mean

# Corpora lutea and implantations incorrectly counted for female 23, excluded for group means.

+ Terminated on grounds for animal welfare on the same day. Two females found dead on Day 38 and the remaining

females for this dose group.

Table 13. 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	13.1	13.1	11.9	11.8	10.7	67.69	89.29	5.93	5.77	8.85	8.54	2.88	2.77	99.4	92.7
	S.D.	2.9	2.9	3.7	3.7	4.3	16.77	28.25	0.52	0.66	1.46	1.64	0.98	1.03	2.2	24.1
	N	10#	10#	11	11	11	11	11	11	10	10	10	10	10	11	11
2	Mean	13.7	13.3	12.4	12.4	12.3	72.02	97.94	5.83	5.81	8.12	7.92	2.28	2.12	100.0	99.3
	S.D.	1.8	1.3	2.3	2.3	2.2	12.18	14.72	0.49	0.72	0.75	0.63	0.51	0.74	0.0	2.1
	N	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10
3	Mean	11.7	11.1	9.6	9.6	9.2	53.77	68.08*	5.85	5.61	7.92	7.47	2.06	1.86	100.0	96.7
	S.D.	2.8	3.2	3.6	3.6	3.5	18.25	21.80	0.40	0.45	1.03	0.96	0.72	0.64	0.0	10.5
	N	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10
4	Mean	14.0	13.0	11.0	11.0
	S.D.	1.4	1.8
	N	9	9	1	1

Dose Levels:

Group 1: 0 (Control), Group 2: 30 mg/kg bw/day, Group 3: 100 mg/kg bw/day, Group 4: 300 mg/kg bw/day

# = female 23 corpora lutes and implantation counts not available due to technician error

S.D. Standard deviation

N Number of animals/litters

* Significantly different from control group p<0.05

Applicant's summary and conclusion

Conclusions:

The oral administration of the test item in a combined repeated dose and reproduction / developmental screening study to rats by gavage, at dose levels of 30, 100 and 300 mg/kg bw/day resulted in the premature termination or early death of all females around the time of parturition at 300 mg/kg bw/day. One female given 100 mg/kg bw/day was also found dead shortly after completing parturition. These deaths were considered to be treatment-related with histopathological changes in the gastrointestinal tract considered to be the primary cause along with secondary changes to the liver, adrenals and lympho-reticular system at the point of parturition when the intrinsic stress on these females was notably increased. The surviving animals of either sex treated with 100 mg/kg bw/day and males given 300 mg/kg bw/day showed adverse changes in the hematology profile which were associated with toxicologically significant alterations in the spleen and sternal bone marrow. The dose level of 30 mg/kg bw/day was considered to be the NOAEL for repeated dose systemic toxicity in animals of either sex.

For toxicity to reproduction/development, no adverse treatment-related effects were detected in mating performance and no differences in fertility of the parental animals. There were also no differences in gestation lengths up to dose 100 mg/kg bw/d of the parental animals. With regard to offspring, there was no adverse effects on the mean number of corpora lutea, implantation sites, pre- and post implantation losses, litter size, offspring viability or sex ratio. There were also no clinical signs of toxicity observed in the offspring. The toxicological importance of observed lower litter weights at 100 mg/kg bw/day on Day 4 post partum as compared to controls remains unclear. There were further no treatment-related macroscopic abnormalities detected in offspring at terminal kill up to a dose level of 100 mg/kg bw/day. The NOEL for reproductive toxicity was considered to be 30 mg/kg bw/day.

Because the lower litter weight was observed only at the dose level causing maternal mortality and other systemic toxic effects it is not justifiable to classify the substance as potentially toxic to reproduction.

Executive summary:

Introduction

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.

Methods

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 30, 100 and 300 mg/kg bw/day. A control group of twelve males and twelve females was dosed with vehicle alone (Arachis oil BP).

On Day 38, two females and one female treated with 300 and 100 mg/kg bw/day respectively, were found dead and an additional female treated with 300 mg/kg bw/day was killed in extremis after showing clinical signs during parturition. The remaining females treated with 300 mg/kg bw/day showed further signs of deterioration and difficulty during parturition, and were terminated early on welfare grounds.

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.

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 surviving dose group on Day 4 post partum. Hematology and blood chemistry were evaluated prior to termination on five selected males and females from each surviving dose group.

Adult males were terminated on Day 43 or 44, followed by the termination of all surviving females and offspring on Day 5 post partum. Any surviving 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.

Results

Adult Responses

Mortality - Two females treated with 300 mg/kg bw/day were found dead around the time of parturition whilst another female was killed in extremis due to clinical signs associated with difficulty in parturition. Another female from 100 mg/kg bw/day dose group was found dead shortly after completing parturition.

Some of the remaining females treated with 300 mg/kg bw/day also showed signs of deterioration and difficulty during parturition and therefore all the females from this dose group were terminated earlier than scheduled (Day 38 of dosing). These deaths were considered to be treatment-related. There were no further unscheduled deaths on the study.

Clinical Observations - There were no clinical signs of toxicity related to the test item in females treated with 300 mg/kg bw/day up to Day 38 (around the time of parturition) or in the remaining animals of either sex throughout the study. On Day 38, clinical signs were evident in females treated with 300 mg/kg bw/day. These included hypothermia, decreased respiratory rate, diarrhoea, hunched posture, pilo-erection and fur staining and were deemed to be due to difficulty in parturition.

Behavioral Assessment - There were no treatment-related effects at any dose level.

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 - Males treated with 300 mg/kg bw/day, showed a statistically significant reduction in group mean body weight gains throughout the study excluding the final week. During Weeks 2 and 5 of treatment, males treated with 100 mg/kg bw/day also showed a statistically significant reduction in group mean body weight gain when compared to controls. Overall body weight

gain in both groups was reduced when compared to controls and this finding was considered to be adverse.

During the pre-pairing phase, females treated with 300 or 100 mg/kg bw/day showed lower group mean body weight gains although not attaining statistical significance. The group mean body weight gains during gestation Days 14 to 20 for these females were lower than controls. The overall effects on body weight development for females treated with 300 mg/kg bw/day were deemed to be adverse. There was no adverse effect on body weight gains during lactation for females treated with 30 mg/kg bw/day.

Food Consumption - During the pre-pairing phase food consumption for both sexes were comparable to controls. For males treated with 300 or 100 mg/kg bw/day, the last two weeks of treatment showed a slight reduction in food intake. Food intake for females treated with 300 or 100 mg/kg bw/day during gestation Days 14 to 20 also showed achieving statistical significance for the former.

Males treated with 300 or 100 mg/kg bw/day generally showed lower food conversion efficiency throughout the study when compared to controls. Food conversion efficiency for the corresponding females was lower than controls during Week 1 of pre-pairing phase and remained low for the 300 mg/kg bw/day females during Week 2. For 30 mg/kg bw/day animals food conversion efficiency was comparable with controls, where measured.

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 treatment related differences in gestation lengths. The distribution for treated females was comparable to controls as gestation lengths were between 22 and 23½ days.

Litter Responses

Offspring Litter Size, Sex Ratio and Viability - Following the early termination of females treated with 300 mg/kg bw/day the following

assessments exclude these females. There was no obvious adverse effect of treatment on the mean number of corpora lutea,

implantation sites, pre- and post implantation losses, litter size, offspring viability or sex ratio.

Offspring Growth and Development - At 100 mg/kg bw/day, offspring body weights on Day 1 post partum were similar to controls,

however, their body weight gains over Days 1 to 4 post partum were lower than controls resulting in statistically significantly lower litter weight on Day 4 post partum for these animals. There were neither any adverse clinical signs for the offspring nor any evidence of

the lack of care by the dams during the lactation period and toxicological importance of this finding remains unclear. There were no clinical signs of toxicity observed in offspring, or any macroscopic abnormalities detected at terminal kill.

Laboratory Investigations

Due to the early termination of females treated with 300 mg/kg bw/day the hematology and blood chemical parameters were not assessed for these females.

Hematology - At all dose levels, hematology evaluations identified increases in hemoglobin, erythrocyte counts hematocrit, reticulocyte counts (females only) and reduced mean corpuscular hemoglobin concentration (males only). Animals of either sex treated with 100 mg/kg bw/day and males receiving 300 mg/kg bw/day showed an increase in lymphocytes count. Taking into consideration the associated histopathology alterations, the hematology changes at 100 mg/kg bw/day were considered to be of toxicological significance.

Blood Chemistry - There were no changes of toxicological significance up to a dose level of 300 mg/kg bw/day in males and up to a dose level of 100 mg/kg bw/day in females.

Pathology

Necropsy - Early decedents: At 300 mg/kg bw/day, some females that were found dead or killed prematurely showed findings of enlarged adrenals, black color contents in the stomach, caecum and ileum and/or yellow color contents in the duodenum, ileum and jejunum. One female had discolored yellow glandular region in the stomach and sloughing of the glandular region. One other female had a mottled appearance of the liver, reddened lungs, a small spleen, and the stomach was discolored yellow on the non glandular region. These findings were considered to be related to treatment.

Terminal Sacrifice: At scheduled termination, one male treated with 300 and 30 mg/kg bw/day and one female each treated with 100 and 30 mg/kg bw/day was shown to have reddened lungs at necropsy. At 30 mg/kg bw/day, one male shown to have small epididymides and testes. One female was shown to have one malformed kidney, being smaller in size, misshapen and pale. These

findings were considered to be incidental and not related to treatment.

Organ Weights - Females treated with 30 or 100 mg/kg bw/day showed a dose-related statistically significant increase in adrenal weights relative to terminal body weight. The relationship for absolute weights was not clear with females treated with 30 mg/kg bw/day showing a marginal but statistically significant reduction in absolute weight but females receiving 100 mg/kg bw/day

showing an increase. The majority of the individual values were within the background control ranges.

There was no histological correlation with the reduced weights in the prostate or seminal vesicles at 300 or 100 mg/kg bw/day and, as the weights were within background ranges this may be linked to the overall body weight reduction. There were no toxicological significant effects were detected in males or females treated with 30 mg/kg bw/day.

Histopathology - Premature Decedents: Minimal to moderate ulceration was present in the glandular area of the stomach in 10/12

animals. Inflammatory change was present in one or more areas of the intestines, most pronounced in the caecum in 7/12 animals. Of these animals 5/12 showed cortical hypertrophy in the adrenal gland.

Hepatocellular atrophy; (presenting as a diffuse change with homogenous, deeply staining cells without normal lobular pattern often concentrated in the centrilobular area occasionally giving the appearance of periportal enlargement) was present in 10/12 animals. Periportal fattype vacuolation was present in one animal.

Hematopoiesis was present in 3/12 animals. Lymphoid depletion or lymphocytolysis was present in the thymus, spleen and lymph nodes of up to 10/12 animals. There was an increase in hematopoiesis in the spleen of some animals. Decreased cellularity was present in the bone marrow (sternum) of 5/12 animals. A further one 100 mg/kg bw/day female had given birth but was found dead. This animal also showed similar changes in the liver, stomach, intestines, bone marrow and lymphoid system. These findings were considered to be of toxicological significance.

Terminal Sacrifice: There was an increase in the amount of hematopoiesis in the spleen and an increased cellularity in the bone marrow (sternum) of all groups treated with the test item when compared to control animals. The change in males treated with 30 mg/kg bw/day was equivocal.

Minimal cortical hypertrophy in the adrenal gland was present in 1/5 and 2/5 females treated with 30 or 100 mg/kg bw/day; respectively. There was hepatocellular atrophy in the liver in 3/5 females treated with 100 mg/kg bw/day. These were low grade changes and did not occur in females treated with 30 mg/kg bw/day.

Thymic lymphoid depletion was present in one female treated with 100 mg/kg bw/day but was not present in females treated with 30 mg/kg bw/day. There were no changes in the stomach, intestines or mesenteric lymph node of females treated with 100 or 30 mg/kg bw/day.

There were no test item-related microscopic findings in the testes, including following the qualitative examination of the stages of spermatogenesis in the testes (no test item-related abnormalities in the integrity of the various cell types present within the different stages of the sperm cycle) or following the evaluation of the uterus or evaluation of follicles and corpora lutea in the ovaries.

Conclusion

The oral administration of CsTreat to rats by gavage, at dose levels of 30, 100 and 300 mg/kg bw/day resulted in the premature termination or early death of all females around the time of parturition at 300 mg/kg bw/day. One female given 100 mg/kg bw/day was also found dead shortly after completing parturition. These deaths were considered to be treatment-related with histopathological changes in the gastrointestinal tract considered to be the primary cause along with secondary changes to the liver, adrenals and lympho-reticular system at the point of parturition when the intrinsic stress on these females was notably increased. The surviving animals of either sex treated with 100 mg/kg bw/day and males given 300 mg/kg bw/day showed adverse changes in hematology profile which were associated with toxicologically significant alterations in the spleen and sternal bone marrow. The ‘No Observed Effect Level’ (NOEL) for reproductive toxicity was considered to be 30 mg/kg bw/day. The substance is not classified as toxic to reproduction.