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Description of key information

A study according to OECD Guideline 422 is a valid investigation of the toxicological effects resulting from repeated oral-gavage administration of the test item to rats (according to OECD 422, GLP compliant). The test item was administered in vehicle (highly purified water) at dosages of 0, 100, 300, and 1000 mg/kg body weight/day, animals in control groups received the vehicle only. Test item was administered to male rats for 28 days and to female rats for 14 days prior to pairing, through the pairing and gestation periods until the F1 generation reached day 4 post partum.


Based on the observed results from a subacute inhalation study on the close structural analogue Pigment Yellow 175, it is concluded that the No Observed Adverse Effect Concentration (NOAEC) of test item was found to be 0.03 mg/L when exposed for 6 hours/day, 5 days/week, for 04 weeks by flow-past nose-only inhalation route to Sprague Dawley rats. The nominal dose of 0.03 mg/L (6 hrs/day, 5 days/week) is the actual exposure concentration in males and females.


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

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
sub-chronic toxicity: oral
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
See Read Across Justification document in chaapter 13
Reason / purpose for cross-reference:
read-across source
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Details on species / strain selection:
Rat is the standard laboratory rodent species used for toxicity assessment and recommended by various regulatory authorities.
The Wistar rat was selected due to the large amount of background data available for this strain.
Sex:
male/female
Details on test animals or test system and environmental conditions:
Hylasco Biotechnology Pvt. Ltd., Plot 4B, AKP, Turkapally Village, Shameerpet Mandal, RR Dist, Telangana 500078
- Females (if applicable) nulliparous and non-pregnant: [yes]
- Age at study initiation: 6 weeks
Body weight range at the start of treatment: Males: 189.68 to 249.91 g & Females : 156.56 to 192.12 g
At the commencement of the treatment, the weight variation of rats used did not exceed ± 20 % of the mean body weight in each sex and group.
Conditions: Rats were housed in an environment controlled room. The temperature maintained during the experiment was between 20 to 24°C and relative humidity was between 49 to 68%. The photoperiod was 12 hours light and 12 hours dark cycle. Adequate fresh air supply of 12-15 air changes/hour was maintained in the experimental room. The maximum and minimum temperature in the experimental room was recorded once daily. The relative humidity in the experimental room was calculated daily from dry and wet bulb temperature recordings.
Housing: Two rats of same sex were housed per cage in sterilized standard polysulfone cages (Size: L 425 x B 266 x H 185 mm), with stainless steel top grill having facilities for pelleted food and drinking water in polycarbonate bottles with stainless steel sipper tubes. The last animal in recovery group of each sex was housed individually. Polycarbonate rat huts were provided to the animals as environmental enrichment objects and changed along with cage at least once a week. During the experimental period, animals were housed in a single experimental room of barrier area.
Bedding: Steam sterilized corn cob was used as bedding and changed along with the cage atleast twice a week.
Diet: Altromin Rat/Mice Maintenance diets manufactured by Altromin Spezialfutter GmbH & Co. KG, Im Seelenkamp 20, 32791 Lage, Germany, was provided ad libitum.
Water: Deep bore-well water passed through activated charcoal filter and exposed to UV rays in Aquaguard on-line water filter-cum-purifier manufactured by Eureka Forbes Ltd., Mumbai 400 001,India, was provided ad libitum to rats in polycarbonate bottles with stainless steel sipper tubes.
Route of administration:
oral: gavage
Details on route of administration:
The dose formulations were administered orally by gavage to specific group of rats once daily at approximately the same time (± 3 hours) each day for a period of 90 consecutive days. Similarly, the vehicle was administered to rats in vehicle control/vehicle control recovery group once daily orally for 90 consecutive days.
The vehicle or the dose formulations were not administered to recovery groups for 28 days following the 90-day treatment period. The dose formulation and the vehicle were administered at an equivolume of 10 mL/kg/day. The dose volume was calculated for individual animals on the first day of treatment and was adjusted according to the most recent body weights recorded during the treatment period
Vehicle:
CMC (carboxymethyl cellulose)
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
For homogeneity and active ingredient (a.i.) concentration analysis, prepared formulation samples were sampled in duplicate sets on Day 1 and during 2nd (Day 34) and 3rd (Day 63) month of the treatment period and analysed in-house. For each set, duplicate samples from top, middle and bottom layers were drawn from each preparation and in case of control, duplicate samples were drawn from middle layer.
The analysis was done as per the method validated under Eurofins Advinus Study No.: G19462. One set of samples was analyzed for concentration. The back up samples were discarded as analysis results of the first set of samples were within the acceptable limits. Formulations were considered acceptable as overall mean results of all the layers and mean of each layer were within ± 15.0 % of the claimed concentration and relative standard deviation (% RSD) was less than 10.0 %.
Duration of treatment / exposure:
90 Days
Frequency of treatment:
Daily
Dose / conc.:
111 mg/kg bw/day (nominal)
Dose / conc.:
333 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10
Control animals:
yes
Details on study design:
No. of groups : 6
Vehicle control (G1)
Low dose (G2)
Mid dose (G3)
High dose (G4)
Vehicle control recovery (G1R)
High dose recovery (G4R)
No. of rats/group: Main groups: 10 males + 10 females
Recovery groups: 5 males + 5 females
Total = 100 (50 males + 50 females)
Observations and examinations performed and frequency:
Observations and examinations performed and frequency
Morbidity and Mortality: All rats were observed for morbidity and mortalities twice daily i.e., once in the morning and once in the afternoon except during holidays wherein the observation was done once daily as there were no clinical signs observed. Clinical Signs: Each rat was observed for checking general clinical signs twice once daily during treatment period once daily during the recovery period. On the days of scheduled detailed clinical examination, clinical signs were included as a part of detailed clinical observations except on Day 1 wherein detailed clinical examination was done prior to the treatment and observations for general clinical signs was done after dosing the animals.
Detailed Clinical Examination: Detailed clinical examination was done prior to the test item administration on Day 1 and at weekly intervals thereafter (± 2 days) during treatment period. During detailed clinical examination, all rats were observed for changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern), changes in gait, posture and response to handling as well as the presen ce of clonic or tonic movements, stereotypies (e.g., excessive grooming, repetitive circling or bizarre behaviour (e.g. self-mutilation, walking backwards). On the days of detailed clinical examination, observation for general clinical signs (first post-dose) was not performed except on Day 1. Ophthalmological Examination: Ophthalmological examination of all animals was performed with an ophthalmoscope prior to start of the treatment, at the end of the treatment period for main groups (Day 84) and at the end of recovery period (Day 117)for recovery groups. Before examination, my driasis was induced using a 1 % solution of Tropicamide.
Functional Observation Battery Tests (FOB)
The following neurological examination was performed during the 12th week (Day 84) of treatment period for main groups and towards the end of recovery period (Day 117) for recovery groups.
Home Cage Observations: Each rat was observed in the home cage for posture and for presence or absence of abnormal vocalizations, tremors and convulsions.
Observations during Removal of Animal from Home Cage and Handling: The objective of this phase of neurological examination was to observe the subject’s response to handling and to conduct other procedures of the FOB that can best be performed when the rat is being held. Each rat was observed for the following examinations:
ease of removal from home cage
handling reactivity
palpebral closure
eye examination
piloerection
lacrimation
salivation
skin/fur examination
perineum wetness
respiration
muscle tone and
extensor thrust response
The observations were recorded using scores/ranks.
Open Field Observation: Rat was placed (one at a time) in an open arena, on a flat surface with a clea
n absorbent paper and observed for at least 2 minutes. Absorbent paper was replaced for each group.
During this observation period, rat was evaluated as it moves about freely/unperturbed and the fol
lowing observations were made and observations were recorded using score/ranks:
gait
posture
tremors
mobility score
arousal level
clonic or tonic movements
stereotypic behaviour
bizarre behaviour
urination
defecation
rearing
abnormal vocalizations
Functional Tests: Functional testing includes motor activity, sensory evaluation, landing hindlimbs footsplay and measurement of grip performance.
Motor Activity: The motor activity of rats was measured using an automated animal activity measuring system (Make: Columbus Instruments) equipped with a computer analyzer. Each rat was individually placed in the activity cages of the instrument. The rats were monitored for 30 minutes. During this motor activity measurement session, parameters viz., the stereotypic time (small movements) in seconds, the ambulatory time (large ambulatory movement) in seconds, horizontal counts, a mbulatory counts were monitored. The Opto-Varimex 4 motor activity measurement system provided the data at 1 minute interval and the data was analyzed in blocks of 10 minutes interval and the same was reported.
Sensory Reactivity Measurements: After the 2 minutes (approximately) observation period, while the rat was in the open field arena, the following tests were conducted. The rat was allowed to move freely in the open field box for these tests but positioned in the box by the observer in order to administer stimulus. During sensory reactivity measurements, rats were observed for following and the observations were recorded using scores/ranks.
approach response
touch response
click response
tail-pinch response
pupil response
aerial righting reflex
Landing Hindlimbs Footsplay: The landing hind limbs foot splay was performed by dropping the rat onto a horizontal surface of the table top from a short height and measuring the distance between the hind feet upon landing. The hind feet of the rat were gently pressed to an ink pad just prior to testing. The rat was suspended in a prone position and then dropped from a height of approximately 30 cm on to a SOP format, which contains the details such as Study no., Animal no, Group and Sex. A clean recording SOP format was used for each rat. A total of 3 readings were recorded for each rat and average of 3 footsplay values is presented in the report along with the individual footsplay values.
Grip Performance: Hindlimbs and forelimbs grip performance was tested using computerized dual grip strength meter (Model: Columbus Instruments). Three trials were conducted for each rat i.e., three trials each for forelimb and hind limbs. Averages of three trials for both forelimb and hindlimbs are calculated and presented in the report along with the individual grip strength values.
Physiological Observations: Body temperature (rectal temperature) was measured in degree Celsius (°C) using digital thermometer. At the end of the functional test, body weight of each rat was measured.
Body Weight: Individual body weights (g) was recorded prior to test item administration on Day 1 and weekly thereafter (± 2 day) for all groups of rats during treatment and recovery period. Fasting body weight was recorded prior to sacrifice for all animals.
Food Consumption: The food consumption was measured at weekly intervals (± 2 days) during treatment and recovery period. The cage wise average food consumption (g/rat/day) was calculated and presented in the report.
Oestrous Cycle Evaluation: Vaginal smear was examined in the female rats and the stage of oestrous cycle was recorded prior to necropsy.
Clinical Pathology Investigations
Blood Collection: At the end of the treatment and recovery periods, all rats were fasted overnight (water allowed) and approximately 4.0 mL of blood was collected under isoflurane anaesthesia, with a fine capillary tube, by retro-orbital sinus puncture: After analysis and data review by the analyst, the residual samples were disposed. Haematology, Coagulation, Clinical Chemistry, Hormone Analysis & Urinalysis Parameters were done as study plan.
Sacrifice and pathology:
All rats from toxicity groups at the end of the scheduled period (Day 91 and 119) were subjected to detailed necropsy (examination of external surfaces of the body, all orifices; cranial, thoracic and abdominal cavities and their contents) and findings were recorded. Terminal fasting body weights were recorded for all animals immediately prior to terminal sacrifice and used in calculation of relative organ weights. All rats sacrificed at term were fasted overnight (water allowed), euthanized with isoflurane (as per the random numbers generated for the study), exsanguinated and subjected for gross examination.
At sacrifice, sperm motility was evaluated using the sperm samples collected from the right vas deferens, immediately after necropsy using Hamilton-Thorne TOX-IVOS sperm analyzer for all rats. For morphological evaluation of sperms, smears were made using semen samples collected from right vas deferens of all rats immediately after necropsy and fixed with acetone for evaluation by manual method. Initially, sperm morphology was assessed for control and high dose rats. The right epididymis was collected and frozen for enumeration of cauda epididymal sperm reserves. As the high dose group did not show any test item-related effect, the analysis was not extended to low, mid dose and recovery rats. Unused frozen samples were discarded at the time of final report preparation On completion of gross pathology examination, the tissues/organs noted in following table were collected from all rats. The below listed organs were weighed from all terminally sacrificed animals. The organ weight ratios (organ to body weight and brain weight) as percentage of fasting body weight
were determined and presented in the report. Paired organs were weighed together, and combined weights were presented.
Histopathological examination was carried out on the preserved organs of vehicle control (G1) and high dose group animals (G4). Examination of the testes also included a qualitative assessment of stages of spermatogenesis. In addition, all gross lesions from all the animals were examined microscopically. In the absence of test item-related changes, the tissues from low (G2), mid dose (G3) and recovery (G1R and G4R) groups were not evaluated.
The tissues were processed for routine paraffin embedding and 4-5-micron sections were stained with Haematoxylin and Eosin stain. In addition, testes were sectioned at 3-4 μm and stained with PAS reagent and haematoxylin to aid in qualitative assessment of spermatogenesis. Unused tissues were archived
Statistics:
For comparative statistics, data was evaluated using the Levene Test for homogeneity of variances and the Shapiro-Wilks Test for normality of distributions. When data found to be homogeneous and of normal distribution, was analysed by analysis of variance (ANOVA), when data found to be no
nhomogeneous or of nonnormal data was subjected for transformation and ANOVA was done on transformed data. When ANOVA was significant, pairwise comparisons of treated groups to the control group was made using a parametric test, Dunnett, to identify statistical differences.
Data captured outside of Provantis™: The statistical analysis of the experimental data was carried out using licensed copies of SYSTAT Statistical package Ver.12.0. All quantitative variables neurological observations (neuromuscular observation/body temperature/body weights) and T3, T4, TSH was tested for normality (Shapiro-Wilk test) and homogeneity of variances (Levene’s test) within the group before performing a one-factor ANOVA modelling by treatment groups. Non-optimal (nonnormal or heteroschedastic) data was transformed, before ANOVA was performed. Comparison of means between treatment groups and control group was done using Dunnett’s test when the overall treatment, ‘F’ test was found significant.
For two groups, the comparisons of the mean between treatment and control group was done using‘t’ test.
Descriptive statistics (Mean, SD & Numbers) was presented by Treatment group and Day. All hypothesis testing were carried out at the 5% (2-sided) significance level. Significant differences are designated throughout the report as below:
*: Statistically significant difference from the control group at p < 0.05
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
There were no clinical signs and mortalities observed throughout the treatment and recovery period in either sex at all the doses tested. Light orange coloured faeces were observed at 111 and 333 mg/kg bwt/day doses and dark orange colour faeces were observed at 1000 mg/kg bwt/day doses in both sexes. This could be due to physical nature of the test item
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Significantly lower body weight gain was observed from Days 22-29 at 333 mg/kg/day and during Days 64-71 at 1000 mg/kg/day in males and significantly higher body weight gain during Days 57-64 at 111 mg/kg/day in females. Significantly higher body weight gain was observed during Days 85-90, 90-97 and 90-118 at 1000 mg/kg/day recovery males and during Days 57-64 and 104-111 at 1000 mg/kg/day recovery females
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
The food consumption was significantly lower in males during Days 1-8 at 333 and during Days 15-22 at 111 and 333 mg/kg/day doses.
In females, the food consumption was significantly lower during Days 15-22 at all the doses in main toxicity groups and during Days 43-50 at 1000 mg/kg/day recovery females. Significantly higher food consumption was observed during Days 64-71 at all the doses in main toxicity groups and during Days 71-77 and 111-118 at 1000 mg/kg/day recovery females.
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
<= 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
behaviour (functional findings)
body weight and weight gain
clinical biochemistry
clinical signs
food consumption and compound intake
gross pathology
haematology
histopathology: non-neoplastic
mortality
ophthalmological examination
organ weights and organ / body weight ratios
urinalysis
Key result
Critical effects observed:
no
Conclusions:
As there were no treatment-related adverse effects observed up to the highest dose the No Observed Adverse Effect Level (NOAEL) for systemic toxicity of the test item C.I. Pigment Orange 36 is considered to be 1000 mg/kg/day under the test conditions and doses employed
Executive summary:

The purpose of this repeated dose toxicity study was to evaluate the systemic toxicity profile of the test item, C.I. Pigment Orange 36 in Wistar rats when administered orally by gavage for a period of 90 consecutive daysand to assess the reversibilityof any effects during a subsequent 28days recovery period. This study was also intended to provide the information on major toxic effects, target organs and an estimation of a No Observed Adverse Effect Level (NOAEL). 


The test item was weighed and suspended in vehicle,i.e.,0.5% Carboxymethylcellulose Sodium salt (medium viscosity) in Milli-Q®Water and administered to rats at the graduated dose levels of 111, 333 and 1000 mg/kg/day for low dose (G2), mid dose (G3) and high dose (G4 )/ high dose recovery (G4R) group rats, respectively. The rats in the vehicle control group (G1)/vehicle control recovery (G1R) groups received vehicle Carboxymethylcellulose alone. The dose volume administered was 10 mL/kg body weight. Each main group in the experiment was comprised of 10 male and 10 female rats and recovery groups comprised of 5 male and 5 female rats.


The identity of the test item was provided by the Sponsor by a Certificate of Analysis (CoA). The authenticity of the test item was not determined at the test facility. The stability of the test item in the vehicle was established separately under Eurofins Advinus Study No. G19462 at 1 and 100 mg/mL. Based on the results, the test item was found to be stable and homogeneous in the vehicle up to 24 hours when stored at room temperature.


During the conduct of this study, the prepared dose formulations and vehicle (Carboxy methylcellulose Sodium salt (medium viscosity) were analyzed for homogeneity and active ingredient (a.i.) concentration on Day 1 and during 2ndmonth (Day 34) and 3rdmonth (Day 63) of the treatment.The results indicated thatthe percent agreement of the analyzed concentrations were in the range, 85% to 115% of the claimed concentrations and the overall % RSD from six replicates at each dose level was<10.0%. This indicates that the prepared dose formulation met the acceptance criteria for concentration and % RSD.


Each rat in the experiment was observed for clinical signs, mortality and morbidity. Ophthalmological examination was carried out for all the rats prior to start of treatment, at the end of treatment for main groups and at the end of recovery period for recovery groups. The body weights and food consumption were measured during in-life phase of the experiment. Neurological examinations were conducted towards the end of treatment (Day 84) for main groups and towards the end of recovery period (Day 117) for recovery groups.The clinical laboratory investigations such as haematology, coagulation, clinical chemistry, hormone analysis and urine analysis were performed at termination. Vaginal smear was examined in the female rats and the stage ofoestrous cycle was recordedprior to necropsy.


All rats in the experiment were subjected to detailed necropsy and the organ weights and their ratios were derived as percent fasting body weights and brain weight. Histopathological examination was carried out on the preserved organs of the vehicle control (G1) and high dose(G4) group animals. Histopathological examination of the testes included a qualitative assessment of stages of spermatogenesis.In addition, gross lesions from all the animals were examined microscopically.There were no test item-related histopathological changes observed in any organ/tissue in high dose group (G4); hence, histopathological evaluation was not carried out in thelower dose (G2and G3) and recovery groups (G1R and G4R).


Under the experimental conditions employed, the following results were obtained:


·        Clinical Signs and Mortality:Orangecolour faecal matter(light to dark) were observed at all the tested doses in both sexes. This could be due to physical nature of the test item. There were no mortality observed at any of the doses tested in both sexes.


·        Ophthalmological Examination:Ophthalmological examination did not reveal any ocular abnormalities.


·        Neurological Findings:No treatment-related neurological abnormalities /dysfunctions were observed at all the doses tested.


·        Body Weights:Treatment did not affect body weight at all the tested doses in either sex.


·        Food Consumption:Treatment did not affect food consumption at all the tested doses in either sex.


·        Haematology, Coagulation, Clinical Chemistry and urine parameters:There were no test item related alterations observed at any of the tested dose levels in either sex.


·        Thyroid Hormone Profile:Thyroid hormone profile (TSH, T4 and T3) was not affected in both sexes across the treated groups when compared to the concurrent vehicle control group.


·        Terminal Fasting Body Weights and Organ Weights:No significant changes in terminal fasting body weights and organ weights attributed to test item were observedat any of the tested dose levels in either sex.


·        Sperm Parameter:There were no test item-related changes in any of the sperm parameters.


·        Gross pathology:There were no test item-related gross pathological changes observed in both sexes. Orange colouration of intestinal contents (ileum, cecum, colon and rectum) observed in both sexes at all doses at the end of treatment period was attributed to the test item colour.


·        Histopathology:There were no test item-related microscopic lesions in any evaluated organs or tissues of male and female rats at the end of treatment period at tested dose levels.


No Observed Adverse Effect Level (NOAEL):


As there were no treatment-related adverse effects observed up to the highest dose the No Observed Adverse Effect Level (NOAEL)for systemic toxicityof the test item C.I. Pigment Orange 36 is considered to be 1000 mg/kg/day under the test conditions and doses employed.

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Details on species / strain selection:
Rat is the standard laboratory rodent species used for toxicity assessment and recommended by various regulatory authorities.
The Wistar rat was selected due to the large amount of background data available for this strain.
Sex:
male/female
Details on test animals or test system and environmental conditions:
Hylasco Biotechnology Pvt. Ltd., Plot 4B, AKP, Turkapally Village, Shameerpet Mandal, RR Dist, Telangana 500078
- Females (if applicable) nulliparous and non-pregnant: [yes]
- Age at study initiation: 6 weeks
Body weight range at the start of treatment: Males: 189.68 to 249.91 g & Females : 156.56 to 192.12 g
At the commencement of the treatment, the weight variation of rats used did not exceed ± 20 % of the mean body weight in each sex and group.
Conditions: Rats were housed in an environment controlled room. The temperature maintained during the experiment was between 20 to 24°C and relative humidity was between 49 to 68%. The photoperiod was 12 hours light and 12 hours dark cycle. Adequate fresh air supply of 12-15 air changes/hour was maintained in the experimental room. The maximum and minimum temperature in the experimental room was recorded once daily. The relative humidity in the experimental room was calculated daily from dry and wet bulb temperature recordings.
Housing: Two rats of same sex were housed per cage in sterilized standard polysulfone cages (Size: L 425 x B 266 x H 185 mm), with stainless steel top grill having facilities for pelleted food and drinking water in polycarbonate bottles with stainless steel sipper tubes. The last animal in recovery group of each sex was housed individually. Polycarbonate rat huts were provided to the animals as environmental enrichment objects and changed along with cage at least once a week. During the experimental period, animals were housed in a single experimental room of barrier area.
Bedding: Steam sterilized corn cob was used as bedding and changed along with the cage atleast twice a week.
Diet: Altromin Rat/Mice Maintenance diets manufactured by Altromin Spezialfutter GmbH & Co. KG, Im Seelenkamp 20, 32791 Lage, Germany, was provided ad libitum.
Water: Deep bore-well water passed through activated charcoal filter and exposed to UV rays in Aquaguard on-line water filter-cum-purifier manufactured by Eureka Forbes Ltd., Mumbai 400 001,India, was provided ad libitum to rats in polycarbonate bottles with stainless steel sipper tubes.
Route of administration:
oral: gavage
Details on route of administration:
The dose formulations were administered orally by gavage to specific group of rats once daily at approximately the same time (± 3 hours) each day for a period of 90 consecutive days. Similarly, the vehicle was administered to rats in vehicle control/vehicle control recovery group once daily orally for 90 consecutive days.
The vehicle or the dose formulations were not administered to recovery groups for 28 days following the 90-day treatment period. The dose formulation and the vehicle were administered at an equivolume of 10 mL/kg/day. The dose volume was calculated for individual animals on the first day of treatment and was adjusted according to the most recent body weights recorded during the treatment period
Vehicle:
CMC (carboxymethyl cellulose)
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
For homogeneity and active ingredient (a.i.) concentration analysis, prepared formulation samples were sampled in duplicate sets on Day 1 and during 2nd (Day 34) and 3rd (Day 63) month of the treatment period and analysed in-house. For each set, duplicate samples from top, middle and bottom layers were drawn from each preparation and in case of control, duplicate samples were drawn from middle layer.
The analysis was done as per the method validated under Eurofins Advinus Study No.: G19462. One set of samples was analyzed for concentration. The back up samples were discarded as analysis results of the first set of samples were within the acceptable limits. Formulations were considered acceptable as overall mean results of all the layers and mean of each layer were within ± 15.0 % of the claimed concentration and relative standard deviation (% RSD) was less than 10.0 %.
Duration of treatment / exposure:
90 Days
Frequency of treatment:
Daily
Dose / conc.:
111 mg/kg bw/day (nominal)
Dose / conc.:
333 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10
Control animals:
yes
Details on study design:
No. of groups : 6
Vehicle control (G1)
Low dose (G2)
Mid dose (G3)
High dose (G4)
Vehicle control recovery (G1R)
High dose recovery (G4R)
No. of rats/group: Main groups: 10 males + 10 females
Recovery groups: 5 males + 5 females
Total = 100 (50 males + 50 females)
Observations and examinations performed and frequency:
Observations and examinations performed and frequency
Morbidity and Mortality: All rats were observed for morbidity and mortalities twice daily i.e., once in the morning and once in the afternoon except during holidays wherein the observation was done once daily as there were no clinical signs observed. Clinical Signs: Each rat was observed for checking general clinical signs twice once daily during treatment period once daily during the recovery period. On the days of scheduled detailed clinical examination, clinical signs were included as a part of detailed clinical observations except on Day 1 wherein detailed clinical examination was done prior to the treatment and observations for general clinical signs was done after dosing the animals.
Detailed Clinical Examination: Detailed clinical examination was done prior to the test item administration on Day 1 and at weekly intervals thereafter (± 2 days) during treatment period. During detailed clinical examination, all rats were observed for changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern), changes in gait, posture and response to handling as well as the presen ce of clonic or tonic movements, stereotypies (e.g., excessive grooming, repetitive circling or bizarre behaviour (e.g. self-mutilation, walking backwards). On the days of detailed clinical examination, observation for general clinical signs (first post-dose) was not performed except on Day 1. Ophthalmological Examination: Ophthalmological examination of all animals was performed with an ophthalmoscope prior to start of the treatment, at the end of the treatment period for main groups (Day 84) and at the end of recovery period (Day 117)for recovery groups. Before examination, my driasis was induced using a 1 % solution of Tropicamide.
Functional Observation Battery Tests (FOB)
The following neurological examination was performed during the 12th week (Day 84) of treatment period for main groups and towards the end of recovery period (Day 117) for recovery groups.
Home Cage Observations: Each rat was observed in the home cage for posture and for presence or absence of abnormal vocalizations, tremors and convulsions.
Observations during Removal of Animal from Home Cage and Handling: The objective of this phase of neurological examination was to observe the subject’s response to handling and to conduct other procedures of the FOB that can best be performed when the rat is being held. Each rat was observed for the following examinations:
ease of removal from home cage
handling reactivity
palpebral closure
eye examination
piloerection
lacrimation
salivation
skin/fur examination
perineum wetness
respiration
muscle tone and
extensor thrust response
The observations were recorded using scores/ranks.
Open Field Observation: Rat was placed (one at a time) in an open arena, on a flat surface with a clea
n absorbent paper and observed for at least 2 minutes. Absorbent paper was replaced for each group.
During this observation period, rat was evaluated as it moves about freely/unperturbed and the fol
lowing observations were made and observations were recorded using score/ranks:
gait
posture
tremors
mobility score
arousal level
clonic or tonic movements
stereotypic behaviour
bizarre behaviour
urination
defecation
rearing
abnormal vocalizations
Functional Tests: Functional testing includes motor activity, sensory evaluation, landing hindlimbs footsplay and measurement of grip performance.
Motor Activity: The motor activity of rats was measured using an automated animal activity measuring system (Make: Columbus Instruments) equipped with a computer analyzer. Each rat was individually placed in the activity cages of the instrument. The rats were monitored for 30 minutes. During this motor activity measurement session, parameters viz., the stereotypic time (small movements) in seconds, the ambulatory time (large ambulatory movement) in seconds, horizontal counts, a mbulatory counts were monitored. The Opto-Varimex 4 motor activity measurement system provided the data at 1 minute interval and the data was analyzed in blocks of 10 minutes interval and the same was reported.
Sensory Reactivity Measurements: After the 2 minutes (approximately) observation period, while the rat was in the open field arena, the following tests were conducted. The rat was allowed to move freely in the open field box for these tests but positioned in the box by the observer in order to administer stimulus. During sensory reactivity measurements, rats were observed for following and the observations were recorded using scores/ranks.
approach response
touch response
click response
tail-pinch response
pupil response
aerial righting reflex
Landing Hindlimbs Footsplay: The landing hind limbs foot splay was performed by dropping the rat onto a horizontal surface of the table top from a short height and measuring the distance between the hind feet upon landing. The hind feet of the rat were gently pressed to an ink pad just prior to testing. The rat was suspended in a prone position and then dropped from a height of approximately 30 cm on to a SOP format, which contains the details such as Study no., Animal no, Group and Sex. A clean recording SOP format was used for each rat. A total of 3 readings were recorded for each rat and average of 3 footsplay values is presented in the report along with the individual footsplay values.
Grip Performance: Hindlimbs and forelimbs grip performance was tested using computerized dual grip strength meter (Model: Columbus Instruments). Three trials were conducted for each rat i.e., three trials each for forelimb and hind limbs. Averages of three trials for both forelimb and hindlimbs are calculated and presented in the report along with the individual grip strength values.
Physiological Observations: Body temperature (rectal temperature) was measured in degree Celsius (°C) using digital thermometer. At the end of the functional test, body weight of each rat was measured.
Body Weight: Individual body weights (g) was recorded prior to test item administration on Day 1 and weekly thereafter (± 2 day) for all groups of rats during treatment and recovery period. Fasting body weight was recorded prior to sacrifice for all animals.
Food Consumption: The food consumption was measured at weekly intervals (± 2 days) during treatment and recovery period. The cage wise average food consumption (g/rat/day) was calculated and presented in the report.
Oestrous Cycle Evaluation: Vaginal smear was examined in the female rats and the stage of oestrous cycle was recorded prior to necropsy.
Clinical Pathology Investigations
Blood Collection: At the end of the treatment and recovery periods, all rats were fasted overnight (water allowed) and approximately 4.0 mL of blood was collected under isoflurane anaesthesia, with a fine capillary tube, by retro-orbital sinus puncture: After analysis and data review by the analyst, the residual samples were disposed. Haematology, Coagulation, Clinical Chemistry, Hormone Analysis & Urinalysis Parameters were done as study plan.
Sacrifice and pathology:
All rats from toxicity groups at the end of the scheduled period (Day 91 and 119) were subjected to detailed necropsy (examination of external surfaces of the body, all orifices; cranial, thoracic and abdominal cavities and their contents) and findings were recorded. Terminal fasting body weights were recorded for all animals immediately prior to terminal sacrifice and used in calculation of relative organ weights. All rats sacrificed at term were fasted overnight (water allowed), euthanized with isoflurane (as per the random numbers generated for the study), exsanguinated and subjected for gross examination.
At sacrifice, sperm motility was evaluated using the sperm samples collected from the right vas deferens, immediately after necropsy using Hamilton-Thorne TOX-IVOS sperm analyzer for all rats. For morphological evaluation of sperms, smears were made using semen samples collected from right vas deferens of all rats immediately after necropsy and fixed with acetone for evaluation by manual method. Initially, sperm morphology was assessed for control and high dose rats. The right epididymis was collected and frozen for enumeration of cauda epididymal sperm reserves. As the high dose group did not show any test item-related effect, the analysis was not extended to low, mid dose and recovery rats. Unused frozen samples were discarded at the time of final report preparation On completion of gross pathology examination, the tissues/organs noted in following table were collected from all rats. The below listed organs were weighed from all terminally sacrificed animals. The organ weight ratios (organ to body weight and brain weight) as percentage of fasting body weight
were determined and presented in the report. Paired organs were weighed together, and combined weights were presented.
Histopathological examination was carried out on the preserved organs of vehicle control (G1) and high dose group animals (G4). Examination of the testes also included a qualitative assessment of stages of spermatogenesis. In addition, all gross lesions from all the animals were examined microscopically. In the absence of test item-related changes, the tissues from low (G2), mid dose (G3) and recovery (G1R and G4R) groups were not evaluated.
The tissues were processed for routine paraffin embedding and 4-5-micron sections were stained with Haematoxylin and Eosin stain. In addition, testes were sectioned at 3-4 μm and stained with PAS reagent and haematoxylin to aid in qualitative assessment of spermatogenesis. Unused tissues were archived
Statistics:
For comparative statistics, data were evaluated using the Levene Test for homogeneity of variances and the Shapiro-Wilks Test for normality of distributions. When data found to be homogeneous and of normal distribution, was analysed by analysis of variance (ANOVA), when data found to be no
nhomogeneous or of nonnormal data was subjected for transformation and ANOVA was done on transformed data. When ANOVA was significant, pairwise comparisons of treated groups to the control group was made using a parametric test, Dunnett, to identify statistical differences.
Data captured outside of Provantis™: The statistical analysis of the experimental data was carried out using licensed copies of SYSTAT Statistical package Ver.12.0. All quantitative variables neurological observations (neuromuscular observation/body temperature/body weights) and T3, T4, TSH was tested for normality (Shapiro-Wilk test) and homogeneity of variances (Levene’s test) within the group before performing a one-factor ANOVA modelling by treatment groups. Non-optimal (nonnormal or heteroschedastic) data was transformed, before ANOVA was performed. Comparison of means between treatment groups and control group was done using Dunnett’s test when the overall treatment, ‘F’ test was found significant.
For two groups, the comparisons of the mean between treatment and control group was done using‘t’ test.
Descriptive statistics (Mean, SD & Numbers) was presented by Treatment group and Day. All hypothesis testing were carried out at the 5% (2-sided) significance level. Significant differences are designated throughout the report as below:
*: Statistically significant difference from the control group at p < 0.05
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
There were no clinical signs and mortalities observed throughout the treatment and recovery period in either sex at all the doses tested. Light orange coloured faeces were observed at 111 and 333 mg/kg bwt/day doses and dark orange colour faeces were observed at 1000 mg/kg bwt/day doses in both sexes. This could be due to physical nature of the test item
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Significantly lower body weight gain was observed from Days 22-29 at 333 mg/kg/day and during Days 64-71 at 1000 mg/kg/day in males and significantly higher body weight gain during Days 57-64 at 111 mg/kg/day in females. Significantly higher body weight gain was observed during Days 85-90, 90-97 and 90-118 at 1000 mg/kg/day recovery males and during Days 57-64 and 104-111 at 1000 mg/kg/day recovery females
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
The food consumption was significantly lower in males during Days 1-8 at 333 and during Days 15-22 at 111 and 333 mg/kg/day doses.
In females, the food consumption was significantly lower during Days 15-22 at all the doses in main toxicity groups and during Days 43-50 at 1000 mg/kg/day recovery females. Significantly higher food consumption was observed during Days 64-71 at all the doses in main toxicity groups and during Days 71-77 and 111-118 at 1000 mg/kg/day recovery females.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Endocrine findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
<= 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
behaviour (functional findings)
body weight and weight gain
clinical biochemistry
clinical signs
food consumption and compound intake
gross pathology
haematology
histopathology: non-neoplastic
mortality
ophthalmological examination
organ weights and organ / body weight ratios
urinalysis
Key result
Critical effects observed:
no
TABLE 2.      Summary of Clinical Signs 





































































Observation Type: All Types



 



Male



 



 



Female



 



From Day 1 (Start Date) to 91 (End Date)



G1


0 mg/kg/day



G2


111 mg/kg/day



G3


333 mg/kg/day



G4


1000 mg/kg/day



G1


0 mg/kg/day



G2


111 mg/kg/day



G3


333 mg/kg/day



G4


1000 mg/kg/day



Normal


Number of Animals Affected 



10



10



10



10



10



10



10



10



First to Last seen 



1 - 91



1 - 1



1 - 1



1 - 1



1 - 91



1 - 1



1 - 1



1 - 1



Faecal colour


Number of Animals Affected 



0



10



10



10



0



10



10



10



First to Last seen 



-



2 - 91



2 - 91



2 - 91



-



2 - 91



2 - 91



2 - 91



TABLE 2 contd. Summary of Clinical Signs 













































Observation Type: All Types



Male



Female



From Day 1 (Start Date) to 119 (End Date)



G1R


0 mg/kg/day



G4R


1000 mg/kg/day



G1R


0 mg/kg/day



G4R


1000 mg/kg/day



Normal


Number of Animals Affected 



5



5



 


5



5



First to Last seen 



1 - 119



1 - 119



1 - 119



1, 92 - 119



Faecal colour


Number of Animals Affected 



 


0



5



0



5



First to Last seen 



-



2 - 91



-



2 - 91



TABLE 3.      Summary of Ophthalmological Findings 
































Observation Type: All Types



 



Male



 



 



Female



 



From Day -5 (Start Date) to 90 (End Date)



G1


0 mg/kg/day



      G2                G3


     111              333


mg/kg/day       mg/kg/day



G4


1000 mg/kg/day



G1


0 mg/kg/day



      G2                G3


     111              333


mg/kg/day       mg/kg/day



G4


1000 mg/kg/day



Normal



10



      10                10



10



10



      10                10



10



TABLE 3 contd. Summary of Ophthalmological Findings















From Day -5 (Start Date) to 119 (End Date)



      G1R             G4R


        0                1000


mg/kg/day      mg/kg/day



     G1R             G4R


        0               1000


mg/kg/day      mg/kg/day



Normal



        5                   5



        5                  5



TABLE 4.    Summary of Functional Observation Battery - Males













































































































































 



Group



G1



G2



G3



G4



Parameters



Dose



0



111



333



1000



 



(mg/kg/day )


No. of rats



10



10



10



10



Neurobehavioral Observations


Home Cage Observations


Posture


Sitting normally, feet tucked in



 


 


 



0



1



0



0



Sitting or Standing alert, watching



 



10



8



8



6



Rears



 



0



1



2



4



 


Abnormal Vocalizations


Absent



 


 


 



10



10



10



10



 


Tremors Absent



 


 


 



10



10



10



10



 


Convulsions - Clonic Movement


Absent



 


 


 



10



10



10



10



 


Convulsions - Tonic Movement


Absent



               


 


 



10



 


10



 


10



 


10



 


Handling Observations


Ease of Removing from Cage


Easy (minimally avoids capture but is not aggressive) 



               


 


  



10



 


10



 


10



 


10



 


Handling Reactivity


Moderately low (slight resistance)



 


 


 



10



10



10



10



 


Palpebral Closure


Eyelids wide open i.e., Normal



 


 


 



10



10



10



10



 


Eye Examination


Normal



 


 


 



10



10



10



10



 


Piloerection


Absent



 


 


 



10



10



10



10



 


Lacrimation


Absent



 


 


 



10



10



10



10



 


Salivation


None (no external salivation observed)



 


 



10



10



10



10



 


 






























































































































 



Group



G1



G2



G3



G4



Parameters



Dose



0



111



333



1000



 



(mg/kg/day )


No. of rats



10



10



10



10



 


Skin/Fur Examination


Normal



 


 


 



10



10



10



10



 


Perineum Wetness


Absent



 


 


 



10



10



10



10



 


Respiration


Normal



 


 


 



10



10



10



10



 


Muscle Tone


Moderate (animal is neither very relaxed nor tense; Normal muscle tone)



 


 


 



10



10



10



10



 


Extensor Thrust Response


Moderate (clearly detectable; Normal)



 


 


 



10



10



10



10



 


Open Field Observations


Gait


Normal gait



 


 


 


 



10



10



10



10



 


Posture


Normal posture



 


 


 



10



10



10



10



 


Tremors Absent



 


 


 



10



10



10



10



 


Mobility Score No impairment



 


 


 



10



10



10



10



 


Arousal Level


Alert (some exploratory movements; normal)



 


 


 



10



10



10



10



 


Clonic Movement


Absent



 


 


 



10



10



10



10



 


Tonic Movement


Absent



 


 



10



10



10



10



TABLE 4 contd. Summary of Functional Observation Battery - Males













































































































































 



Group



G1



G2



G3



G4



Parameters



Dose



0



111



333



1000



 



(mg/kg/day )


No. of rats



10



10



10



10



 


Stereotypic Behavior


Absent



 


 


 



10



10



10



10



 


Bizarre Behavior


Absent



 


 


 



10



10



10



10



 


Urination Absent



 


 


 



8



8



7



7



Normal



 



2



2



3



3



 


Defecation


Absent



 



7



8



7



8



Normal



 



3



2



3



2



 


Abnormal Vocalization


Absent



 


 


 



10



10



10



10



 


Rearing



 



79



75



87



78



Sensory Reactivity Measurements


Approach Response


Normal response (approaches towards the object)



 



10



10



10



10



Touch Response


Normal Response (spontaneously lifts tail when touched)



 



10



10



10



10



Click Response


Normal Response (clear movements of the head, neck and ears)



 



10



10



10



10



Tail-Pinch Response


Normal Response (Flinches, moves rapidly and/or vocalizes)



 



10



10



10



10



Pupil Response


Normal



 



10



10



10



10



Aerial Righting Reflex


Normal



 



10



10



10



10



 


 














































































































 



Group



G1



G2



G3



G4



Parameters



Dose



0



111



333



1000



 



(mg/kg/day )


No. of rats



10



10



10



10



Physiological Observation


Body Temperature (°C)



Mean



37.55



37.66



37.86



37.58



 



SD



0.58



0.41



0.28



0.50



Body Weight (g)



Mean



538.50



504.77



516.38



518.73



 



SD



49.90



57.70



42.90



38.18



Hindlimbs Footsplay (mm)


Average



Mean



80.70



80.30



84.40



64.17*



 



SD



11.88



12.77



7.82



12.07



Grip Strength (gf)


Forelimbs Grip Strength


Average



Mean



1056.60



1050.43



1051.30



1055.83



 



SD



21.83



18.48



11.88



19.60



Hindlimbs Grip Strength


Average



Mean



832.37



842.43



829.10



832.43



 



SD



19.75



24.51



20.25



20.33



*: Statistically significant difference from the control group at p < 0.05


TABLE 4 contd. Summary of Functional Observation Battery - Males 













































































































































































Group



 


 



Ambulatory


(s)



Ambulatory


(s)



Ambulatory


(s)



Ambulatory


(s)



Stereotypic


(s)



Stereotypic


(s)



Stereotypic


(s)



Stereotypic



Dose



(s)



(mg/kg/day)



 



1



2



3



Total



1



2



3



Total



G1



Mean


SD


N



562.48


39.85


10



496.00


87.62


10



473.16


67.89


10



1531.64


173.34


10



27.81


32.61


10



57.76


47.67


10



52.81


26.99


10



138.38



0



76.38



 



10



 



 



 



 



 



 



 



 



 



 



G2



Mean


SD



547.48


43.20



466.52


71.06



440.67


70.16



1454.67


163.88



30.53


21.69



76.73


42.33



92.52


55.78



199.78



111



99.14



 



N



10



10



10



10



10



10



10



10



 



 



 



 



 



 



 



 



 



 



G3



Mean


SD



577.55


20.35



527.13


57.62



451.99


98.02



1556.67


139.04



15.25


15.29



41.06


38.03



66.07


38.71



122.38



333



66.33



 



N



10



10



10



10



10



10



10



10



 



 



 



 



 



 



 



 



 



 



G4



Mean


SD



572.77


22.98



514.99


33.43



441.96


62.63



1529.72


70.77



15.01


12.63



42.36


23.84



78.80


35.14



136.17



1000



44.04



 



N



10



10



10



10



10



10



10



10























































































































































































Group



 


 



Resting


(s)



Resting


(s)



Resting


(s)



Resting


(s)



Distance


(cm)



Distance


(cm)



Distance


(cm)



Distance



Dose



(cm)



(mg/kg/day)



 



1



2



3



Total



1



2



3



Total



G1



Mean


SD



9.71


9.23



46.24


47.39



73.48


55.35



129.43


99.64



1564.47


298.45



856.78


283.03



737.11


189.83



3158.36



0



720.22



 



N



10



10



10



10



10



10



10



10



 



 



 



 



 



 



 



 



 



 



G2



Mean


SD



21.99 25.19



56.75 43.75



66.79 36.01



145.53


98.51



1207.77*


351.84



746.01


235.08



628.11


211.42



2581.89



111



743.02



 



N



10



10



10



10



10



10



10



10



 



 



 



 



 



 



 



 



 



 



G3



Mean


SD



7.20 6.73



31.81 21.33



81.94 71.96



120.95


86.23



1742.36


378.46



1009.42


288.97



691.11


253.88



3442.89



333



828.09



 



N



10



10



10



10



10



10



10



10



 



 



 



 



 



 



 



 



 



 



G4



Mean


SD



12.22 10.82



42.65 20.82



79.12 43.05



133.99


48.11



1410.36


232.95



831.16


144.06



624.91


116.49



2866.43



1000



416.98



 



N



10



10



10



10



10



10



10



10



 


*: Statistically significant difference from the control group at p < 0.05


 


 






































































































 



Group



G1R



G4R



Parameters



Dose



0



1000



 



(mg/kg/day )


No. of rats



5



5



Neurobehavioral Observations


Home Cage Observations


Posture


Sitting or Standing alert, watching



 


 


 



5



5



 


Abnormal Vocalizations


Absent



 


 


 



5



5



 


Tremors Absent



 


 


 



5



5



 


Convulsions - Clonic Movement


Absent



 


 


 



5



5



 


Convulsions - Tonic Movement


Absent



               


 


 



5



 


5



 


Handling Observations


Ease of Removing from Cage


Easy (minimally avoids capture but is not aggressive) 



               


 


  



5



 


5



 


Handling Reactivity


Moderately low (slight resistance)



 


 


 



5



5



 


Palpebral Closure


Eyelids wide open i.e., Normal



 


 


 



5



5



 


Eye Examination


Normal



 


 


 



5



5



 


Piloerection


Absent



 


 


 



5



5



 


Lacrimation


Absent



 


 


 



5



5



 


Salivation


None (no external salivation observed)



 


 


 



5



5



 


Skin/Fur Examination


Normal



 


 



5



5



 


 


 
































































































 



Group



G1R



G4R



Parameters



Dose



0



1000



 



(mg/kg/day )


No. of rats



5



5



 


Perineum Wetness


Absent



 


 


 



5



5



 


Respiration


Normal



 


 


 



5



5



 


Muscle Tone


Moderate (animal is neither very relaxed nor tense; Normal muscle tone)



 


  



5



5



 


Extensor Thrust Response


Moderate (clearly detectable; Normal)



 


 


 



5



5



 


Open Field Observations


Gait


Normal gait



 


 


 


 



5



5



 


Posture


Normal posture



 


 


 



5



5



 


Tremors Absent



 


 


 



5



5



 


Mobility Score No impairment



 


 


 



5



5



 


Arousal Level


Alert (some exploratory movements; normal)



 


 


 



5



5



 


Clonic Movement


Absent



 


 


 



5



5



 


Tonic Movement


Absent



 


 


 



5



5



 


Stereotypic Behavior


Absent



 


 



5



5



 


 


 






































































































 



Group



G1R



G4R



Parameters



Dose



0



1000



 



(mg/kg/day )


No. of rats



5



5



 


Bizarre Behavior


Absent



 


 


 



5



5



 


Urination Absent



 


 


 



2



2



Normal



 



3



3



 


Defecation


Absent



 



1



1



Normal



 



4



4



 


Abnormal Vocalization


Absent



 


 


 



5



5



 


Rearing



 



33



30



Sensory Reactivity Measurements


Approach Response


Normal response (approaches towards the object)



 



5



5



Touch Response


Normal Response (spontaneously lifts tail when touched)



 



5



5



Click Response


Normal Response (clear movements of the head, neck and ears)



 



5



5



Tail-Pinch Response


Normal Response (Flinches, moves rapidly and/or vocalizes)



 



5



5



Pupil Response


Normal



 



5



5



Aerial Righting Reflex


Normal



 



5



5



 


 




















































































 



Group



G1R



G4R



Parameters



Dose



0



1000



 



(mg/kg/day )


No. of rats



5



5



Physiological Observation


Body Temperature (°C)



Mean



37.40



37.52



 



SD



0.61



0.55



Body Weight (g)



Mean



555.67



590.44



 



SD



40.61



64.78



Hindlimbs Footsplay (mm)


Average



Mean



62.33



74.87



 



SD



9.46



22.12



Grip Strength (gf)


Forelimbs Grip Strength


Average



Mean



1103.13



1068.00



 



SD



49.30



31.47



Hindlimbs Grip Strength


Average



Mean



776.40



836.80



 



SD



102.26



23.67



 


 























































































Group


Dose



 


 



Ambulatory


(s)



Ambulatory


(s)



Ambulatory


(s)



Ambulatory


(s)



Stereotypic


(s)



Stereotypic


(s)



Stereotypic


(s)



Stereotypic



(s)



(mg/kg/day)



 



1



2



3



Total



1



2



3



Total



G1R



Mean



518.76



379.62



313.86



1212.24



55.62



113.76



137.26



306.64



0


 



SD


N



33.48


5



61.99


5



102.78


5



160.87


5



34.84


5



35.57


5



65.65


5



113.65



5



 


G4R


1000



 


Mean


SD



 


566.16


34.47



 


458.08


142.91



 


315.38


92.22



 


1339.62


198.39



 


15.98*


14.36



 


57.46


45.63



 


96.52


29.14



 


169.96



69.72



 



N



5



5



5



5



5



5



5



5



 






















































































Group



 


 



Resting


(s)



Resting


(s)



Resting


(s)



Resting


(s)



Distance


(cm)



Distance


(cm)



Distance


(cm)



Distance



Dose



(cm)



(mg/kg/day)



 



1



2



3



Total



1



2



3



Total



G1R



Mean


SD



25.62


14.49



106.62


33.33



148.88


85.80



281.12


104.81



1177.02


281.74



534.03


107.09



392.82


187.51



2103.87



0



440.71



 



N



5



5



5



5



5



5



5



5



 



 


Mean


SD


N



 


17.86


20.34


5



 


84.46


103.72


5



 


188.10


96.49


5



 


290.42


161.83


5



 


1445.34


187.64


5



 


796.46


283.20


5



 


480.82


222.72


5



 



G4R



2722.63



1000



479.13



 



5



 


*: Statistically significant difference from the control group at p < 0.05


TABLE 5. Summary of Functional Observation Battery - Females 













































































































































 



Group



G1



G2



G3



G4



Parameters



Dose



0



111



333



1000



 



(mg/kg/day )


No. of rats



10



10



10



10



Neurobehavioral Observations


Home Cage Observations


Posture


Sitting normally, feet tucked in



 


 


 



0



2



0



0



Sitting or Standing alert, watching



 



6



7



7



7



Rears



 



4



1



3



3



 


Abnormal Vocalizations


Absent



 


 


 



10



10



10



10



 


Tremors Absent



 


 


 



10



10



10



10



 


Convulsions - Clonic Movement


Absent



 


 


 



10



10



10



10



 


Convulsions - Tonic Movement


Absent



               


 


 



10



 


10



 


10



 


10



 


Handling Observations


Ease of Removing from Cage


Easy (minimally avoids capture but is not aggressive) 



               


 


  



10



 


10



 


10



 


10



 


Handling Reactivity


Moderately low (slight resistance)



 


 


 



10



10



10



10



 


Palpebral Closure


Eyelids wide open i.e., Normal



 


 


 



10



10



10



10



 


Eye Examination


Normal



 


 


 



10



10



10



10



 


Piloerection


Absent



 


 


 



10



10



10



10



 


Lacrimation


Absent



 


 


 



10



10



10



10



 


Salivation


None (no external salivation observed)



 


 



10



10



10



10



 


 






























































































































 



Group



G1



G2



G3



G4



Parameters



Dose



0



111



333



1000



 



(mg/kg/day )


No. of rats



10



10



10



10



 


Skin/Fur Examination


Normal



 


 


 



10



10



10



10



 


Perineum Wetness


Absent



 


 


 



10



10



10



10



 


Respiration


Normal



 


 


 



10



10



10



10



 


Muscle Tone


Moderate (animal is neither very relaxed nor tense; Normal muscle tone)



 


  



10



10



10



10



 


Extensor Thrust Response


Moderate (clearly detectable; Normal)



 


 


 



10



10



10



10



 


Open Field Observations


Gait


Normal gait



 


 


 


 



10



10



10



10



 


Posture


Normal posture



 


 


 



10



10



10



10



 


Tremors Absent



 


 


 



10



10



10



10



 


Mobility Score No impairment



 


 


 



10



10



10



10



 


Arousal Level


Alert (some exploratory movements; normal)



 


 


 



10



10



10



10



 


Clonic Movement


Absent



 


 


 



10



10



10



10



 


Tonic Movement


Absent



 


 



10



10



10



10



TABLE 5 contd. Summary of Functional Observation Battery - Females 













































































































































 



Group



G1



G2



G3



G4



Parameters



Dose



0



111



333



1000



 



(mg/kg/day )


No. of rats



10



10



10



10



 


Stereotypic Behavior


Absent



 


 


 



10



10



10



10



 


Bizarre Behavior


Absent



 


 


 



10



10



10



10



 


Urination Absent



 


 


 



8



6



8



10



Normal



 



2



4



2



0



 


Defecation


Absent



 



7



7



9



7



Normal



 



3



3



1



3



 


Abnormal Vocalization


Absent



 


 


 



10



10



10



10



 


Rearing



 



81



101



99



88



Sensory Reactivity Measurements


Approach Response


Normal response (approaches towards the object)



 



10



10



10



10



Touch Response


Normal Response (spontaneously lifts tail when touched)



 



10



10



10



10



Click Response


Normal Response (clear movements of the head, neck and ears)



 



10



10



10



10



Tail-Pinch Response


Normal Response (Flinches, moves rapidly and/or vocalizes)



 



10



10



10



10



Pupil Response


Normal



 



10



10



10



10



Aerial Righting Reflex


Normal



 



10



10



10



10



 














































































































 



Group



G1



G2



G3



G4



Parameters



Dose



0



111



333



1000



 



(mg/kg/day )


No. of rats



10



10



10



10



Physiological Observation


Body Temperature (°C)



Mean



37.61



37.76



37.68



37.79



 



SD



0.71



0.46



0.58



0.53



Body Weight (g)



Mean



299.72



307.34



296.45



307.67



 



SD



22.55



25.46



19.18



27.51



Hindlimbs Footsplay (mm)


Average



Mean



63.87



67.67



70.53



74.60



 



SD



12.85



10.88



12.01



7.54



Grip Strength (gf)


Forelimbs Grip Strength


Average



Mean



947.90



951.23



950.20



950.40



 



SD



21.48



18.06



17.12



21.27



Hindlimbs Grip Strength


Average



Mean



758.63



760.00



754.77



763.77



 



SD



16.38



15.35



16.53



23.18



TABLE 5 contd. Summary of Functional Observation Battery - Females 






























































































































































Group


Dose


(mg/kg/day)



 


 


 



Ambulatory


(s)


1



Ambulatory


(s)


2



Ambulatory


(s)


3



Ambulatory


(s)


Total



Stereotypic


(s)


1



Stereotypic


(s)


2



Stereotypic


(s)


3



Stereotypic



(s)



Total



G1


0


 



Mean


SD


N



580.56


9.73


10



539.54


43.98


10



490.11


62.84


10



1610.21


103.97


10



10.25


5.81


10



33.25


31.16


10



57.97


35.43


10



101.47



58.76



10



 



 



 



 



 



 



 



 



 



 



G2



Mean



587.18



566.80



500.52



1654.50



7.96



22.78



54.54



85.28



111


 



SD


N



9.40


10



30.44


10



65.62


10



80.05


10



7.92


10



25.53


10



41.34


10



51.89



10



 



 



 



 



 



 



 



 



 



 



G3 333



Mean


SD



585.38


8.05



549.03


41.21



467.99


140.35



1602.40


160.70



8.36 2.64



25.29 23.24



37.62 31.45



71.27



45.03



 



N



10



10



10



10



10



10



10



10



 



 



 



 



 



 



 



 



 



 



G4 1000



Mean


SD



588.20


7.70



570.03


17.37



518.42


61.46



1676.65


56.57



6.57


6.02



17.50


14.53



39.14


26.23



63.21



24.21



 



N



10



10



10



10



10



10



10



10



 






















































































































































































Group



 


 



Resting


(s)



Resting


(s)



Resting


(s)



Resting


(s)



Distance


(cm)



Distance


(cm)



Distance


(cm)



Distance



Dose



(cm)



(mg/kg/day)



 



1



2



3



Total



1



2



3



Total



G1



Mean


SD



9.19


6.67



27.21


19.83



51.92


35.39



88.32


53.92



1838.31


271.63



1186.30


234.07



956.16


229.56



3980.76



0



653.60



 



N



10



10



10



10



10



10



10



10



 



 



 



 



 



 



 



 



 



 



G2



Mean


SD



4.86


2.64



10.42


8.01



44.93


37.05



60.21


37.41



1785.44


291.63



1358.14


412.32



1028.15


395.26



4171.73



111



976.26



 



N



10



10



10



10



10



10



10



10



 



 



 



 



 



 



 



 



 



 



G3



Mean


SD



6.26 7.10



25.68 24.27



94.39


143.33



126.33


158.22



1776.21


291.05



1313.50


537.21



966.97


225.28



4056.68



333



823.08



 



N



10



10



10



10



10



10



10



10



 



 



 



 



 



 



 



 



 



 



G4



Mean


SD



5.23 2.90



12.47


7.85



42.44 46.07



60.14 44.50



1913.65


275.02



1227.80


256.27



1095.84


305.09



4237.28



1000



744.67



 



N



10



10



10



10



10



10



10



10



 


 






































































































 



Group



G1R



G4R



Parameters



Dose



0



1000



 



(mg/kg/day )


No. of rats



5



5



Neurobehavioral Observations


Home Cage Observations


Posture


Sitting or Standing alert, watching



 


 


 



4



3



Rears



 



1



2



 


Abnormal Vocalizations


Absent



 


 


 



5



5



 


Tremors Absent



 


 


 



5



5



 


Convulsions - Clonic Movement


Absent



 


 


 



5



5



 


Convulsions - Tonic Movement


Absent



               


 


 



5



 


5



 


Handling Observations


Ease of Removing from Cage


Easy (minimally avoids capture but is not aggressive) 



               


 


  



5



 


5



 


Handling Reactivity


Moderately low (slight resistance)



 


 


 



5



5



 


Palpebral Closure


Eyelids wide open i.e., Normal



 


 


 



5



5



 


Eye Examination


Normal



 


 


 



5



5



 


Piloerection


Absent



 


 


 



5



5



 


Lacrimation


Absent



 


 


 



5



5



 


Salivation


None (no external salivation observed)



 


 



5



5



 


 


 


 
































































































 



Group



G1R



G4R



Parameters



Dose



0



1000



 



(mg/kg/day )


No. of rats



5



5



 


Skin/Fur Examination


Normal



 


 


 



5



5



 


Perineum Wetness


Absent



 


 


 



5



5



 


Respiration


Normal



 


 


 



5



5



 


Muscle Tone


Moderate (animal is neither very relaxed nor tense; Normal muscle tone)



 


  



5



5



 


Extensor Thrust Response


Moderate (clearly detectable; Normal)



 


 


 



5



5



 


Open Field Observations


Gait


Normal gait



 


 


 


 



5



5



 


Posture


Normal posture



 


 


 



5



5



 


Tremors Absent



 


 


 



5



5



 


Mobility Score No impairment



 


 


 



5



5



 


Arousal Level


Alert (some exploratory movements; normal)



 


 


 



5



5



 


Clonic Movement


Absent



 


 


 



5



5



 


Tonic Movement


Absent



 


 



5



5



 


 


 


 


 


 












































































































 



Group



G1R



G4R



Parameters



Dose



0



1000



 



(mg/kg/day )


No. of rats



5



5



 


Stereotypic Behavior


Absent



 


 


 



5



5



 


Bizarre Behavior


Absent



 


 


 



5



5



 


Urination Absent



 


 


 



3



4



Normal



 



2



1



 


Defecation


Absent



 



2



2



Normal



 



3



3



 


Abnormal Vocalization


Absent



 


 


 



5



5



 


Rearing



 



70



65



Sensory Reactivity Measurements


Approach Response


Normal response (approaches towards the object)



 



5



5



Touch Response


Normal Response (spontaneously lifts tail when touched)



 



5



5



Click Response


Normal Response (clear movements of the head, neck and ears)



 



5



5



Tail-Pinch Response


Normal Response (Flinches, moves rapidly and/or vocalizes)



 



5



5



Pupil Response


Normal



 



5



5



Aerial Righting Reflex


Normal



 



5



5



 


 




















































































 



Group



G1R



G4R



Parameters



Dose



0



1000



 



(mg/kg/day )


No. of rats



5



5



Physiological Observation


Body Temperature (°C)



Mean



37.62



37.54



 



SD



0.72



0.61



Body Weight (g)



Mean



317.27



332.23



 



SD



21.16



27.29



Hindlimbs Footsplay (mm)


Average



Mean



59.67



61.13



 



SD



8.92



18.04



Grip Strength (gf)


Forelimbs Grip Strength


Average



Mean



959.87



967.00



 



SD



21.90



9.45



Hindlimbs Grip Strength


Average



Mean



776.87



755.80



 



SD



8.37



29.40



TABLE 5 contd. Summary of Functional Observation Battery - Females


































































































Group



 



Ambulatory



Ambulatory



Ambulatory



Ambulatory



Stereotypic



Stereotypic



Stereotypic



Stereotypic



Dose (mg/kg/day)



 


 



(s)


1



(s)


2



(s)


3



(s) Total



(s)


1



(s)


2



(s)


3



(s)



Total



G1R



Mean



574.32



489.90



378.70



1442.92



11.64



56.02



94.34



162.00



0


 



SD


N



7.81


5



48.21


5



83.12


5



118.25


5



2.82


5



41.81


5



43.51


5



72.46



5



 



 



 



 



 



 



 



 



 



 



G4R


1000



Mean


SD



588.42*


10.95



509.68


74.72



467.60


80.69



1565.70


145.94



7.54


7.26



37.02


38.02



68.00


49.62



112.56



72.33



 



N



5



5



5



5



5



5



5



5



 




























































































Group



 


 



Resting


(s)



Resting


(s)



Resting    Resting


    (s)            (s)



Distance


(cm)



Distance


(cm)



Distance


(cm)



Distance



Dose



(cm)



(mg/kg/day)



 



1



2



Total


195.04


71.26         64.81



240.77



279.15



3



Total



G1R



Mean


SD



14.04


6.28



54.08


21.21



741.31


287.32



3622.98



0



687.48



 



N



5



5



     5               5



5



5



5



5



 



 



 



 



                



 



 



 



 



G4R



Mean


SD



4.04*


4.11



53.30


41.51



64.40        121.74


46.19         88.53



2081.03


451.03



1322.80


499.57



1060.48


537.62



4464.31



1000



1404.21



 



N



5



5



     5               5



5



5



5



5



*: Statistically significant difference from the control group at p < 0.05


TABLE 6.      Summary of Body Weights (g) - Males













































































































Sex: Male


Day(s) Relative to Start Date



G1


0 mg/kg/day



G2


111 mg/kg/day



G3


333 mg/kg/day



G4


1000 mg/kg/day



Body


Weight


(g)



1 [a]



Mean


SD


N



218.08   14.07  


10       



220.54   14.92  


10       



218.80   12.99  


10       



217.74   16.30  


10       



8 [a]



Mean


SD


N



277.13   17.26  


10       



275.45   20.10  


10       



276.74   14.06  


10       



278.07   19.28  


10       



15 [a]



Mean


SD


N



324.70   23.99  


10       



313.65   28.16  


10       



320.05   17.34  


10       



321.51   20.76  


10       



22 [a]



Mean


SD


N



357.34   32.77  


10       



345.26   33.93  


10       



351.54   20.79  


10       



352.58   25.08  


10       



29 [a]



Mean


SD


N



386.64   37.82  


10       



370.53   37.61  


10       



374.07   23.87  


10       



380.39   24.28  


10       



36 [a]



Mean


SD


N



413.80   41.11  


10       



394.37   42.01  


10       



397.91   27.13  


10       



398.95   26.52  


10       



43 [a]



Mean


SD


N



436.51   40.21  


10       



417.85   45.34  


10       



423.64   28.30  


10       



425.45   28.89  


10       



50 [a]



Mean


SD


N



455.15   41.42  


10       



437.33   47.17  


10       



443.68   30.34  


10       



449.49   31.90  


10       



57 [a]



Mean


SD


N



474.55   43.08  


10       



452.63   50.71  


10       



462.34   32.89  


10       



462.07   32.70  


10       



64 [a]



Mean


SD


N



493.42   44.76  


10       



465.77   51.27  


10       



475.57   35.96  


10       



482.16   32.91  


10       



71 [a]



Mean SD


N



517.22   48.83  


10       



484.75   53.08  


10       



492.49   40.32  


10       



497.64   35.47  


10       



77 [a]



Mean


SD


N



523.90   48.88  


10       



493.83   54.56  


10       



503.73   41.62  


10       



505.86   33.72  


10       



[a] Anova & Dunnett






























Sex: Male


Day(s) Relative to Start Date



G1


0 mg/kg/day



G2


111 mg/kg/day



G3


333 mg/kg/day



G4


1000 mg/kg/day



Body


Weight


(g)



85 [a]



Mean


SD


N



538.82   50.88  


10       



507.26   56.70  


10       



514.63   40.20  


10       



521.97   39.20  


10       



90 [a]



Mean


SD


N



538.16   48.63  


10       



506.79   54.33  


10       



519.48   39.13  


10       



523.66   39.81  


10       



[a] Anova & Dunnett


TABLE 6 contd. Summary of Body Weights (g) - Males








































































Sex: Male


Da



y(s) Relative to Start Date



G1R


0 mg/kg/day



G4R


1000 mg/kg/day



Body


Weight


(g)



1 [p]



Mean


SD


N



221.02  


19.58  


5       



218.50  


18.56  


5       



8 [p]



Mean


SD


N



272.49  


22.71  


5       



278.52  


11.47  


5       



15 [p]



Mean


SD


N



317.80  


26.64  


5       



327.93  


12.56  


5       



22 [p]



Mean


SD


N



350.26  


27.96  


5       



363.61  


16.86  


5       



29 [p]



Mean


SD


N



377.76  


24.61  


5       



389.58  


20.27  


5       



36 [p]



Mean


SD


N



401.23  


36.12  


5       



416.43  


22.48  


5       



43 [p]



Mean


SD


N



423.69  


35.36  


5       



441.87  


34.00  


5       



50 [p]



Mean


SD


N



443.31  


35.95  


5       



464.16  


38.13  


5       



57 [p]



Mean


SD


N



460.31  


37.92  


5       



477.88  


40.87  


5       



64 [p]



Mean


SD


N



478.10  


39.77  


5       



500.82  


46.14  


5       



[p] T-Test: [G1RvsG4R]





























































Sex: Male


Da



y(s) Relative to Start Date



G1R


0 mg/kg/day



G4R


1000 mg/kg/day



Body


Weight


(g)



71 [p]



Mean


SD


N



495.97  


44.86  


5       



513.65  


46.56  


5       



77 [p]



Mean


SD


N



506.80  


40.67  


5       



523.71  


47.60  


5       



85 [p]



Mean


SD


N



521.03  


36.89  


5       



533.97  


55.30  


5       



90 [p]



Mean


SD


N



519.20  


36.56  


5       



540.16  


54.70  


5       



97 [p]



Mean


SD


N



526.24  


30.84  


5       



561.16  


51.99  


5       



104 [p]



Mean


SD


N



537.57  


38.08  


5       



582.51  


54.74  


5       



111 [p]



Mean


SD


N



540.87  


41.76  


5       



585.11  


59.04  


5       



118 [p]



Mean


SD


N



551.30  


41.60  


5       



594.92  


62.02  


5       



[p] T-Test: [G1RvsG4R]


TABLE 7.     Summary of Body Weights (g) - Females 













































































































Sex: Female


Day(s) Relative to Start Date



G1


0 mg/kg/day



G2


111 mg/kg/day



G3


333 mg/kg/day



G4


1000 mg/kg/day



Body


Weight


(g)



1 [a]



Mean


SD


N



172.08   10.16  


10       



172.02  


7.86   10       



172.96  


9.79   10       



173.15  


9.66   10       



8 [a]



Mean


SD


N



196.82   10.70  


10       



197.77   13.96  


10       



198.57  


9.60   10       



198.02   11.43  


10       



15 [a]



Mean


SD


N



212.31   14.04  


10       



216.29   13.49  


10       



212.78   14.71  


10       



213.99   15.68  


10       



22 [a]



Mean


SD


N



226.06   18.40  


10       



228.50   16.36  


10       



231.73   17.81  


10       



230.27   12.92  


10       



29 [a]



Mean


SD


N



243.52   18.19  


10       



242.32   20.46  


10       



239.73   14.78  


10       



246.53   16.41  


10       



36 [a]



Mean


SD


N



254.04   17.59  


10       



255.20   22.73  


10       



251.97   17.06  


10       



260.70   18.95  


10       



43 [a]



Mean


SD


N



257.81   18.07  


10       



261.73   24.36  


10       



260.23   16.31  


10       



265.95   22.02  


10       



50 [a]



Mean


SD


N



265.97   23.38  


10       



268.53   21.76  


10       



269.04   17.85  


10       



273.33   19.03  


10       



57 [a]



Mean


SD


N



279.85   23.84  


10       



275.92   22.77  


10       



276.82   20.23  


10       



286.57   21.62  


10       



64 [a]



Mean


SD


N



284.67   25.48  


10       



289.40   22.78  


10       



282.16   17.18  


10       



290.01   23.48  


10       



71 [a]



Mean SD


N



286.41   22.56  


10       



295.32   24.30  


10       



289.17   17.53  


10       



300.62   28.02  


10       



77 [a]



Mean


SD


N



292.72   24.07  


10       



296.91   25.79  


10       



293.26   20.10  


10       



304.96   25.68  


10       



[a] Anova & Dunnett






























Sex: Female


Day(s) Relative to Start Date



G1


0 mg/kg/day



G2


111 mg/kg/day



G3


333 mg/kg/day



G4


1000 mg/kg/day



Body


Weight


(g)



85 [a]



Mean


SD


N



298.17   23.87  


10       



303.26   24.33  


10       



293.32   15.93  


10       



308.37   26.22  


10       



90 [a]



Mean


SD


N



296.83   24.63  


10       



304.10   26.35  


10       



296.94   17.12  


10       



309.35   29.99  


10       



[a] Anova & Dunnett


TABLE 7 contd. Summary of Body Weights (g) - Females







































































Sex: Female


Day(s) Relative to Start Date



G1R


0 mg/kg/day



G4R


1000 mg/kg/day



Body


Weight


(g)



1 [p]



Mean


SD


N



174.99   9.03  


5       



172.75   9.34  


5       



8 [p]



Mean


SD


N



200.39   8.61  


5       



198.83  


16.22  


5       



15 [p]



Mean


SD


N



221.36   9.33  


5       



219.11  


16.04  


5       



22 [p]



Mean


SD


N



236.17  


15.63  


5       



236.14  


20.70  


5       



29 [p]



Mean


SD


N



245.95  


11.50  


5       



254.24  


19.94  


5       



36 [p]



Mean


SD


N



261.74  


14.01  


5       



265.01  


18.92  


5       



43 [p]



Mean


SD


N



265.04  


15.70  


5       



271.90  


21.29  


5       



50 [p]



Mean


SD


N



273.64  


14.74  


5       



281.20  


23.32  


5       



57 [p]



Mean


SD


N



287.58  


15.43  


5       



289.35  


25.88  


5       



64 [p]



Mean


SD


N



289.42  


15.55  


5       



297.77  


27.16  


5       



[p] T-Test: [G1RvsG4R]




























































Sex: Female


Day(s) Relative to Start Date



G1R


0 mg/kg/day



G4R


1000 mg/kg/day



Body


Weight


(g)



71 [p]



Mean


SD


N



298.17  


19.97  


5       



303.90  


26.63  


5       



77 [p]



Mean


SD


N



300.72  


19.80  


5       



311.95  


24.64  


5       



85 [p]



Mean


SD


N



304.10  


18.82  


5       



318.13  


26.27  


5       



90 [p]



Mean


SD


N



302.68  


20.51  


5       



317.65  


27.65  


5       



97 [p1]



Mean


SD


N



314.60  


19.98  


5       



325.45  


29.24  


5       



104 [p1]



Mean


SD


N



312.44  


17.22  


5       



327.57  


30.50  


5       



111 [p1]



Mean


SD


N



309.30  


17.85  


5       



328.40  


28.31  


5       



118 [p1]



Mean


SD


N



311.68  


20.69  


5       



332.15  


24.24  


5       



[p] - T-Test: [G1RvsG4R]


[p1] - Wilcoxon(Rank): [G1RvsG4R]


TABLE 8.     Summary of Body Weight Gains (g) - Males 













































































































Sex: Male


Day(s) Relative to Start Date



G1


0 mg/kg/day



G2


111 mg/kg/day



G3


333 mg/kg/day



G4


1000 mg/kg/day



Absolute 


Weight 


Gain


(g)



1 → 8 [a]



Mean


SD


N



59.05  


7.08   10       



54.91  


6.94   10       



57.94  


3.67   10       



60.34  


5.84   10       



8 → 15 [a]



Mean


SD


N



47.57  


9.73   10       



38.20  


8.82   10       



43.31  


6.14   10       



43.43  


5.25   10       



15 → 22 [a]



Mean


SD


N



32.65  


9.73   10       



31.61  


6.80   10       



31.49  


5.54   10       



31.07  


6.58   10       



22 → 29 [a1]



Mean


SD


N



29.30  


6.29   10       



25.27  


6.44   10       



22.53 *


5.02   10       



27.81  


2.46   10       



29 → 36 [a]



Mean


SD


N



27.15  


5.27   10       



23.85  


8.67   10       



23.84  


5.39   10       



18.55  


8.70   10       



36 → 43 [a]



Mean


SD


N



22.71  


5.22   10       



23.48  


5.70   10       



25.73  


2.89   10       



26.50  


5.21   10       



43 → 50 [a]



Mean


SD


N



18.64  


5.74   10       



19.48  


5.41   10       



20.04  


4.35   10       



24.04  


7.33   10       



50 → 57 [a1]



Mean


SD


N



19.40  


7.06   10       



15.30  


5.91   10       



18.66  


4.96   10       



12.58   12.02  


10       



57 → 64 [a]



Mean


SD


N



18.87  


8.78   10       



13.14  


5.26   10       



13.23  


8.62   10       



20.09  


9.47   10       



64 → 71 [a]



Mean


SD


N



23.80  


7.36   10       



18.98  


5.04   10       



16.92  


7.28   10       



15.48 *


5.94   10       



71 → 77 [a1]



Mean SD


N



6.68   7.35   10       



9.08   5.22   10       



11.24   4.73   10       



8.22   5.41   10       



77 → 85 [a]



Mean


SD


N



14.92  


6.70   10       



13.43  


7.60   10       



10.90  


3.96   10       



16.11  


7.68   10       



[a] - Anova & Dunnett: * = p < 0.05


[a1] - Anova & Dunnett(Rank): * = p < 0.05







































Sex: Male


Day(s) Relative to Start Date



G1


0 mg/kg/day



G2


111 mg/kg/day



G3


333 mg/kg/day



G4


1000 mg/kg/day



Absolute 


Weight 


Gain


(g)



85 → 90 [a]



Mean


SD


N



-0.66  


7.56   10       



-0.47  


5.86   10       



4.85  


6.31   10       



1.70  


6.48   10       



1 → 90 [a]



Mean


SD


N



320.08   40.81  


10       



286.25   42.26  


10       



300.68   33.90  


10       



305.93   29.17  


10       



Total %


Weight 


Gain



1 → 90 [a]



Mean


SD


N



146.83   16.75  


10       



129.47   13.37  


10       



137.67   15.89  


10       



140.80   12.88  


10       



[a] - Anova & Dunnett; [a1] - Anova & Dunnett(Log); [a2] - Anova & Dunnett(Rank) [a] - Anova & Dunnett


TABLE 8 contd. Summary of Body Weight Gains (g) - Males







































































Sex: Male


Day(s) Relative to Start Date



G1R


0 mg/kg/day



G4R


1000 mg/kg/day



Absolute 


Weight 


Gain


(g)



1 → 8 [p]



Mean


SD


N



51.47   9.03  


5       



60.02  


12.71  


5       



8 → 15 [p]



Mean


SD


N



45.31   6.85  


5       



49.41  


12.40  


5       



15 → 22 [p]



Mean


SD


N



32.46   5.55  


5       



35.68  


10.11  


5       



22 → 29 [p]



Mean


SD


N



27.50   6.65  


5       



25.97   5.59  


5       



29 → 36 [p]



Mean


SD


N



23.47  


12.55  


5       



26.85   3.65  


5       



36 → 43 [p1]



Mean


SD


N



22.46   3.12  


5       



25.44  


12.30  


5       



43 → 50 [p]



Mean


SD


N



19.62   2.73  


5       



22.29   5.20  


5       



50 → 57 [p]



Mean


SD


N



17.01   5.83  


5       



13.72   7.19  


5       



57 → 64 [p1]



Mean


SD


N



17.78   3.21  


5       



22.94  


11.89  


5       



64 → 71 [p]



Mean


SD


N



17.87  


14.16  


5       



12.83   3.26  


5       



[p] - T-Test: [G1RvsG4R]


[p1] - Wilcoxon(Rank): [G1RvsG4R]









































































Sex: Male


Day(s) Relative to Start Date



G1R


0 mg/kg/day



G4R


1000 mg/kg/day



Absolute 


Weight 


Gain


(g)



71 → 77 [p]



Mean


SD


N



10.83   7.07  


5       



10.06   2.83  


5       



77 → 85 [p]



Mean


SD


N



14.23   5.51  


5       



10.26  


10.41  


5       



85 → 90 [p]



Mean


SD


N



-1.83   2.04  


5       



6.19 * 3.06  


5       



1 → 90 [p]



Mean


SD


N



298.18  


32.10  


5       



321.66  


69.20  


5       



90 → 97 [p]



Mean


SD


N



7.04   8.00  


5       



21.00 * 10.02  


5       



97 → 104 [p1]



Mean


SD


N



11.33   8.64  


5       



21.35  


19.80  


5       



104 → 111 [p]



Mean


SD


N



3.30   4.84  


5       



2.60   6.40  


5       



111 → 118 [p]



Mean


SD


N



10.43  


10.50  


5       



9.82   7.84  


5       



90 → 118 [p2]



Mean


SD


N



32.10   8.14  


5       



54.76 * 21.68  


5       



Total %


Weight 


Gain



1 → 90 [p]



Mean


SD


N



135.75  


18.68  


5       



149.73  


41.14  


5       



[p] - T-Test: [G1RvsG4R]


        {Absolute Wei}{85-90} G1RvsG4R: * = p < 0.05


        {Absolute Wei}{90-97} G1RvsG4R: * = p < 0.05


[p1] - Wilcoxon(Rank): [G1RvsG4R]


[p2] - T-Test(Log): [G1RvsG4R]


    {90-118} G1RvsG4R: * = p < 0.05


























Sex: Male


Da



y(s) Relative to Start Date



G1R


0 mg/kg/day



G4R


1000 mg/kg/day



Total %


Weight 


Gain



90 → 118 [p]



Mean


SD


N



14.63   3.94  


5       



25.18   9.93  


5       


     

[p] - T-Test: [G1RvsG4R]


TABLE 9.      Summary of Body Weight Gains (g) - Females 













































































































Sex: Female


Day(s) Relative to Start Date



G1


0 mg/kg/day



G2


111 mg/kg/day



G3


333 mg/kg/day



G4


1000 mg/kg/day



Absolute 


Weight 


Gain


(g)



1 → 8 [a]



Mean


SD


N



24.74  


4.99   10       



25.75  


6.95   10       



25.61  


4.36   10       



24.87  


8.14   10       



8 → 15 [a]



Mean


SD


N



15.48  


8.12   10       



18.52  


3.54   10       



14.21  


9.95   10       



15.96  


8.72   10       



15 → 22 [a]



Mean


SD


N



13.75   11.23  


10       



12.21  


8.40   10       



18.96   10.08  


10       



16.28   11.95  


10       



22 → 29 [a1]



Mean


SD


N



17.46  


2.86   10       



13.82   10.86  


10       



7.99  


9.54   10       



16.27  


7.86   10       



29 → 36 [a]



Mean


SD


N



10.53  


6.45   10       



12.87  


6.53   10       



12.25  


9.49   10       



14.16  


5.52   10       



36 → 43 [a1]



Mean


SD


N



3.77  


5.11   10       



6.54  


4.76   10       



8.26  


4.67   10       



5.25   11.77  


10       



43 → 50 [a]



Mean


SD


N



8.16   10.55  


10       



6.79  


6.26   10       



8.81  


7.74   10       



7.38   10.27  


10       



50 → 57 [a]



Mean


SD


N



13.88  


6.43   10       



7.39   10.57  


10       



7.78  


5.24   10       



13.24  


7.88   10       



57 → 64 [a1]



Mean


SD


N



4.82  


6.57   10       



13.48 *


6.24   10       



5.34  


6.50   10       



3.44  


7.11   10       



64 → 71 [a1]



Mean


SD


N



1.74  


4.66   10       



5.93  


3.34   10       



7.01  


4.83   10       



10.62  


9.49   10       



71 → 77 [a]



Mean SD


N



6.31   6.77   10       



1.58   4.05   10       



4.09   7.81   10       



4.34   6.72   10       



77 → 85 [a1]



Mean


SD


N



5.45  


4.22   10       



6.36  


4.80   10       



0.06  


8.05   10       



3.41  


2.94   10       



[a] - Anova & Dunnett


[a1] - Anova & Dunnett(Rank): * = p < 0.05







































Sex: Female


Day(s) Relative to Start Date



G1


0 mg/kg/day



G2


111 mg/kg/day



G3


333 mg/kg/day



G4


1000 mg/kg/day



Absolute 


Weight 


Gain


(g)



85 → 90 [a]



Mean


SD


N



-1.34  


4.53   10       



0.84  


5.85   10       



3.61  


4.78   10       



0.98  


6.41   10       



1 → 90 [a]



Mean


SD


N



124.75   18.50  


10       



132.09   19.48  


10       



123.98   10.78  


10       



136.19   24.37  


10       



Total %


Weight 


Gain



1 → 90 [a]



Mean


SD


N



72.47  


9.56   10       



76.54  


9.01   10       



71.78  


5.91   10       



78.55   12.65  


10       



[a] - Anova & Dunnett


TABLE 9 contd. Summary of Body Weight Gains (g) - Females 






































Sex: Female


Day(s) Relative to Start Date



G1


0 mg/kg/day



G2


111 mg/kg/day



G3


333 mg/kg/day



G4


1000 mg/kg/day



Absolute 


Weight 


Gain


(g)



85 → 90 [a]



Mean


SD


N



-1.34  


4.53   10       



0.84  


5.85   10       



3.61  


4.78   10       



0.98  


6.41   10       



1 → 90 [a]



Mean


SD


N



124.75   18.50  


10       



132.09   19.48  


10       



123.98   10.78  


10       



136.19   24.37  


10       



Total %


Weight 


Gain



1 → 90 [a]



Mean


SD


N



72.47  


9.56   10       



76.54  


9.01   10       



71.78  


5.91   10       



78.55   12.65  


10       



[a] - Anova & Dunnett








































































Sex: Female


Day(s) Relative to Start Date



G1R


0 mg/kg/day



G4R


1000 mg/kg/day



Absolute 


Weight 


Gain


(g)



1 → 8 [p]



Mean


SD


N



25.39   4.18  


5       



26.08   8.15  


5       



8 → 15 [p]



Mean


SD


N



20.97   8.07  


5       



20.28   5.18  


5       



15 → 22 [p]



Mean


SD


N



14.81   8.55  


5       



17.03   9.95  


5       



22 → 29 [p]



Mean


SD


N



9.78  


12.34  


5       



18.11  


21.89  


5       



29 → 36 [p]



Mean


SD


N



15.79  


12.74  


5       



10.76  


13.82  


5       



36 → 43 [p]



Mean


SD


N



3.31   6.77  


5       



6.89   9.41  


5       



43 → 50 [p]



Mean


SD


N



8.59   3.52  


5       



9.30   5.79  


5       



50 → 57 [p]



Mean


SD


N



13.95   8.67  


5       



8.15   5.36  


5       



57 → 64 [p]



Mean


SD


N



1.84   4.81  


5       



8.41 * 3.01  


5       



64 → 71 [p]



Mean


SD


N



8.75   6.43  


5       



6.13   8.26  


5       



[p] - T-Test: [G1RvsG4R]


        {57-64} G1RvsG4R: * = p < 0.05









































































Sex: Female


Day(s) Relative to Start Date



G1R


0 mg/kg/day



G4R


1000 mg/kg/day



Absolute 


Weight 


Gain


(g)



71 → 77 [p]



Mean


SD


N



2.55   7.36  


5       



8.05   5.08  


5       



77 → 85 [p]



Mean


SD


N



3.39   2.10  


5       



6.18   3.01  


5       



85 → 90 [p]



Mean


SD


N



-1.42   5.80  


5       



-0.48   3.81  


5       



1 → 90 [p]



Mean


SD


N



127.69  


15.27  


5       



144.90  


24.64  


5       



90 → 97 [p]



Mean


SD


N



11.92   6.85  


5       



7.79   6.56  


5       



97 → 104 [p]



Mean


SD


N



-2.16   4.40  


5       



2.12   7.75  


5       



104 → 111 [p1]



Mean


SD


N



-3.14   1.63  


5       



0.84 * 4.36  


5       



111 → 118 [p]



Mean


SD


N



2.38   5.14  


5       



3.75   9.38  


5       



90 → 118 [p]



Mean


SD


N



9.00   9.61  


5       



14.50   5.16  


5       



Total %


Weight 


Gain



1 → 90 [p]



Mean


SD


N



72.99   8.00  


5       



83.97  


13.56  


5       



[p] - T-Test: [G1RvsG4R]


[p1] - Wilcoxon(Rank): [G1RvsG4R]


        {104-111} G1RvsG4R: * = p < 0.05

























Sex: Female


Day(s) Relative to Start Date



G1R


0 mg/kg/day



G4R


1000 mg/kg/day



Total %


Weight 


Gain



90 → 118 [p]



Mean


SD


N



5.06   5.48  


5       



8.43   3.10  


5       


     

[p] - T-Test: [G1RvsG4R]


TABLE 10. Summary of Food Consumption (g/rat/day) - Males 













































































































Sex: Male


Day(s) Relative to Start Date



G1


0 mg/kg/day



G2


111 mg/kg/day



G3


333 mg/kg/day



G4


1000 mg/kg/day



Food


Consumption


(g/animal/day)



1 → 8 [a]



Mean


SD


N



30.07  


0.59   10       



29.58  


1.07   10       



28.98 *


0.81   10       



29.94  


0.65   10       



8 → 15 [a]



Mean


SD


N



29.67  


1.96   10       



28.48  


1.69   10       



29.21  


1.48   10       



30.29  


0.74   10       



15 → 22 [a]



Mean


SD


N



29.70  


1.14   10       



28.14 *


1.82   10       



28.07 *


0.82   10       



29.19  


1.26   10       



22 → 29 [a]



Mean


SD


N



29.39  


1.63   10       



29.21  


1.38   10       



29.48  


1.32   10       



30.23  


0.60   10       



29 → 36 [a]



Mean


SD


N



30.18  


1.30   10       



29.28  


1.68   10       



29.01  


1.13   10       



30.67  


1.19   10       



36 → 43 [a]



Mean


SD


N



30.19  


1.55   10       



29.25  


0.95   10       



29.72  


0.76   10       



30.18  


1.24   10       



43 → 50 [a1]



Mean


SD


N



30.19  


1.30   10       



28.68  


1.57   10       



29.51  


1.05   10       



30.66  


1.74   10       



50 → 57 [a]



Mean


SD


N



31.86  


1.06   10       



31.18  


2.10   10       



31.64  


1.65   10       



31.68  


1.44   10       



57 → 64 [a1]



Mean


SD


N



32.16  


1.41   10       



31.08  


2.28   10       



31.20  


1.26   10       



32.31  


1.94   10       



64 → 71 [a]



Mean


SD


N



33.18  


1.36   10       



31.70  


2.15   10       



31.49  


1.32   10       



31.55  


0.78   10       



71 → 77 [a]



Mean SD


N



32.00   1.53   10       



31.02   1.31   10       



30.81   1.23   10       



31.38   1.46   10       



77 → 85 [a]



Mean


SD


N



32.92  


1.63   10       



33.54  


1.01   10       



32.59  


0.90   10       



33.16  


0.49   10       



[a] - Anova & Dunnett(Rank): * = p < 0.05


[a1] Anova & Dunnett































Sex: Male


Day(s) Relative to Start Date



G1


0 mg/kg/day



G2


111 mg/kg/day



G3


333 mg/kg/day



G4


1000 mg/kg/day



Food


Consumption


(g/animal/day)



85 → 90 [a]



Mean


SD


N



31.12  


1.60   10       



31.04  


1.25   10       



31.75  


1.95   10       



30.82  


2.09   10       


       

[a] - Anova & Dunnett(Rank)


TABLE 10 contd. Summary of Food Consumption (g/rat/day) - Males







































































Sex: Male


Day(s) Relative to Start Date



G1R


0 mg/kg/day



G4R


1000 mg/kg/day



Food


Consumption


(g/animal/day)



1 → 8 [p]



Mean


SD


N



29.24   0.42  


5       



29.23   0.93  


5       



8 → 15 [p]



Mean


SD


N



30.09   0.70  


5       



30.50   1.32  


5       



15 → 22 [p]



Mean


SD


N



28.86   0.89  


5       



29.19   0.99  


5       



22 → 29 [p]



Mean


SD


N



28.55   0.95  


5       



28.62   1.39  


5       



29 → 36 [p]



Mean


SD


N



29.13   0.65  


5       



29.58   1.19  


5       



36 → 43 [p1]



Mean


SD


N



29.03   0.24  


5       



30.05   1.21  


5       



43 → 50 [p1]



Mean


SD


N



29.36   0.73  


5       



28.72   0.78  


5       



50 → 57 [p1]



Mean


SD


N



31.67   0.27  


5       



30.78   0.93  


5       



57 → 64 [p]



Mean


SD


N



30.44   1.12  


5       



30.75   1.55  


5       



64 → 71 [p1]



Mean


SD


N



31.22   2.22  


5       



30.61   0.44  


5       



[p] - T-Test: [G1RvsG4R]


[p1] Wilcoxon(Rank): [G1RvsG4R]






















































Sex: Male


Day(s) Relative to Start Date



G1R


0 mg/kg/day



G4R


1000 mg/kg/day



Food


Consumption


(g/animal/day)



71 → 77 [p]



Mean


SD


N



31.02   1.19  


5       



30.23   1.12  


5       



77 → 85 [p1]



Mean


SD


N



32.82   0.79  


5       



32.50   0.32  


5       



85 → 90 [p1]



Mean


SD


N



30.79   1.00  


5       



31.29   1.48  


5       



90 → 97 [p]



Mean


SD


N



29.35   0.92  


5       



30.10   0.89  


5       



97 → 104 [p1]



Mean


SD


N



31.51   1.19  


5       



31.50   2.21  


5       



104 → 111 [p1]



Mean


SD


N



30.74   2.04  


5       



31.68   2.20  


5       



111 → 118 [p1]



Mean


SD


N



30.91   1.63  


5       



31.77   1.79  


5       



[p] - T-Test: [G1RvsG4R]


[p1] Wilcoxon(Rank): [G1RvsG4R]


TABLE 11. Summary of Food Consumption (g/rat/day) - Females 













































































































Sex: Female


Day(s) Relative to Start Date



G1


0 mg/kg/day



G2


111 mg/kg/day



G3


333 mg/kg/day



G4


1000 mg/kg/day



Food


Consumption


(g/animal/day)



1 → 8 [a]



Mean


SD


N



19.86  


0.73   10       



19.86  


0.82   10       



20.55  


0.95   10       



20.49  


0.84   10       



8 → 15 [a]



Mean


SD


N



20.07  


0.59   10       



19.96  


1.35   10       



20.69  


0.88   10       



20.59  


0.90   10       



15 → 22 [a]



Mean


SD


N



20.74  


0.15   10       



19.93 *


0.85   10       



20.27 *


0.41   10       



19.99 *


0.35   10       



22 → 29 [a1]



Mean


SD


N



20.81  


0.56   10       



20.65  


0.83   10       



20.60  


0.49   10       



21.01  


0.67   10       



29 → 36 [a]



Mean


SD


N



21.19  


0.40   10       



20.58  


0.91   10       



21.43  


0.48   10       



20.93  


0.39   10       



36 → 43 [a]



Mean


SD


N



21.06  


0.49   10       



20.87  


0.88   10       



20.40  


1.64   10       



20.14  


0.96   10       



43 → 50 [a]



Mean


SD


N



21.30  


0.23   10       



20.82  


1.46   10       



20.64  


0.99   10       



20.91  


0.88   10       



50 → 57 [a]



Mean


SD


N



22.09  


0.33   10       



21.81  


1.12   10       



22.39  


0.21   10       



22.55  


0.29   10       



57 → 64 [a1]



Mean


SD


N



22.57  


0.96   10       



22.67  


0.96   10       



22.63  


0.43   10       



23.35  


0.81   10       



64 → 71 [a]



Mean


SD


N



21.12  


0.39   10       



21.79 *


1.41   10       



22.17 *


0.80   10       



22.60 *


0.69   10       



71 → 77 [a]



Mean SD


N



22.27   0.55   10       



21.76   1.24   10       



21.43   0.98   10       



22.11   0.69   10       



77 → 85 [a]



Mean


SD


N



23.46  


0.10   10       



23.13  


0.82   10       



23.62  


0.66   10       



23.48  


0.76   10       



[a] - Anova & Dunnett(Rank): * = p < 0.05


[a1] Anova & Dunnett































Sex: Female


Day(s) Relative to Start Date



G1


0 mg/kg/day



G2


111 mg/kg/day



G3


333 mg/kg/day



G4


1000 mg/kg/day



Food


Consumption


(g/animal/day)



85 → 90 [a]



Mean


SD


N



22.91  


0.40   10       



22.15  


1.30   10       



22.16  


1.13   10       



22.81  


0.95   10       


       

[a] - Anova & Dunnett(Rank)








































































Sex: Female


Day(s) Relative to Start Date



G1R


0 mg/kg/day



G4R


1000 mg/kg/day



Food


Consumption


(g/animal/day)



1 → 8 [p]



Mean


SD


N



20.12   0.99  


5       



19.66   0.22  


5       



8 → 15 [p]



Mean


SD


N



20.88   0.29  


5       



20.25   1.34  


5       



15 → 22 [p1]



Mean


SD


N



20.00   0.63  


5       



19.65   0.78  


5       



22 → 29 [p]



Mean


SD


N



21.41   0.78  


5       



20.41   1.01  


5       



29 → 36 [p]



Mean


SD


N



21.63   0.05  


5       



21.16   0.45  


5       



36 → 43 [p]



Mean


SD


N



20.62   0.60  


5       



20.70   0.46  


5       



43 → 50 [p1]



Mean


SD


N



21.87   0.63  


5       



20.71 * 0.93  


5       



50 → 57 [p1]



Mean


SD


N



22.21   0.43  


5       



22.17   0.53  


5       



57 → 64 [p]



Mean


SD


N



22.99   1.17  


5       



22.95   0.48  


5       



64 → 71 [p]



Mean


SD


N



21.95   0.73  


5       



21.97   1.33  


5       



[p] - Wilcoxon(Rank): [G1RvsG4R]


[p1] - T-Test: [G1RvsG4R]


        {43-50} G1RvsG4R: * = p < 0.05






















































Sex: Female


Day(s) Relative to Start Date



G1R


0 mg/kg/day



G4R


1000 mg/kg/day



Food


Consumption


(g/animal/day)



71 → 77 [p]



Mean


SD


N



21.30   0.90  


5       



22.29 * 0.37  


5       



77 → 85 [p]



Mean


SD


N



23.11   0.59  


5       



23.22   1.17  


5       



85 → 90 [p1]



Mean


SD


N



22.74   1.01  


5       



21.75   1.50  


5       



90 → 97 [p1]



Mean


SD


N



22.64   0.39  


5       



22.30   0.64  


5       



97 → 104 [p]



Mean


SD


N



22.12   0.51  


5       



21.95   0.29  


5       



104 → 111 [p]



Mean


SD


N



22.75   0.75  


5       



22.88   0.15  


5       



111 → 118 [p]



Mean


SD


N



21.15   1.18  


5       



22.65 * 0.14  


5       



[p] - Wilcoxon(Rank): [G1RvsG4R]


        {71-77} G1RvsG4R: * = p < 0.05


        {111-118} G1RvsG4R: * = p < 0.05


TABLE 12. Summary of Oestrous Cycle 
















































Sex: Female


Day(s) Relative to Start Date



G1


0 mg/kg/day



G2


111 mg/kg/day



G3


333 mg/kg/day



G4


1000 mg/kg/day



Stage of Estrus is E



91



N+ve 



2  



2  



2  



1  



Stage of Estrus is M



                 91



N+ve 



0  



1  



0  



1  



Stage of Estrus is D



                 91



N+ve 



5  



5  



7  



4  



Stage of Estrus is P



91



N+ve 



3  



2  



1  



4  



[p1] - T-Test: [G1RvsG4R]







































Sex: Female


Day(s) Relative to Start Date



G1R


0 mg/kg/day



G4R


1000 mg/kg/day



Stage of Estrus is E



119



N+ve 



1  





Stage of Estrus is M



                119



N+ve  



0  



              0  



Stage of Estrus is P



119



N+ve  



3  



2  



Stage of Estrus is D



                119



N+ve  



              1  



2  



 







































Sex: Female


Day(s) Relative to Start Date



G1R


0 mg/kg/day



G4R


1000 mg/kg/day



Stage of Estrus is E



119



N+ve 



1  





Stage of Estrus is M



                119



N+ve  



0  



              0  



Stage of Estrus is P



119



N+ve  



3  



2  



Stage of Estrus is D



                119



N+ve  



              1  



2  



Appendix 15, TABLE 1. Summary of Haematology and Coagulation Parameters on Day 91 – Males











































































Sex: Male


Day(s) Relative to Start Date



G1


0 mg/kg/day



G2


111 mg/kg/day



G3


333 mg/kg/day



G4


1000 mg/kg/day



RBC


(10^12/L)



91 [a]



Mean


SD


N


%Diff



9.26  


0.36  


10  


-  



9.21  


0.31  


10   -0.57  



9.28  


0.41  


10  


0.21  



9.38  


0.34  


10  


1.26  



HGB


(g/L)



91 [a]



Mean


SD


N


%Diff



158  


6  


10  


-  



159  


3  


10   1  



161  


4  


10   2  



161  


5  


10   2  



HCT


(L/L)



91 [a]



Mean


SD


N


%Diff



0.482  


0.020  


10  


.  



0.495  


0.016  


10  


2.782  



0.496  


0.015  


10  


3.073  



0.516 * 0.019  


10  


7.081  



MCV


(fL)



91 [a]



Mean


SD


N


%Diff



52.0  


1.8  


10  


-  



53.8  


1.2  


10  


3.4  



53.5  


1.5  


10  


2.9  



55.0 * 2.0  


10  


5.8  



MCH


(pg)



91 [a]



Mean


SD


N


%Diff



17.0  


0.6  


10  


-  



17.3  


0.5  


10  


1.8  



17.4  


0.5  


10  


2.1  



17.2  


0.8  


10  


0.9  



MCHC


(g/L)



91 [a]



Mean


SD


N


%Diff



327  


4  


10  


-  



322  


8  


10  


-2  



325  


4  


10  


-1  



312 *


8  


10  


-5  



Retic A (10^12/L)



91 [a]



Mean


SD


N


%Diff



0.170  


0.027  


10  


-  



0.151  


0.020  


10   -11.019  



0.152  


0.024  


10   -10.725  



0.170  


0.023  


10  


0.412  



[a] - Anova & Dunnett: *: Significantly different from vehicle control group at p < 0.05












































































Sex: Male


Day(s) Relative to Start Date



G1


0 mg/kg/day



G2


111 mg/kg/day



G3


333 mg/kg/day



G4


1000 mg/kg/day



Retic (%)



91 [a]



Mean


SD


N


%Diff



1.84  


0.30  


10  


-  



1.65  


0.24  


10   -10.45  



1.64  


0.30  


10   -10.78  



1.82  


0.25  


10   -0.98  



RDW


(%)



91 [a]



Mean


SD


N


%Diff



12.3  


0.5  


10  


-  



12.6  


0.8  


10  


2.9  



12.4  


0.4  


10  


0.9  



13.1  


0.9  


10  


6.5  



HDW


(g/L)



91 [a]



Mean


SD


N


%Diff



23.5  


1.3  


10  


-  



24.1  


2.1  


10  


2.3  



24.0  


1.4  


10  


2.2  



24.7  


1.6  


10  


5.0  



Hyper (%)



91 [a1]



Mean


SD


N


%Diff



0.6  


0.3  


10  


.  



0.6  


0.6  


10  


6.7  



0.5  


0.3  


10   -10.0  



0.3  


0.2  


10   -43.3  



Hypo (%)



91 [a1]



Mean


SD


N


%Diff



0.4  


0.2  


10  


.  



0.8  


1.1  


10  


92.9  



0.6  


0.4  


10  


42.9  



4.3 * 4.8  


10   911.9  



Macro (%)



91 [a1]



Mean


SD


N


%Diff



0.0  


0.0  


10  


.  



0.0  


0.1  


10  


.  



0.0  


0.0  


10  


.  



0.0  


0.0  


10  


.  



Micro (%)



91 [a1]



Mean


SD


N


%Diff



0.0  


0.0  


10  


.  



0.0 n 0.0  


10  


.  



0.0 n 0.0  


10  


.  



0.0 n 0.0  


10  


.  



[a] - Anova & Dunnett


[a1] - Anova & Dunnett(Rank): *: Significantly different from vehicle control group at p < 0.05;  n - Inappropriate for statistics












































































Sex: Male


Day(s) Relative to Start Date



G1


0 mg/kg/day



G2


111 mg/kg/day



G3


333 mg/kg/day



G4


1000 mg/kg/day



RBC Frag- ments


(10^12/L)



91 [a]



Mean


SD


N


%Diff



0.14  


0.02  


10  


-  



0.17 * 0.02  


10  


19.58  



0.17 * 0.01  


10  


18.88  



0.17 * 0.02  


10  


16.78  



RBC Ghosts (10^12/L)



91 [a]



Mean


SD


N


%Diff



0.08  


0.01  


10  


-  



0.09  


0.01  


10  


7.59  



0.09  


0.01  


10  


7.59  



0.09  


0.01  


10  


11.39  



Plat (10^9/L)



91 [a]



Mean


SD


N


%Diff



1614  


109  


10  


-  



1766 *


138  


10   9  



1769 *


105   10  


10  



1710  


173  


10   6  



MPV


(fL)



91 [a1]



Mean


SD


N


%Diff



11.2  


0.4  


10  


-  



12.5 * 0.6  


10  


11.6  



12.4 * 0.3  


10  


10.3  



13.0 * 0.3  


10  


15.4  



WBC


(10^9/L)



91 [a]



Mean


SD


N


%Diff



10.78  


1.90  


10  


-  



12.01  


2.60  


10  


11.38  



10.12  


1.62  


10   -6.12  



12.03  


1.75  


10  


11.54  



Neut A (10^9/L)



91 [a]



Mean


SD


N


%Diff



3.22  


0.98  


10  


-  



2.81  


0.65  


10   -12.72  



2.67  


0.65  


10   -17.22  



3.19  


0.70  


10   -0.96  



Lymp A


(10^9/L)



91 [a]



Mean


SD


N


%Diff



6.81  


0.99  


10  


-  



8.40  


2.06  


10  


23.36  



6.83  


1.42  


10  


0.29  



8.05  


1.61  


10  


18.13  



[a] - Anova & Dunnett: *: Significantly different from vehicle control group at p < 0.05


[a1] - Anova & Dunnett(Rank): *: Significantly different from vehicle control group at p < 0.05


























































Sex: Male


Day(s) Relative to Start Date



G1


0 mg/kg/day



G2


111 mg/kg/day



G3


333 mg/kg/day



G4


1000 mg/kg/day



Mono A


(10^9/L)



91 [a]



Mean


SD


N


%Diff



0.41  


0.10  


10  


-  



0.43  


0.12  


10  


4.40  



0.34  


0.05  


10   -16.14  



0.43  


0.11  


10  


4.40  



Baso A (10^9/L)



91 [a]



Mean


SD


N


%Diff



0.03  


0.01  


10  


-  



0.03  


0.02  


10  


6.25  



0.02  


0.01  


10   -31.25  



0.03  


0.01  


10   -6.25  



Eosi A (10^9/L)



91 [a1]



Mean


SD


N


%Diff



0.27  


0.10  


10  


-  



0.29  


0.10  


10  


7.55  



0.23  


0.07  


10   -15.09  



0.29  


0.12  


10  


8.30  



Prothrombin


Time


(seconds)



91 [a2]



Mean


SD


N


%Diff



18.6  


0.9  


10  


.  



18.6  


1.1  


10   -0.2  



18.2  


0.8  


10   -2.1  



18.6  


1.1  


10  


0.0  



APTT


(seconds)



91 [a]



Mean


SD


N


%Diff



9.0  


1.9  


10  


-  



9.0  


1.7  


10  


0.4  



9.2  


1.0  


10  


2.5  



9.1  


1.7  


10  


1.9  



[a] - Anova & Dunnett


[a1]  Anova & Dunnett(Log)


[a2] Anova & Dunnett(Rank)


 


Appendix 15, TABLE 2. Summary of Haematology and Coagulation Parameters on Day 119 – Males



























































Sex: Male


Day(s) Relative to Start Date



G1R


0 mg/kg/day



G4R


1000 mg/kg/day



RBC


(10^12/L)



119 [p]



Mean


SD


N


%Diff



8.77  


1.20  


5  


-  



9.16  


0.25  


5  


4.47  



HGB


(g/L)



119 [p]



Mean


SD


N


%Diff



147  


21  


5  


-  



159  


2  


5  


8  



HCT


(L/L)



119 [p]



Mean


SD


N


%Diff



0.467  


0.060  


5  


.  



0.500  


0.007  


5  


6.889  



MCV


(fL)



119 [p1]



Mean


SD


N


%Diff



53.4  


0.9  


5  


-  



54.5  


0.9  


5  


2.1  



MCH


(pg)



119 [p1]



Mean


SD


N


%Diff



16.7  


0.3  


5  


-  



17.4 * 0.4  


5  


3.8  



MCHC


(g/L)



119 [p1]



Mean


SD


N


%Diff



313  


8  


5  


-  



318  


5  


5  


2  



Retic A (10^12/L)



119 [p2]



Mean


SD


N


%Diff



0.276  


0.068  


5  


-  



0.202 * 0.019  


5  


-26.633  



[p] - Wilcoxon(Rank): [G1RvsG4R]


[p1] - T-Test: [G1RvsG4R]


      {MCH} G1RvsG4R: *: Significantly different from vehicle control group at p < 0.05


[p2]  T-Test(Log): [G1RvsG4R]


         G1RvsG4R: *: Significantly different from vehicle control group at p < 0.05





























































Sex: Male


Da



y(s) Relative to Start Date



G1R


0 mg/kg/day



G4R


1000 mg/kg/day



Retic (%)



119 [p]



Mean


SD


N


%Diff



3.29  


1.43  


5  


-  



2.21 * 0.25  


5   -32.68  



RDW


(%)



119 [p]



Mean


SD


N


%Diff



15.3  


3.9  


5  


-  



13.1  


0.7  


5   -14.6  



HDW


(g/L)



119 [p1]



Mean


SD


N


%Diff



26.2  


2.5  


5  


-  



25.2  


1.7  


5   -3.9  



Hyper (%)



119 [p1]



Mean


SD


N


%Diff



0.4  


0.1  


5  


.  



0.5  


0.2  


5  


25.0  



Hypo (%)



119 [p2]



Mean


SD


N


%Diff



2.7  


2.2  


5  


.  



1.5  


0.9  


5   -44.5  



Macro (%)



119 [p]



Mean


SD


N


%Diff



0.6  


1.3  


5  


.  



0.0  


0.0  


5   -100.0  



Micro (%)



119 [p]



Mean


SD


N


%Diff



0.0  


0.0  


5  


.  



0.0  


0.0  


5  


.  



[p] - Wilcoxon(Rank): [G1RvsG4R]


      {Retic} G1RvsG4R: *: Significantly different from vehicle control group at p < 0.05


[p1]  T-Test: [G1RvsG4R]


[p2] T-Test(Log): [G1RvsG4R]




























































Sex: Male


Day(s) Relative to Start Date



G1R


0 mg/kg/day



G4R


1000 mg/kg/day



RBC Frag- ments


(10^12/L)



119 [p]



Mean


SD


N


%Diff



0.16  


0.01  


5  


-  



0.16  


0.01  


5  


0.00  



RBC Ghosts (10^12/L)



119 [p]



Mean


SD


N


%Diff



0.07  


0.01  


5  


-  



0.09 * 0.01  


5  


27.78  



Plat (10^9/L)



119 [p]



Mean


SD


N


%Diff



1667  


105  


5  


-  



1665  


126  


5  


0  



MPV


(fL)



119 [p]



Mean


SD


N


%Diff



11.4  


0.6  


5  


-  



12.0  


0.4  


5  


5.1  



WBC


(10^9/L)



119 [p]



Mean


SD


N


%Diff



10.33  


3.16  


5  


-  



9.22  


0.90  


5   -10.73  



Neut A (10^9/L)



119 [p]



Mean


SD


N


%Diff



4.06  


2.46  


5  


-  



2.60  


1.07  


5   -35.89  



Lymp A


(10^9/L)



119 [p]



Mean


SD


N


%Diff



5.65  


1.16  


5  


-  



6.08  


1.45  


5  


7.69  



[p] - T-Test: [G1RvsG4R]


   {RBC Ghosts} G1RvsG4R: *: Significantly different from vehicle control group at p < 0.05














































Sex: Male


Day(s) Relative to Start Date



G1R


0 mg/kg/day



G4R


1000 mg/kg/day



Mono A


(10^9/L)



119 [p]



Mean


SD


N


%Diff



0.27  


0.11  


5  


-  



0.28  


0.06  


5  


2.22  



Baso A (10^9/L)



119 [p1]



Mean


SD


N


%Diff



0.02  


0.02  


5  


-  



0.02  


0.00  


5   -8.33  



Eosi A (10^9/L)



119 [p2]



Mean


SD


N


%Diff



0.30  


0.24  


5  


-  



0.20  


0.06  


5   -32.43  



Prothrombin


Time


(seconds)



119 [p1]



Mean


SD


N


%Diff



14.7  


0.5  


5  


.  



15.6 * 0.3  


5  


5.8  



APTT


(seconds)



119 [p]



Mean


SD


N


%Diff



7.5  


0.9  


5  


-  



9.0 * 0.8  


5  


20.6  



[p] - T-Test: [G1RvsG4R]


     {APTT} G1RvsG4R: *: Significantly different from vehicle control group at p < 0.05


[p1] - Wilcoxon(Rank): [G1RvsG4R]


             {Prothrombin} G1RvsG4R: *: Significantly different from vehicle control group at p < 0.05 [p2]  T-Test(Log): [G1RvsG4R]


Appendix 15, TABLE 3. Summary of Haematology and Coagulation Parameters on Day 91 - Females











































































Sex: Female


Day(s) Relative to Start Date



G1


0 mg/kg/day



G2


111 mg/kg/day



G3


333 mg/kg/day



G4


1000 mg/kg/day



RBC


(10^12/L)



91 [a]



Mean


SD


N


%Diff



8.64  


0.37  


10  


-  



8.83  


0.31  


10  


2.23  



8.65  


0.30  


10  


0.16  



8.45  


0.34  


10   -2.13  



HGB


(g/L)



91 [a]



Mean


SD


N


%Diff



156  


6  


10  


-  



158  


4  


10   1  



158  


5  


10   1  



156  


5  


10   0  



HCT


(L/L)



91 [a]



Mean


SD


N


%Diff



0.473  


0.016  


10  


.  



0.490  


0.012  


10  


3.443  



0.489  


0.016  


10  


3.338  



0.494 * 0.020  


10  


4.246  



MCV


(fL)



91 [a]



Mean


SD


N


%Diff



54.8  


1.3  


10  


-  



55.5  


1.7  


10  


1.2  



56.6  


1.9  


10  


3.1  



58.5 * 2.4  


10  


6.6  



MCH


(pg)



91 [a]



Mean


SD


N


%Diff



18.1  


0.5  


10  


-  



18.0  


0.6  


10   -0.9  



18.3  


0.7  


10  


1.0  



18.4  


0.7  


10  


1.8  



MCHC


(g/L)



91 [a]



Mean


SD


N


%Diff



330  


3  


10  


-  



323 *


3  


10  


-2  



323 *


5  


10  


-2  



316 *


7  


10  


-4  



Retic A (10^12/L)



91 [a]



Mean


SD


N


%Diff



0.182  


0.042  


10  


-  



0.140  


0.030  


10   -22.984  



0.168  


0.044  


10   -8.009  



0.170  


0.046  


10   -6.967  



[a] - Anova & Dunnett: *: Significantly different from vehicle control group at p < 0.05


 












































































Sex: Female


Day(s) Relative to Start Date



G1


0 mg/kg/day



G2


111 mg/kg/day



G3


333 mg/kg/day



G4


1000 mg/kg/day



Retic (%)



91 [a]



Mean


SD


N


%Diff



2.13  


0.54  


10  


-  



1.59  


0.33  


10   -25.18  



1.95  


0.54  


10   -8.33  



2.02  


0.58  


10   -5.04  



RDW


(%)



91 [a]



Mean


SD


N


%Diff



11.2  


0.5  


10  


-  



11.5  


0.5  


10  


2.6  



11.3  


0.5  


10  


1.3  



11.6  


0.3  


10  


3.9  



HDW


(g/L)



91 [a]



Mean


SD


N


%Diff



20.2  


1.1  


10  


-  



20.2  


0.8  


10   -0.2  



19.9  


1.0  


10   -1.4  



20.8  


0.6  


10  


2.7  



Hyper (%)



91 [a1]



Mean


SD


N


%Diff



0.3  


0.2  


10  


.  



0.2  


0.1  


10   -50.0  



0.1 * 0.1  


10   -66.7  



0.1 * 0.2  


10   -70.0  



Hypo (%)



91 [a1]



Mean


SD


N


%Diff



0.2  


0.1  


10  


.  



0.3  


0.2  


10  


17.4  



0.2  


0.1  


10   -8.7  



1.8 * 1.4  


10   700.0  



Macro (%)



91 [a1]



Mean


SD


N


%Diff



0.0  


0.0  


10  


.  



0.0 n 0.0  


10  


.  



0.0 n 0.0  


10  


.  



0.0 n 0.0  


10  


.  



Micro (%)



91 [a1]



Mean


SD


N


%Diff



0.0  


0.0  


10  


.  



0.0 n 0.0  


10  


.  



0.0 n 0.0  


10  


.  



0.0 n 0.0  


10  


.  



[a] - Anova & Dunnett


[a1] - Anova & Dunnett(Rank): *: Significantly different from vehicle control group at p < 0.05;  n - Inappropriate for statistics


 












































































Sex: Female


Day(s) Relative to Start Date



G1


0 mg/kg/day



G2


111 mg/kg/day



G3


333 mg/kg/day



G4


1000 mg/kg/day



RBC Frag- ments


(10^12/L)



91 [a]



Mean


SD


N


%Diff



0.16  


0.01  


10  


-  



0.17  


0.01  


10  


8.39  



0.17  


0.01  


10  


6.45  



0.16  


0.01  


10  


5.81  



RBC Ghosts (10^12/L)



91 [a]



Mean


SD


N


%Diff



0.09  


0.01  


10  


-  



0.10  


0.01  


10  


2.13  



0.10  


0.01  


10  


2.13  



0.10  


0.01  


10  


9.57  



Plat (10^9/L)



91 [a]



Mean


SD


N


%Diff



1690   86  


10  


-  



1819 *


123  


10   8  



1847 *


110  


10   9  



1914 *


113   10  


13  



MPV


(fL)



91 [a]



Mean


SD


N


%Diff



11.8  


0.5  


10  


-  



12.6 * 0.6  


10  


6.9  



13.0 * 0.6  


10  


10.0  



12.8 * 0.5  


10  


9.0  



WBC


(10^9/L)



91 [a1]



Mean


SD


N


%Diff



6.42  


1.31  


10  


-  



6.51  


1.12  


10  


1.37  



6.36  


1.06  


10   -0.83  



6.75  


2.64  


10  


5.19  



Neut A (10^9/L)



91 [a]



Mean


SD


N


%Diff



1.60  


0.44  


10  


-  



1.53  


0.40  


10   -4.68  



1.51  


0.26  


10   -5.74  



1.46  


0.43  


10   -8.98  



Lymp A


(10^9/L)



91 [a1]



Mean


SD


N


%Diff



4.44  


1.16  


10  


-  



4.55  


0.83  


10  


2.48  



4.49  


0.90  


10  


1.26  



4.97  


2.25  


10  


11.97  



[a] - Anova & Dunnett: *: Significantly different from vehicle control group at p < 0.05 [a1] Anova & Dunnett(Rank)


























































Sex: Female


Day(s) Relative to Start Date



G1


0 mg/kg/day



G2


111 mg/kg/day



G3


333 mg/kg/day



G4


1000 mg/kg/day



Mono A


(10^9/L)



91 [a]



Mean


SD


N


%Diff



0.19  


0.09  


10  


-  



0.19  


0.08  


10   -2.58  



0.18  


0.06  


10   -9.79  



0.15  


0.06  


10   -21.13  



Baso A (10^9/L)



91 [a1]



Mean


SD


N


%Diff



0.01  


0.01  


10  


-  



0.01  


0.00  


10   -21.43  



0.01  


0.00  


10   -50.00  



0.01  


0.01  


10   -14.29  



Eosi A (10^9/L)



91 [a]



Mean


SD


N


%Diff



0.14  


0.05  


10  


-  



0.20  


0.10  


10  


42.55  



0.15  


0.05  


10  


8.51  



0.14  


0.06  


10   -3.55  



Prothrombin


Time


(seconds)



91 [a2]



Mean


SD


N


%Diff



19.8  


1.0  


10  


.  



17.8 * 1.2  


10   -10.2  



18.4 * 0.7  


10   -7.3  



19.0  


1.2  


10   -4.1  



APTT


(seconds)



91 [a]



Mean


SD


N


%Diff



10.1  


1.4  


10  


-  



9.9  


2.7  


10   -2.1  



8.6  


1.1  


10   -15.1  



9.3  


1.1  


10   -7.3  



[a] - Anova & Dunnett(Log)


[a1] - Anova & Dunnett(Rank)


[a2] Anova & Dunnett: *: Significantly different from vehicle control group at p < 0.05


Appendix 15, TABLE 4. Summary of Haematology and Coagulation Parameters on Day 119 – Females



























































Sex: Female


Day(s) Relative to Start Date



G1R


0 mg/kg/day



G4R


1000 mg/kg/day



RBC


(10^12/L)



119 [p]



Mean


SD


N


%Diff



8.50  


0.30  


5  


-  



8.44  


0.46  


5   -0.80  



HGB


(g/L)



119 [p1]



Mean


SD


N


%Diff



152  


7  


5  


-  



154  


3  


5  


2  



HCT


(L/L)



119 [p]



Mean


SD


N


%Diff



0.476  


0.018  


5  


.  



0.482  


0.009  


5  


1.260  



MCV


(fL)



119 [p]



Mean


SD


N


%Diff



56.0  


2.4  


5  


-  



57.3  


3.0  


5  


2.2  



MCH


(pg)



119 [p]



Mean


SD


N


%Diff



17.9  


0.8  


5  


-  



18.3  


1.0  


5  


2.5  



MCHC


(g/L)



119 [p]



Mean


SD


N


%Diff



319  


4  


5  


-  



320  


3  


5  


0  



Retic A (10^12/L)



119 [p]



Mean


SD


N


%Diff



0.195  


0.030  


5  


-  



0.200  


0.042  


5  


2.669  



 




























































Sex: Female


Day(s) Relative to Start Date



G1R


0 mg/kg/day



G4R


1000 mg/kg/day



Retic (%)



119 [p]



Mean


SD


N


%Diff



2.29  


0.37  


5  


-  



2.36  


0.44  


5  


3.05  



RDW


(%)



119 [p]



Mean


SD


N


%Diff



11.9  


0.4  


5  


-  



11.8  


0.7  


5   -1.3  



HDW


(g/L)



119 [p]



Mean


SD


N


%Diff



20.0  


0.9  


5  


-  



21.0  


2.0  


5  


5.0  



Hyper (%)



119 [p]



Mean


SD


N


%Diff



0.1  


0.1  


5  


.  



0.2  


0.2  


5   175.0  



Hypo (%)



119 [p]



Mean


SD


N


%Diff



0.7  


0.3  


5  


.  



0.8  


0.6  


5  


13.5  



Macro (%)



119 [p1]



Mean


SD


N


%Diff



0.0  


0.0  


5  


.  



0.0  


0.0  


5  


.  



Micro (%)



119 [p1]



Mean


SD


N


%Diff



0.0  


0.0  


5  


.  



0.0  


0.0  


5  


.  



 




























































Sex: Female


Day(s) Relative to Start Date



G1R


0 mg/kg/day



G4R


1000 mg/kg/day



RBC Frag- ments


(10^12/L)



119 [p]



Mean


SD


N


%Diff



0.16  


0.01  


5  


-  



0.15  


0.03  


5   -2.56  



RBC Ghosts (10^12/L)



119 [p]



Mean


SD


N


%Diff



0.09  


0.02  


5  


-  



0.10  


0.02  


5  


6.38  



Plat (10^9/L)



119 [p]



Mean


SD


N


%Diff



1699  


63  


5  


-  



1675  


138  


5  


-1  



MPV


(fL)



119 [p]



Mean


SD


N


%Diff



12.5  


0.6  


5  


-  



12.6  


0.7  


5  


0.8  



WBC


(10^9/L)



119 [p]



Mean


SD


N


%Diff



4.81  


0.71  


5  


-  



5.34  


2.13  


5  


11.01  



Neut A (10^9/L)



119 [p1]



Mean


SD


N


%Diff



1.26  


0.18  


5  


-  



1.51  


0.69  


5  


19.30  



Lymp A


(10^9/L)



119 [p]



Mean


SD


N


%Diff



3.29  


0.82  


5  


-  



3.52  


1.54  


5  


7.18  



 














































Sex: Female


Day(s) Relative to Start Date



G1R


0 mg/kg/day



G4R


1000 mg/kg/day



Mono A


(10^9/L)



119 [p]



Mean


SD


N


%Diff



0.14  


0.06  


5  


-  



0.10  


0.04  


5   -30.99  



Baso A (10^9/L)



119 [p]



Mean


SD


N


%Diff



0.00  


0.01  


5  


-  



0.01  


0.01  


5  


150.00  



Eosi A (10^9/L)



119 [p]



Mean


SD


N


%Diff



0.09  


0.03  


5  


-  



0.19  


0.14  


5  


104.35  



Prothrombin


Time


(seconds)



119 [p]



Mean


SD


N


%Diff



16.0  


1.4  


5  


.  



16.4  


0.5  


5  


2.2  



APTT


(seconds)



119 [p]



Mean


SD


N


%Diff



10.7  


1.9  


5  


-  



8.2 * 0.8  


5   -23.6  



T-Test: [G1RvsG4R]


 {APTT} G1RvsG4R: *: Significantly different from vehicle control group at p < 0.05


Appendix 15, TABLE 5. Summary of Clinical Chemistry Parameters on Day 91 – Males











































































Sex: Male


Day(s) Relative to Start Date



G1


0 mg/kg/day



G2


111 mg/kg/day



G3


333 mg/kg/day



G4


1000 mg/kg/day



Glu (mmol/L)



91 [a]



Mean


SD


N


%Diff



6.67  


0.30  


10  


.  



6.29  


0.57  


10   -5.61  



6.56  


0.45  


10   -1.61  



6.24  


0.49  


10   -6.42  



BUN


(mmol/L)



91 [a]



Mean


SD


N


%Diff



5.09  


0.81  


10  


.  



5.34  


0.72  


10  


4.91  



4.93  


0.70  


10   -3.30  



5.17  


0.61  


10  


1.43  



Creat (µmol/L)



91 [a]



Mean


SD


N


%Diff



26  


6  


10  


-  



24  


4  


10  


-6  



29  


3   10  


11  



24  


3  


10  


-7  



AST


(U/L)



91 [a]



Mean


SD


N


%Diff



93  


8  


10  


.  



93  


9  


10   0  



94  


10  


10   2  



92  


9  


10  


-1  



ALT


(U/L)



91 [a]



Mean


SD


N


%Diff



32  


6  


10  


.  



38  


5   10  


20  



34  


8  


10   9  



38  


10  


10  


21  



GGT


(U/L)



91 [a]



Mean


SD


N


%Diff



3  


1  


10  


.  



3  


1   10  


19  



3  


1   10  


12  



3  


1  


10   0  



Alp (U/L)



91 [a]



Mean


SD


N


%Diff



71  


10  


10  


.  



63  


11  


10  


-11  



61  


11  


10  


-14  



75  


15  


10   7  



[a] - Anova & Dunnett


 












































































Sex: Male


Day(s) Relative to Start Date



G1


0 mg/kg/day



G2


111 mg/kg/day



G3


333 mg/kg/day



G4


1000 mg/kg/day



T.Bil (µmol/L)



91 [a]



Mean


SD


N


%Diff



2.71  


0.61  


10  


.  



2.62  


0.95  


10   -3.03  



1.99 * 0.31  


10   -26.36  



2.17  


0.35  


10   -19.70  



T.Chol (mmol/L)



91 [a1]



Mean


SD


N


%Diff



2.34  


0.45  


10  


.  



2.39  


0.38  


10  


2.09  



2.36  


0.40  


10  


0.98  



2.25  


0.42  


10   -3.72  



HDL        


Cholesterol


(mmol/L)



           91 [a1]



Mean


SD


N


%Diff



2.11  


0.42  


10  


.  



2.01  


0.33  


10   -4.60  



2.05  


0.35  


10   -2.61  



2.09  


0.46  


10   -0.95  



LDL


Cholesterol


(mmol/L)



91 [a1]



Mean


SD


N


%Diff



0.04  


0.12  


10  


.  



0.18 * 0.10  


10   413.64  



0.12  


0.10  


10   238.07  



-0.06  


0.14  


10   -265.91  



Trig (mmol/L)



91 [a2]



Mean


SD


N


%Diff



0.98  


0.38  


10  


.  



0.99  


0.35  


10  


0.20  



0.96  


0.42  


10   -2.95  



1.12  


0.42  


10  


13.52  



T.Pro (g/L)



91 [a1]



Mean


SD


N


%Diff



68.3  


1.3  


10  


-  



67.8  


2.0  


10   -0.7  



68.8  


2.1  


10  


0.7  



67.8  


1.7  


10   -0.7  



ALB


(g/L)



91 [a1]



Mean


SD


N


%Diff



34.4  


1.0  


10  


-  



34.0  


1.6  


10   -1.2  



34.4  


0.9  


10  


0.1  



34.2  


1.1  


10   -0.8  



[a] - Anova & Dunnett(Rank): *: Significantly different from vehicle control group at p < 0.05


[a1] - Anova & Dunnett: *: Significantly different from vehicle control group at p < 0.05 [a2] - Anova & Dunnett(Log)












































































Sex: Male


Day(s) Relative to Start Date



G1


0 mg/kg/day



G2


111 mg/kg/day



G3


333 mg/kg/day



G4


1000 mg/kg/day



GLOB


(g/L)



91 [a]



Mean


SD


N


%Diff



33.8  


1.4  


10  


-  



33.8  


0.9  


10   -0.3  



34.3  


1.5  


10  


1.4  



33.7  


1.2  


10   -0.6  



A/G


(ratio)



91 [a]



Mean


SD


N


%Diff



1.02  


0.06  


10  


-  



1.01  


0.05  


10   -1.09  



1.00  


0.04  


10   -1.42  



1.02  


0.05  


10   -0.26  



Pi


(mmol/L)



91 [a1]



Mean


SD


N


%Diff



1.85  


0.17  


10  


.  



1.93  


0.14  


10  


4.82  



1.78  


0.17  


10   -3.79  



1.94  


0.12  


10  


5.37  



Ca (mmol/L)



91 [a]



Mean


SD


N


%Diff



2.90  


0.08  


10  


.  



2.81  


0.13  


10   -2.83  



2.49 * 0.12  


10   -13.88  



2.86  


0.06  


10   -1.14  



Na (mEq/L)



91 [a]



Mean


SD


N


%Diff



146.2  


0.9  


10  


-  



146.8  


0.8  


10  


0.4  



147.6 *


1.4  


10  


1.0  



146.7  


1.1  


10  


0.4  



K (mEq/L)



91 [a1]



Mean


SD


N


%Diff



3.74  


0.22  


10  


-  



3.94  


0.21  


10  


5.24  



4.08 * 0.30  


10  


9.00  



3.91  


0.25  


10  


4.49  



Cl (mEq/L)



91 [a]



Mean


SD


N


%Diff



95.7  


0.8  


10  


-  



95.8  


1.1  


10  


0.2  



96.0  


0.9  


10  


0.4  



95.7  


1.0  


10  


0.1  



[a] - Anova & Dunnett: *: Significantly different from vehicle control group at p < 0.05


[a1] - Anova & Dunnett(Rank): *: Significantly different from vehicle control group at p < 0.05


Appendix 15, TABLE 6. Summary of Clinical Chemistry Parameters on Day 119 – Males



























































Sex: Male


Day(s) Relative to Start Date



G1R


0 mg/kg/day



G4R


1000 mg/kg/day



Glu (mmol/L)



119 [p]



Mean


SD


N


%Diff



8.29  


0.51  


5  


.  



6.66 * 0.63  


5   -19.69  



BUN


(mmol/L)



119 [p]



Mean


SD


N


%Diff



5.92  


0.98  


5  


.  



5.62  


0.47  


5   -5.00  



Creat (µmol/L)



119 [p]



Mean


SD


N


%Diff



36   6  


5  


-  



36   5  


5  


-1  



AST


(U/L)



119 [p1]



Mean


SD


N


%Diff



177  


117  


5  


.  



99 *


13  


5   -44  



ALT


(U/L)



119 [p1]



Mean


SD


N


%Diff



109  


127  


5  


.  



35 *


3  


5   -68  



GGT


(U/L)



119 [p]



Mean


SD


N


%Diff



3  


1  


5  


.  



2  


0  


5   -21  



Alp (U/L)



119 [p]



Mean


SD


N


%Diff



71  


15  


5  


.  



63  


22  


5   -11  



[p] - T-Test: [G1RvsG4R]


{Glu} G1RvsG4R: *: Significantly different from vehicle control group at p < 0.05


[p1] - Wilcoxon(Rank): [G1RvsG4R]


{AST} G1RvsG4R: *: Significantly different from vehicle control group at p < 0.05


{ALT} G1RvsG4R: *: Significantly different from vehicle control group at p < 0.05


 




























































Sex: Male


Day(s) Relative to Start Date



G1R


0 mg/kg/day



G4R


1000 mg/kg/day



T.Bil (µmol/L)



119 [p]



Mean


SD


N


%Diff



2.15  


0.58  


5  


.  



1.84  


0.39  


5   -14.33  



T.Chol (mmol/L)



119 [p]



Mean


SD


N


%Diff



2.99  


0.42  


5  


.  



2.82  


0.59  


5   -5.43  



HDL        


Cholesterol


(mmol/L)



          119 [p]



Mean


SD


N


%Diff



2.43  


0.36  


5  


.  



2.25  


0.54  


5   -7.34  



LDL


Cholesterol


(mmol/L)



119 [p]



Mean


SD


N


%Diff



0.41  


0.12  


5  


.  



0.45  


0.09  


5  


8.03  



Trig (mmol/L)



119 [p]



Mean


SD


N


%Diff



0.73  


0.26  


5  


.  



0.65  


0.16  


5   -11.72  



T.Pro (g/L)



119 [p]



Mean


SD


N


%Diff



67.3  


2.0  


5  


-  



67.0  


0.5  


5   -0.5  



ALB


(g/L)



119 [p]



Mean


SD


N


%Diff



30.5  


1.4  


5  


-  



30.5  


1.8  


5   -0.2  



[p] - T-Test: [G1RvsG4R]




























































Sex: Male


Day(s) Relative to Start Date



G1R


0 mg/kg/day



G4R


1000 mg/kg/day



GLOB


(g/L)



119 [p]



Mean


SD


N


%Diff



36.8  


1.3  


5  


-  



36.5  


2.1  


5   -0.8  



A/G


(ratio)



119 [p]



Mean


SD


N


%Diff



0.83  


0.05  


5  


-  



0.84  


0.10  


5  


1.00  



Pi


(mmol/L)



119 [p]



Mean


SD


N


%Diff



1.85  


0.11  


5  


.  



1.88  


0.14  


5  


1.19  



Ca (mmol/L)



119 [p]



Mean


SD


N


%Diff



2.91  


0.03  


5  


.  



2.83 * 0.04  


5   -2.61  



Na (mEq/L)



119 [p]



Mean


SD


N


%Diff



145.8  


1.0  


5  


-  



146.3  


1.8  


5  


0.3  



K (mEq/L)



119 [p]



Mean


SD


N


%Diff



3.88  


0.34  


5  


-  



3.81  


0.43  


5   -1.70  



Cl (mEq/L)



119 [p]



Mean


SD


N


%Diff



97.4  


1.4  


5  


-  



97.4  


0.6  


5  


0.0  



[p] - T-Test: [G1RvsG4R]


{Ca} G1RvsG4R: *: Significantly different from vehicle control group at p < 0.05


Appendix 15, TABLE 7. Summary of Clinical Chemistry Parameters on Day 91 – Females











































































Sex: Female


Day(s) Relative to Start Date



G1


0 mg/kg/day



G2


111 mg/kg/day



G3


333 mg/kg/day



G4


1000 mg/kg/day



Glu (mmol/L)



91 [a]



Mean


SD


N


%Diff



6.41  


0.59  


10  


.  



6.35  


0.46  


10   -1.00  



6.69  


0.84  


10  


4.34  



6.46  


0.80  


10  


0.80  



BUN


(mmol/L)



91 [a]



Mean


SD


N


%Diff



5.38  


1.06  


10  


.  



5.75  


0.89  


10  


6.97  



5.43  


0.85  


10  


0.87  



5.61  


0.72  


10  


4.37  



Creat (µmol/L)



91 [a]



Mean


SD


N


%Diff



36  


5  


10  


-  



32  


4  


10  


-12  



34  


4  


10  


-5  



30 *


3  


10  


-19  



AST


(U/L)



91 [a1]



Mean


SD


N


%Diff



102   14  


10  


.  



142   76  


10  


40  



100   20  


10  


-1  



97  


10  


10  


-5  



ALT


(U/L)



91 [a1]



Mean


SD


N


%Diff



31  


6  


10  


.  



50  


54  


10  


60  



29  


9  


10  


-9  



27  


6  


10  


-14  



GGT


(U/L)



91 [a1]



Mean


SD


N


%Diff



2  


0  


10  


.  



3  


0   10  


17  



2  


0  


10   0  



3  


1   10  


17  



Alp (U/L)



91 [a]



Mean


SD


N


%Diff



38  


4  


10  


.  



32 *


7  


10  


-16  



37  


5  


10  


-3  



34  


4  


10  


-10  



[a] - Anova & Dunnett: *: Significantly different from vehicle control group at p < 0.05 [a1] - Anova & Dunnett(Rank)


 












































































Sex: Female


Day(s) Relative to Start Date



G1


0 mg/kg/day



G2


111 mg/kg/day



G3


333 mg/kg/day



G4


1000 mg/kg/day



T.Bil (µmol/L)



91 [a]



Mean


SD


N


%Diff



2.77  


1.13  


10  


.  



2.38  


0.54  


10   -14.06  



2.73  


0.66  


10   -1.23  



2.63  


0.55  


10   -5.13  



T.Chol (mmol/L)



91 [a]



Mean


SD


N


%Diff



2.25  


0.32  


10  


.  



2.36  


0.52  


10  


4.66  



2.48  


0.36  


10  


10.04  



2.38  


0.32  


10  


5.51  



HDL        


Cholesterol


(mmol/L)



            91 [a]



Mean


SD


N


%Diff



1.70  


0.23  


10  


.  



2.04  


0.44  


10  


20.09  



2.04  


0.26  


10  


19.98  



1.94  


0.25  


10  


14.50  



LDL


Cholesterol


(mmol/L)



91 [a1]



Mean


SD


N


%Diff



0.46  


0.13  


10  


.  



0.18 * 0.14  


10   -59.51  



0.32  


0.16  


10   -29.23  



0.33  


0.18  


10   -28.75  



Trig (mmol/L)



91 [a1]



Mean


SD


N


%Diff



0.49  


0.08  


10  


.  



0.67  


0.21  


10  


36.11  



0.60  


0.23  


10  


20.69  



0.54  


0.13  


10  


9.33  



T.Pro (g/L)



91 [a]



Mean


SD


N


%Diff



68.7  


3.5  


10  


-  



70.0  


2.9  


10  


1.9  



70.0  


1.8  


10  


1.8  



69.8  


2.7  


10  


1.5  



ALB


(g/L)



91 [a2]



Mean


SD


N


%Diff



36.6  


3.0  


10  


-  



37.8  


2.8  


10  


3.1  



38.1  


1.8  


10  


3.9  



37.4  


1.7  


10  


2.1  



[a] - Anova & Dunnett


[a1] - Anova & Dunnett(Log): *: Significantly different from vehicle control group at p < 0.05 [a2] - Anova & Dunnett(Rank)


 












































































Sex: Female


Day(s) Relative to Start Date



G1


0 mg/kg/day



G2


111 mg/kg/day



G3


333 mg/kg/day



G4


1000 mg/kg/day



GLOB


(g/L)



91 [a]



Mean


SD


N


%Diff



32.1 


1.3 


10 




32.2 


1.3 


10 


0.4 



31.9 


0.9 


10 


-0.6



32.4 


1.5 


10 


0.8 



A/G


(ratio)



91 [a]



Mean


SD


N


%Diff



1.14 


0.10 


10 




1.18 


0.11 


10 


2.81 



1.20 


0.07 


10 


4.56 



1.16 


0.05 


10 


1.28 



Pi


(mmol/L)



91 [a]



Mean


SD


N


%Diff



1.75 


0.19 


10 




1.65 


0.17 


10 


-5.66



1.63 


0.15 


10 


-7.14



1.74 


0.11 


10 


-0.51



Ca (mmol/L)



91 [a]



Mean


SD


N


%Diff



2.80 


0.11 


10 




2.79 


0.10 


10 


-0.43



2.71 


0.16 


10 


-3.39



2.87 


0.08 


10 


2.32 



Na (mEq/L)



91 [a]



Mean


SD


N


%Diff



143.2 


1.6 


10 




144.7 * 1.5 


10 


1.1 



145.1 * 1.3 


10 


1.3 



144.4 


0.9 


10 


0.9 



K (mEq/L)



91 [a]



Mean


SD


N


%Diff



3.53 


0.40 


10 




3.98 * 0.39 


10 


12.84 



3.81 


0.25 


10 


7.88 



3.56 


0.26 


10 


0.82 



Cl (mEq/L)



91 [a1]



Mean


SD


N


%Diff



95.1 


0.9 


10 




95.3 


1.3 


10 


0.2 



95.5 


0.8 


10 


0.4 



95.6 


0.7 


10 


0.5 



[a] - Anova & Dunnett: *: Significantly different from vehicle control group at p < 0.05 [a1] - Anova & Dunnett(Rank)


Appendix 15, TABLE 8. Summary of Clinical Chemistry Parameters on Day 119 – Females



























































Sex: Female


Day(s) Relative to Start Date



G1R


0 mg/kg/day



G4R


1000 mg/kg/day



Glu (mmol/L)



119 [p]



Mean


SD


N


%Diff



7.62  


0.81  


5  


.  



7.75  


0.53  


5  


1.65  



BUN


(mmol/L)



119 [p]



Mean


SD


N


%Diff



4.98  


0.68  


5  


.  



5.89  


1.07  


5  


18.32  



Creat (µmol/L)



119 [p]



Mean


SD


N


%Diff



44   3  


5  


-  



49   8  


5   11  



AST


(U/L)



119 [p1]



Mean


SD


N


%Diff



122  


52  


5  


.  



126  


7  


5  


4  



ALT


(U/L)



119 [p]



Mean


SD


N


%Diff



33   7  


5  


.  



42  


13  


5   27  



GGT


(U/L)



119 [p]



Mean


SD


N


%Diff



2  


0  


5  


.  



2  


1  


5   22  



Alp (U/L)



119 [p]



Mean


SD


N


%Diff



27   7  


5  


.  



33   9  


5   21  



[p] - T-Test: [G1RvsG4R]


[p1] - Wilcoxon(Rank): [G1RvsG4R]




























































Sex: Female


Day(s) Relative to Start Date



G1R


0 mg/kg/day



G4R


1000 mg/kg/day



T.Bil (µmol/L)



119 [p]



Mean


SD


N


%Diff



1.96  


0.89  


5  


.  



2.14  


0.49  


5  


9.61  



T.Chol (mmol/L)



119 [p]



Mean


SD


N


%Diff



2.75  


0.30  


5  


.  



2.62  


0.28  


5   -4.59  



HDL        


Cholesterol


(mmol/L)



          119 [p]



Mean


SD


N


%Diff



2.10  


0.24  


5  


.  



2.08  


0.22  


5   -0.67  



LDL


Cholesterol


(mmol/L)



119 [p]



Mean


SD


N


%Diff



0.54  


0.09  


5  


.  



0.43  


0.08  


5   -19.66  



Trig (mmol/L)



119 [p]



Mean


SD


N


%Diff



0.55  


0.12  


5  


.  



0.52  


0.15  


5   -5.42  



T.Pro (g/L)



119 [p]



Mean


SD


N


%Diff



72.6  


3.4  


5  


-  



70.5  


3.6  


5   -2.9  



ALB


(g/L)



119 [p]



Mean


SD


N


%Diff



40.1  


2.8  


5  


-  



38.3  


4.2  


5   -4.4  



[p] - T-Test: [G1RvsG4R]




























































Sex: Female


Day(s) Relative to Start Date



G1R


0 mg/kg/day



G4R


1000 mg/kg/day



GLOB


(g/L)



119 [p]



Mean


SD


N


%Diff



32.5  


0.9  


5  


-  



32.2  


1.4  


5   -0.9  



A/G


(ratio)



119 [p]



Mean


SD


N


%Diff



1.23  


0.07  


5  


-  



1.19  


0.17  


5   -3.17  



Pi


(mmol/L)



119 [p]



Mean


SD


N


%Diff



1.47  


0.14  


5  


.  



1.38  


0.24  


5   -6.27  



Ca (mmol/L)



119 [p]



Mean


SD


N


%Diff



2.86  


0.12  


5  


.  



2.86  


0.13  


5  


0.07  



Na (mEq/L)



119 [p]



Mean


SD


N


%Diff



143.5  


1.4  


5  


-  



146.2 *


1.1  


5  


1.9  



K (mEq/L)



119 [p]



Mean


SD


N


%Diff



3.73  


0.64  


5  


-  



3.26  


0.47  


5   -12.70  



Cl (mEq/L)



119 [p]



Mean


SD


N


%Diff



95.5  


1.5  


5  


-  



96.3  


1.5  


5  


0.8  



[p] - T-Test: [G1RvsG4R]


{Na} G1RvsG4R: *: Significantly different from vehicle control group at p < 0.05


Appendix 15, TABLE 9. Summary of Thyroid Hormone Profile on Day 91 – Males








































Sex: Male


Da



y(s) Relative to Start Date



G1


0 mg/kg/day



G2


111 mg/kg/day



G3


333 mg/kg/day



G4


1000 mg/kg/day



TSH


(ng/mL)



91 [a]



Mean


SD


N


%Diff



0.62  


0.30  


9a      


.  



0.64  


0.47  


9a       


1.78  



0.70  


0.29  


9a       


12.28  



0.63  


0.37  


10       


1.21  



T3 (ng/mL)



91 [a1]



Mean


SD


N


%Diff



0.35  


0.07  


10       


.  



0.39  


0.04  


10       


11.53  



0.37  


0.05  


10       


5.48  



0.36  


0.04  


10       


4.90  



T4 (ng/mL)



91 [a1]



Mean


SD


N


%Diff



16.36  


3.61  


10       


.  



17.44  


3.43  


10       


6.60  



16.85  


2.17  


10       


2.96  



16.95  


2.51  


10       


3.58  



 


 a: Values below LLOQ (Lower Limit of Quantification for TSH is below 0.1 ng/mL) were not considered for analysis


[a] - Anova & Dunnett(Log)


[a1] Anova & Dunnett


Page 231/475


Appendix 15, TABLE 10. Summary of Thyroid Hormone Profile on Day 119 – Males
































Sex: Male


Da



y(s) Relative to Start Date



G1R


0 mg/kg/day



G4R


1000 mg/kg/day



TSH


(ng/mL)



119 [p]



Mean


SD


N


%Diff



1.17   0.26  


5        .  



1.15   0.60  


5       


-2.05  



T3 (ng/mL)



119 [p1]



Mean


SD


N


%Diff



0.56   0.04  


5        .  



0.53   0.11  


5       


-6.05  



T4 (ng/mL)



119 [p]



Mean


SD


N


%Diff



21.39   4.58  


5        .  



19.95   8.81  


5       


-6.73  



[p] - T-Test: [G1RvsG4R]


[p1] Wilcoxon(Rank): [G1RvsG4R]


Appendix 15, TABLE 11. Summary of Thyroid Hormone Profile on Day 91 – Females







































Sex: Female


Day(s) Relative to Start Date



G1


0 mg/kg/day



G2


111 mg/kg/day



G3


333 mg/kg/day



G4


1000 mg/kg/day



TSH


(ng/mL)



91 [a]



Mean


SD


N


%Diff



0.53  


0.26  


7a       


.  



0.48  


0.24  


7a       


-8.70  



0.47  


0.19  


9a       


-11.02  



0.53  


0.24  


9a       


1.24  



T3 (ng/mL)



91 [a1]



Mean


SD


N


%Diff



0.33  


0.06  


10       


.  



0.36  


0.06  


10       


8.87  



0.36  


0.06  


10       


9.79  



0.36  


0.04  


9a       


10.77  



T4 (ng/mL)



91 [a]



Mean


SD


N


%Diff



10.85  


2.14  


10       


.  



11.87  


2.57  


10       


9.40  



10.80  


2.57  


10       


-0.43  



10.01  


2.42  


10       


-7.76  



 


a: Values below LLOQ (Lower Limit of Quantification for TSH is below 0.1 ng/mL and for T3 is below 0.2 ng/mL) were not considered for analysis


Appendix 15, TABLE 12. Summary of Thyroid Hormone Profile on Day 119 – Females
































Sex: Female


Da



y(s) Relative to Start Date



G1R


0 mg/kg/day



G4R


1000 mg/kg/day



TSH


(ng/mL)



119 [p]



Mean


SD


N


%Diff



0.91  


0.67  


4a       


.  



0.86   0.31  


5       


-6.19  



T3 (ng/mL)



119 [p1]



Mean


SD


N


%Diff



0.49   0.13  


5        .  



0.51  


0.08  


4a       


5.02  



T4 (ng/mL)



119 [p]



Mean


SD


N


%Diff



11.78   4.38  


5        .  



13.06   5.05  


5       


10.88  



 a: Values below LLOQ (Lower Limit of Quantification for TSH is below 0.1 ng/mL and for T3 is below 0.2 ng/mL) were not considered for analysis


[p] - T-Test: [G1RvsG4R]


[p1] Wilcoxon(Rank): [G1RvsG4R]


Appendix 15, TABLE 13. Summary of Clinical Analysis of Urine on Day 91 – Males







































































































Group No.



G1



G2



G3



G4



Dose (mg/kg/day)



0



111



333



1000



No. of rats



10



10



10



10



Volume (mL) 1



27.5


 



38.2


 



29.0


 



34.8


 



 


Glucose2



0


 



0


 



0


 



0


 



 


Bilirubin2



0


 



0


 



0


 



0


 



 


Ketone Bodies2



0


 



0


 



0


 



1


 



 


Specific gravity 1



1.012



1.009



1.012


 



1.011


 



 


pH  1



 


9.0



 


8.9



9.0


 



9.0


 



 


Proteins2



 


5



 


4



7


 



7


 



 


Urobilinogen2,3



 


0



 


0



0


 



0


 



 


Nitrite2



 


0



 


2



0


 



0


 



 


Erythrocytes2 Ery/µL



 


1



 


0



0


 



0


 



 


Leukocytes2 Leu/µL



 


7



 


6



8



8



1: Mean value    2: Incidences of findings 


3: Values less than or equal to 3.2 are normal and hence not considered as positive findings/incidence.


Appendix 15, TABLE 14. Summary of Clinical Analysis of Urine on Day 119 – Males











































































Group No.



G1R



G4R



Dose (mg/kg/day)



0



1000



No. of rats



5



5



Volume (mL) 1



12.9


 



17.4


 



 


Glucose2



0


 



0


 



 


Bilirubin2



0


 



0


 



 


Ketone Bodies2



3


 



1


 



 


Specific gravity 1



1.024


 



1.024


 



 


pH  1



9.0


 



8.9


 



 


Proteins2



4


 



5


 



 


Urobilinogen2,3



0


 



2


 



 


Nitrite2



2


 



1


 



 


Erythrocytes2 Ery/µL



0


 



0


 



 


Leukocytes2 Leu/µL



4



5



1: Mean value    2: Incidences of findings 


3: Values less than or equal to 3.2 are normal and hence not considered as positive findings/incidence.


Appendix 15, TABLE 15. Summary of Clinical Analysis of Urine on Day 91 – Females







































































































Group No.



G1



G2



G3



G4



Dose (mg/kg/day)



0



111



333



1000



No. of rats



10



10



10



10



Volume (mL) 1



24.6


 



20.5


 



17.8


 



15.7


 



 


Glucose2



0


 



0


 



0


 



0


 



 


Bilirubin2



0


 



0


 



0


 



0


 



 


Ketone Bodies2



0


 



0


 



0


 



0


 



 


Specific gravity 1



1.011



1.011



1.014


 



1.016


 



 


pH  1



 


9.0



 


9.0



8.9


 



9.0


 



 


Proteins2



 


3



 


4



7


 



7


 



 


Urobilinogen2,3



 


0



 


0



0


 



0


 



 


Nitrite2



 


1



 


2



2


 



2


 



 


Erythrocytes2 Ery/µL



 


0



 


0



0


 



0


 



 


Leukocytes2 Leu/µL



 


4



 


4



3



4



1: Mean value    2: Incidences of findings 


3: Values less than or equal to 3.2 are normal and hence not considered as positive findings/incidence.


Appendix 15, TABLE 16. Summary of Clinical Analysis of Urine on Day 119 – Females











































































Group No.



G1R



G4R



Dose (mg/kg/day)



0



1000



No. of rats



5



5



Volume (mL) 1



17.0


 



11.2


 



 


Glucose2



0


 



0


 



 


Bilirubin2



0


 



2


 



 


Ketone Bodies2



0


 



0


 



 


Specific gravity 1



1.017


 



1.024


 



 


pH  1



9.0


 



9.0


 



 


Proteins2



4


 



5


 



 


Urobilinogen2,3



2


 



3


 



 


Nitrite2



1


 



1


 



 


Erythrocytes2 Ery/µL



0


 



1


 



 


Leukocytes2 Leu/µL



3



5



1: Mean value    2: Incidences of findings 


3: Values less than or equal to 3.2 are normal and hence not considered as positive findings/incidence.


Appendix 15, TABLE 17. Summary of Terminal Fasting Body Weights and Organ Weights on Day 91 – Males











































































Sex: Male


Day(s) Relative to Start Date



G1


0 mg/kg/day



G2


111 mg/kg/day



G3


333 mg/kg/day



G4


1000 mg/kg/day



Terminal


Fasting BW


(g)



91 [a]



Mean


SD


N


%Diff



509.88  


49.34  


10  


-  



478.23  


55.04  


10   -6.21  



491.21  


37.99  


10   -3.66  



495.90  


38.71  


10   -2.74  



Adrenals (g)



91 [a]



Mean


SD


N


%Diff



0.0759  


0.0101  


10  


-  



0.0686  


0.0101  


10   -9.6719  



0.0693  


0.0102  


10   -8.6441  



0.0738  


0.0146  


10   -2.7540  



Brain (g)



91 [a]



Mean


SD


N


%Diff



2.1871  


0.0848  


10  


-  



2.1804  


0.0952  


10   -0.3050  



2.2186  


0.0756  


10  


1.4398  



2.1833  


0.0823  


10   -0.1747  



Epididymides


(g)



91 [a]



Mean


SD


N


%Diff



1.6373  


0.1344  


10  


-  



1.5911  


0.1162  


10   -2.8217  



1.5410  


0.1319  


10   -5.8852  



1.6232  


0.1804  


10   -0.8630  



Heart (g)



91 [a]



Mean


SD


N


%Diff



1.4513  


0.1114  


10  


-  



1.4222  


0.1513  


10   -2.0065  



1.4791  


0.1170  


10  


1.9169  



1.4579  


0.1070  


10  


0.4541  



Kidneys


(g)



91 [a]



Mean


SD


N


%Diff



3.0645  


0.2430  


10  


-  



2.7847  


0.3149  


10   -9.1316  



2.9595  


0.3123  


10   -3.4286  



3.0083  


0.2526  


10   -1.8332  



Liver (g)



91 [a]



Mean


SD


N


%Diff



13.2163  


1.3875  


10  


-  



12.4174  


1.6967  


10   -6.0453  



12.9404  


1.1575  


10   -2.0878  



13.0536  


1.7073  


10   -1.2309  



[a] - Anova & Dunnett












































































Sex: Male


Day(s) Relative to Start Date



G1


0 mg/kg/day



G2


111 mg/kg/day



G3


333 mg/kg/day



G4


1000 mg/kg/day



Pituitary (g)



103b [a]



Mean


SD


N


%Diff



0.0152  


0.0014  


10  


-  



0.0149  


0.0026  


10   -2.3653  



0.0147  


0.0012  


10   -3.2194  



0.0146  


0.0013  


10   -4.1393  



Prostate (g)



91 [a1]



Mean


SD


N


%Diff



1.3174  


0.2153  


10  


.  



1.3204  


0.2125  


10  


0.2277  



1.3592  


0.1831  


10  


3.1698  



1.3574  


0.2370  


10  


3.0355  



Seminal vesicles


& coagulating glands


(g)



          91 [a1]



Mean


SD


N


%Diff



1.8548  


0.3517  


10  


-  



1.8582  


0.2892  


10  


0.1865  



1.7220  


0.2682  


10   -7.1599  



1.6366  


0.2224  


10   -11.7642  



Spleen (g)



91 [a]



Mean


SD


N


%Diff



1.1633  


0.3505  


10  


-  



0.9371  


0.2472  


10   -19.4385  



0.9102  


0.0729  


10   -21.7518  



0.9859  


0.1282  


10   -15.2502  



Testes (g)



91 [a]



Mean


SD


N


%Diff



3.9520  


0.2849  


10  


-  



4.0709  


0.3147  


10  


3.0073  



4.4664  


0.9630  


10  


13.0143  



4.1772  


0.9146  


10  


5.6971  



Thymus


(g)



91 [a1]



Mean


SD


N


%Diff



0.4240  


0.0807  


10  


.  



0.4419  


0.0954  


10  


4.2266  



0.4314  


0.0884  


10  


1.7595  



0.4439  


0.0984  


10  


4.7054  



Thyroid with parathyroids


(g)



103b [a1]



Mean


SD


N


%Diff



0.0345  


0.0039  


10  


-  



0.0348  


0.0075  


10  


0.9576  



0.0366  


0.0053  


10  


6.2101  



0.0367  


0.0069  


10  


6.4423  



[a] - Anova & Dunnett(Rank);            b: Weighed after fixation


[a1] Anova & Dunnett


Appendix 15, TABLE 18. Summary of Terminal Fasting Body Weights and Organ to Body Weight  Ratios on Day 91 – Males











































































Sex: Male


Day(s) Relative to Start Date



G1


0 mg/kg/day



G2


111 mg/kg/day



G3


333 mg/kg/day



G4


1000 mg/kg/day



Terminal


Fasting BW


(g)



91 [a]



Mean


SD


N


%Diff



509.88  


49.34  


10  


-  



478.23  


55.04  


10   -6.21  



491.21  


37.99  


10   -3.66  



495.90  


38.71  


10   -2.74  



Adrenals (%)



91 [a1]



Mean


SD


N


%Diff



0.0150  


0.0021  


10  


.  



0.0144  


0.0020  


10   -3.7578  



0.0141  


0.0018  


10   -5.5958  



0.0150  


0.0032  


10  


0.0158  



Brain (%)



91 [a]



Mean


SD


N


%Diff



0.4319  


0.0375  


10  


.  



0.4599  


0.0422  


10  


6.4924  



0.4541  


0.0380  


10  


5.1272  



0.4420  


0.0286  


10  


2.3426  



Epididymides (%)



91 [a]



Mean


SD


N


%Diff



0.3226  


0.0287  


10  


.  



0.3354  


0.0337  


10  


3.9506  



0.3148  


0.0294  


10   -2.4277  



0.3298  


0.0490  


10  


2.2104  



Heart (%)



91 [a]



Mean


SD


N


%Diff



0.2859  


0.0233  


10  


.  



0.2982  


0.0215  


10  


4.2923  



0.3018  


0.0219  


10  


5.5438  



0.2945  


0.0163  


10  


2.9834  



Kidneys (%)



91 [a]



Mean


SD


N


%Diff



0.6037  


0.0507  


10  


.  



0.5837  


0.0463  


10   -3.3054  



0.6028  


0.0479  


10   -0.1498  



0.6072  


0.0348  


10  


0.5810  



Liver (%)



91 [a]



Mean


SD


N


%Diff



2.5931  


0.1411  


10  


.  



2.5957  


0.1589  


10  


0.0993  



2.6367  


0.1752  


10  


1.6800  



2.6237  


0.1589  


10  


1.1796  



[a] - Anova & Dunnett


[a1] Anova & Dunnett(Log)












































































Sex: Male


Day(s) Relative to Start Date



G1


0 mg/kg/day



G2


111 mg/kg/day



G3


333 mg/kg/day



G4


1000 mg/kg/day



Pituitary (%)



103b [a]



Mean


SD


N


%Diff



0.0030  


0.0004  


10  


.  



0.0031  


0.0006  


10  


4.4058  



0.0030  


0.0003  


10  


0.2903  



0.0029  


0.0002  


10   -2.0435  



Prostate (%)



91 [a1]



Mean


SD


N


%Diff



0.2618  


0.0565  


10  


.  



0.2770  


0.0415  


10  


5.8307  



0.2779  


0.0394  


10  


6.1614  



0.2763  


0.0600  


10  


5.5593  



Seminal vesicles


& coagulating glands


(%)



          91 [a2]



Mean


SD


N


%Diff



0.3657  


0.0686  


10  


.  



0.3935  


0.0800  


10  


7.5991  



0.3535  


0.0716  


10   -3.3203  



0.3319  


0.0507  


10   -9.2462  



Spleen (%)



91 [a]



Mean


SD


N


%Diff



0.2294  


0.0722  


10  


.  



0.1983  


0.0579  


10   -13.5662  



0.1863  


0.0212  


10   -18.7774  



0.1999  


0.0289  


10   -12.8569  



Testes (%)



91 [a]



Mean


SD


N


%Diff



0.7815  


0.0947  


10  


.  



0.8577  


0.0818  


10  


9.7452  



0.9055 * 0.1463  


10  


15.8598  



0.8503  


0.2251  


10  


8.7921  



Thymus (%)



91 [a2]



Mean


SD


N


%Diff



0.0828  


0.0112  


10  


.  



0.0938  


0.0252  


10  


13.2315  



0.0881  


0.0185  


10  


6.4465  



0.0897  


0.0204  


10  


8.3523  



Thyroid with parathyroids


(%)



103b [a]



Mean


SD


N


%Diff



0.0068  


0.0007  


10  


.  



0.0074  


0.0019  


10  


9.0421  



0.0075  


0.0013  


10  


10.7394  



0.0074  


0.0013  


10  


9.2319  



[a] - Anova & Dunnett(Rank): *: Significantly different from vehicle control group at p < 0.05 [a1] - Anova & Dunnett;            b: Weighed after fixation


[a2] Anova & Dunnett(Log)


Appendix 15, TABLE 19. Summary of Brain Weights and Organ to Brain Weight Ratios on Day 91 – Males











































































Sex: Male


Day(s) Relative to Start Date



G1


0 mg/kg/day



G2


111 mg/kg/day



G3


333 mg/kg/day



G4


1000 mg/kg/day



Brain (g)



91 [a]



Mean


SD


N


%Diff



2.1871  


0.0848  


10  


-  



2.1804  


0.0952  


10   -0.3050  



2.2186  


0.0756  


10  


1.4398  



2.1833  


0.0823  


10   -0.1747  



Adrenals (%)



91 [a]



Mean


SD


N


%Diff



3.4790  


0.5169  


10  


.  



3.1474  


0.4738  


10   -9.5312  



3.1298  


0.4766  


10   -10.0379  



3.3800  


0.6560  


10   -2.8465  



Epididymides (%)



91 [a]



Mean


SD


N


%Diff



74.9358  


6.4603  


10  


.  



73.0190  


5.0640  


10   -2.5580  



69.5946  


7.2156  


10   -7.1278  



74.4686  


9.0070  


10   -0.6235  



Heart (%)



91 [a]



Mean


SD


N


%Diff



66.3512  


4.2154  


10  


.  



65.1155  


4.9748  


10   -1.8623  



66.8264  


7.0001  


10  


0.7162  



66.7903  


4.4849  


10  


0.6618  



Kidneys (%)



91 [a]



Mean


SD


N


%Diff



140.3386  


12.7030  


10  


.  



127.5292  


11.3593  


10   -9.1275  



133.3486  


12.6854  


10   -4.9808  



137.7556  


10.0786  


10   -1.8406  



Liver (%)



91 [a]



Mean


SD


N


%Diff



603.5938  


50.4892  


10  


.  



568.8458  


65.9076  


10   -5.7568  



583.9523  


57.7208  


10   -3.2541  



597.6385  


73.0185  


10   -0.9866  



Pituitary (%)



103b [a1]



Mean


SD


N


%Diff



0.6965  


0.0651  


10  


.  



0.6809  


0.1094  


10   -2.2338  



0.6647  


0.0594  


10   -4.5662  



0.6680  


0.0527  


10   -4.0882  



[a] - Anova & Dunnett;            b: Weighed after fixation


[a1] Anova & Dunnett(Rank)



































































Sex: Male


Day(s) Relative to Start Date



G1


0 mg/kg/day



G2


111 mg/kg/day



G3


333 mg/kg/day



G4


1000 mg/kg/day



Prostate (%)



91 [a]



Mean


SD


N


%Diff



60.1040  


8.8678  


10  


.  



60.5553  


9.3413  


10  


0.7508  



61.4339  


9.3932  


10  


2.2126  



62.2769  


11.3167  


10  


3.6152  



Seminal vesicles


& coagulating glands


(%)



           91 [a]



Mean


SD


N


%Diff



84.5021  


13.9828  


10  


.  



85.6124  


15.5667  


10  


1.3139  



77.5066  


10.8812  


10   -8.2785  



74.9204  


9.3218  


10   -11.3390  



Spleen (%)



91 [a1]



Mean


SD


N


%Diff



53.3096  


16.7113  


10  


.  



42.8106  


10.7724  


10   -19.6943  



41.0098 * 2.6829  


10   -23.0725  



45.1351  


5.2933  


10   -15.3340  



Testes (%)



91 [a1]



Mean


SD


N


%Diff



180.6955  


10.7228  


10  


.  



186.7867  


12.9311  


10  


3.3710  



201.3298  


42.7738  


10  


11.4194  



191.5330  


42.5190  


10  


5.9977  



Thymus (%)



91 [a]



Mean


SD


N


%Diff



19.4011  


3.7557  


10  


.  



20.2604  


4.3009  


10  


4.4290  



19.5627  


4.6970  


10  


0.8330  



20.3386  


4.5300  


10  


4.8318  



Thyroid with parathyroids


(%)



103b [a1]



Mean


SD


N


%Diff



1.5764  


0.1795  


10  


.  



1.5965  


0.3402  


10  


1.2736  



1.6487  


0.2197  


10  


4.5865  



1.6786  


0.3001  


10  


6.4834  



[a] - Anova & Dunnett;            b: Weighed after fixation


[a1] Anova & Dunnett(Rank): *: Significantly different from vehicle control group at p < 0.05


Appendix 15, TABLE 20. Summary of Terminal Fasting Body Weights and Organ Weights on Day 119  – Males



























































Sex: Male


Day(s) Relative to Start Date



G1R


0 mg/kg/day



G4R


1000 mg/kg/day



Terminal


Fasting BW


(g)



Day 119 [p]



Mean


SD


N


%Diff



521.68  


40.03  


5  


-  



560.05  


63.66  


5  


7.36  



Adrenals (g)



Day 119 [p]



Mean


SD


N


%Diff



0.0682  


0.0126  


5  


-  



0.0775  


0.0145  


5  


13.6324  



Brain (g)



Day 119 [p]



Mean


SD


N


%Diff



2.1785  


0.0994  


5  


-  



2.2669  


0.0962  


5  


4.0606  



Epididymides


(g)



Day 119 [p]



Mean


SD


N


%Diff



1.6050  


0.1204  


5  


-  



1.7387  


0.1539  


5  


8.3341  



Heart (g)



Day 119 [p]



Mean


SD


N


%Diff



1.5181  


0.1572  


5  


-  



1.6032  


0.1118  


5  


5.6029  



Kidneys


(g)



Day 119 [p]



Mean


SD


N


%Diff



3.0056  


0.4252  


5  


-  



3.3120  


0.1087  


5  


10.1930  



Liver (g)



Day 119 [p]



Mean


SD


N


%Diff



14.0219  


1.2716  


5  


-  



14.1928  


1.3253  


5  


1.2191  



[p] - T-Test: [G1RvsG4R]


 




























































Sex: Male


Day(s) Relative to Start Date



G1R


0 mg/kg/day



G4R


1000 mg/kg/day



Pituitary (g)



Day 124b [p]



Mean


SD


N


%Diff



0.0145  


0.0012  


5  


-  



0.0156  


0.0006  


5  


7.4278  



Prostate (g)



Day 119 [p]



Mean


SD


N


%Diff



1.2863  


0.2097  


5  


.  



1.2834  


0.1934  


5  


-0.2270  



Seminal vesicles


& coagulating glands


(g)



       Day 119 [p]



Mean


SD


N


%Diff



2.1859  


0.4301  


5  


-  



2.5391  


0.2215  


5  


16.1541  



Spleen (g)



Day 119 [p]



Mean


SD


N


%Diff



0.9333  


0.0951  


5  


-  



0.9920  


0.1330  


5  


6.2941  



Testes (g)



Day 119 [p]



Mean


SD


N


%Diff



4.0280  


0.6371  


5  


-  



4.4491  


0.8062  


5  


10.4532  



Thymus


(g)



Day 119 [p]



Mean


SD


N


%Diff



0.3881  


0.0831  


5  


.  



0.3699  


0.1002  


5  


-4.7047  



Thyroid with parathyroids


(g)



Day 124b [p]



Mean


SD


N


%Diff



0.0296  


0.0023  


5  


-  



0.0311  


0.0028  


5  


4.9966  



[p] - T-Test: [G1RvsG4R] ;            b: Weighed after fixation


Appendix 15, TABLE 21. Summary of Terminal Fasting Body Weights and Organ to Body Weight  Ratios on Day 119 – Males



























































Sex: Male


Day(s) Relative to Start Date



G1R


0 mg/kg/day



G4R


1000 mg/kg/day



Terminal


Fasting BW


(g)



Day 119 [p]



Mean


SD


N


%Diff



521.68  


40.03  


5  


-  



560.05  


63.66  


5  


7.36  



Adrenals (%)



Day 119 [p]



Mean


SD


N


%Diff



0.0131  


0.0021  


5  


.  



0.0140  


0.0030  


5  


6.7155  



Brain (%)



Day 119 [p]



Mean


SD


N


%Diff



0.4186  


0.0203  


5  


.  



0.4082  


0.0406  


5  


-2.4909  



Epididymides (%)



Day 119 [p]



Mean


SD


N


%Diff



0.3093  


0.0352  


5  


.  



0.3138  


0.0465  


5  


1.4529  



Heart (%)



Day 119 [p]



Mean


SD


N


%Diff



0.2911  


0.0217  


5  


.  



0.2878  


0.0223  


5  


-1.1221  



Kidneys (%)



Day 119 [p]



Mean


SD


N


%Diff



0.5747  


0.0539  


5  


.  



0.5963  


0.0585  


5  


3.7572  



Liver (%)



Day 119 [p]



Mean


SD


N


%Diff



2.6866  


0.0817  


5  


.  



2.5395 * 0.0847  


5  


-5.4741  



[p] - T-Test: [G1RvsG4R]


{Liver.} G1RvsG4R: *: Significantly different from vehicle control group at p < 0.05




























































Sex: Male


Day(s) Relative to Start Date



G1R


0 mg/kg/day



G4R


1000 mg/kg/day



Pituitary (%)



Day 124b [p]



Mean


SD


N


%Diff



0.0028  


0.0003  


5  


.  



0.0028  


0.0003  


5  


0.5069  



Prostate (%)



Day 119 [p]



Mean


SD


N


%Diff



0.2465  


0.0364  


5  


.  



0.2288  


0.0175  


5  


-7.1964  



Seminal vesicles


& coagulating glands


(%)



       Day 119 [p]



Mean


SD


N


%Diff



0.4205  


0.0854  


5  


.  



0.4575  


0.0632  


5  


8.7997  



Spleen (%)



Day 119 [p]



Mean


SD


N


%Diff



0.1789  


0.0134  


5  


.  



0.1774  


0.0179  


5  


-0.8544  



Testes (%)



Day 119 [p]



Mean


SD


N


%Diff



0.7729  


0.1173  


5  


.  



0.8072  


0.2077  


5  


4.4404  



Thymus (%)



Day 119 [p]



Mean


SD


N


%Diff



0.0746  


0.0158  


5  


.  



0.0656  


0.0150  


5  


-12.0535  



Thyroid with parathyroids


(%)



Day 124b [p1]



Mean


SD


N


%Diff



0.0057  


0.0004  


5  


.  



0.0056  


0.0003  


5  


-2.0224  



[p] - T-Test: [G1RvsG4R] ;            b: Weighed after fixation


[p1] - Wilcoxon(Rank): [G1RvsG4R]


Appendix 15, TABLE 22. Summary of Brain Weights and Organ to Brain Weight Ratios on Day 119 –  Males



























































Sex: Male


Day(s) Relative to Start Date



G1R


0 mg/kg/day



G4R


1000 mg/kg/day



Brain (g)



Day 119 [p]



Mean


SD


N


%Diff



2.1785  


0.0994  


5  


-  



2.2669  


0.0962  


5  


4.0606  



Adrenals (%)



Day 119 [p]



Mean


SD


N


%Diff



3.1328  


0.5596  


5  


.  



3.4341  


0.6901  


5  


9.6183  



Epididymides (%)



Day 119 [p]



Mean


SD


N


%Diff



73.7359  


5.4465  


5  


.  



76.8589  


8.1876  


5  


4.2354  



Heart (%)



Day 119 [p]



Mean


SD


N


%Diff



69.5539  


4.2952  


5  


.  



70.8565  


6.1340  


5  


1.8729  



Kidneys (%)



Day 119 [p]



Mean


SD


N


%Diff



137.7725  


16.7897  


5  


.  



146.4011  


9.6973  


5  


6.2629  



Liver (%)



Day 119 [p]



Mean


SD


N


%Diff



643.1001  


39.6597  


5  


.  



626.5726  


58.1752  


5  


-2.5700  



Pituitary (%)



Day 124b [p]



Mean


SD


N


%Diff



0.6681  


0.0536  


5  


.  



0.6895  


0.0251  


5  


3.2152  



[p] - T-Test: [G1RvsG4R] ;            b: Weighed after fixation


 





















































Sex: Male


Day(s) Relative to Start Date



G1R


0 mg/kg/day



G4R


1000 mg/kg/day



Prostate (%)



Day 119 [p]



Mean


SD


N


%Diff



58.8403  


7.4497  


5  


.  



56.6770  


8.6437  


5  


-3.6766  



Seminal vesicles


& coagulating glands


(%)



       Day 119 [p]



Mean


SD


N


%Diff



100.0448  


16.7022  


5  


.  



112.4500  


14.0003  


5  


12.3997  



Spleen (%)



Day 119 [p]



Mean


SD


N


%Diff



42.7643  


2.7556  


5  


.  



43.7411  


5.3876  


5  


2.2841  



Testes (%)



Day 119 [p]



Mean


SD


N


%Diff



184.3709  


23.1031  


5  


.  



197.3571  


42.8986  


5  


7.0435  



Thymus (%)



Day 119 [p]



Mean


SD


N


%Diff



17.8647  


3.9596  


5  


.  



16.2224  


3.9026  


5  


-9.1929  



Thyroid with parathyroids


(%)



Day 124b [p]



Mean


SD


N


%Diff



1.3584  


0.0555  


5  


.  



1.3733  


0.1256  


5  


1.0992  



[p] - T-Test: [G1RvsG4R] ;            b: Weighed after fixation


Appendix 15, TABLE 23. Summary of Terminal Fasting Body Weights and Organ Weights on Day 91 –  Females











































































Sex: Female


Day(s) Relative to Start Date



G1


0 mg/kg/day



G2


111 mg/kg/day



G3


333 mg/kg/day



G4


1000 mg/kg/day



Terminal


Fasting BW


(g)



91 [a]



Mean


SD


N


%Diff



275.98  


22.96  


10  


-  



283.90  


22.39  


10  


2.87  



274.09  


16.62  


10   -0.68  



288.64  


27.41  


10  


4.59  



Adrenals (g)



91 [a1]



Mean


SD


N


%Diff



0.0887  


0.0083  


10  


-  



0.0895  


0.0161  


10  


0.9129  



0.0933  


0.0061  


10  


5.1505  



0.0942  


0.0102  


10  


6.1760  



Brain (g)



91 [a]



Mean


SD


N


%Diff



2.0429  


0.0704  


10  


-  



2.0222  


0.0982  


10   -1.0123  



2.0281  


0.0700  


10   -0.7274  



2.0674  


0.0795  


10  


1.2002  



Heart (g)



91 [a1]



Mean


SD


N


%Diff



0.9845  


0.0374  


10  


-  



1.0288  


0.0660  


10  


4.4945  



1.0019  


0.0798  


10  


1.7602  



1.0110  


0.0932  


10  


2.6886  



Kidneys


(g)



91 [a]



Mean


SD


N


%Diff



1.7205  


0.1478  


10  


-  



1.7287  


0.1405  


10  


0.4731  



1.6658  


0.1587  


10   -3.1839  



1.7996  


0.1636  


10  


4.5945  



Liver (g)



91 [a]



Mean


SD


N


%Diff



7.5040  


0.7841  


10  


-  



7.9984  


0.9514  


10  


6.5882  



7.3846  


0.5394  


10   -1.5917  



7.6663  


0.4731  


10  


2.1634  



Ovaries (g)



91 [a]



Mean


SD


N


%Diff



0.1118  


0.0141  


10  


-  



0.1269  


0.0144  


10  


13.5009  



0.1122  


0.0181  


10  


0.3937  



0.1113  


0.0177  


10   -0.4295  



[a] - Anova & Dunnett


[a1] Anova & Dunnett(Rank)


 


























































Sex: Female


Day(s) Relative to Start Date



G1


0 mg/kg/day



G2


111 mg/kg/day



G3


333 mg/kg/day



G4


1000 mg/kg/day



Pituitary (g)



103b [a]



Mean


SD


N


%Diff



0.0177  


0.0025  


10  


-  



0.0185  


0.0030  


10  


4.2301  



0.0173  


0.0040  


10   -2.6509  



0.0173  


0.0029  


10   -2.4817  



Spleen (g)



91 [a]



Mean


SD


N


%Diff



0.6368  


0.0974  


10  


-  



0.6698  


0.0656  


10  


5.1824  



0.6868  


0.0836  


10  


7.8631  



0.6569  


0.0907  


10  


3.1660  



Thymus


(g)



91 [a]



Mean


SD


N


%Diff



0.3346  


0.0471  


10  


.  



0.3354  


0.0702  


10  


0.2660  



0.3772  


0.0606  


10  


12.7485  



0.3455  


0.0612  


10  


3.2611  



Thyroid with parathyroids


(g)



103b [a1]



Mean


SD


N


%Diff



0.0298  


0.0063  


10  


-  



0.0296  


0.0036  


10   -0.5373  



0.0306  


0.0057  


10  


2.8543  



0.0270  


0.0031  


10   -9.3015  



Uterus with cervix


(g)



91 [a2]



Mean


SD


N


%Diff



0.7268  


0.1673  


10  


.  



0.6877  


0.0893  


10   -5.3704  



0.9847  


0.4090  


10  


35.4973  



0.7409  


0.2852  


10  


1.9497  



Anova & Dunnett;           


Anova & Dunnett(Log)


Anova & Dunnett(Rank)


Appendix 15, TABLE 24. Summary of Terminal Fasting Body Weights and Organ to Body Weight Ratios on Day 91 – Females











































































Sex: Female


Day(s) Relative to Start Date



G1


0 mg/kg/day



G2


111 mg/kg/day



G3


333 mg/kg/day



G4


1000 mg/kg/day



Terminal


Fasting BW


(g)



91 [a]



Mean


SD


N


%Diff



275.98  


22.96  


10  


-  



283.90  


22.39  


10  


2.87  



274.09  


16.62  


10   -0.68  



288.64  


27.41  


10  


4.59  



Adrenals (%)



91 [a1]



Mean


SD


N


%Diff



0.0322  


0.0030  


10  


.  



0.0316  


0.0054  


10   -2.0225  



0.0342  


0.0033  


10  


5.9907  



0.0329  


0.0043  


10  


1.9291  



Brain (%)



91 [a]



Mean


SD


N


%Diff



0.7440  


0.0541  


10  


.  



0.7158  


0.0589  


10   -3.7890  



0.7424  


0.0519  


10   -0.2155  



0.7224  


0.0775  


10   -2.8947  



Heart (%)



91 [a]



Mean


SD


N


%Diff



0.3593  


0.0364  


10  


.  



0.3639  


0.0304  


10  


1.2928  



0.3659  


0.0268  


10  


1.8429  



0.3518  


0.0349  


10   -2.0858  



Kidneys (%)



91 [a2]



Mean


SD


N


%Diff



0.6251  


0.0503  


10  


.  



0.6098  


0.0355  


10   -2.4416  



0.6099  


0.0717  


10   -2.4368  



0.6256  


0.0541  


10  


0.0835  



Liver (%)



91 [a]



Mean


SD


N


%Diff



2.7205  


0.1903  


10  


.  



2.8136  


0.1908  


10  


3.4192  



2.6963  


0.1623  


10   -0.8929  



2.6766  


0.2985  


10   -1.6168  



Ovaries (%)



91 [a]



Mean


SD


N


%Diff



0.0406  


0.0048  


10  


.  



0.0448  


0.0050  


10  


10.3760  



0.0409  


0.0055  


10  


0.6560  



0.0390  


0.0078  


10   -4.0506  



Anova & Dunnett Anova & Dunnett(Rank)


Anova & Dunnett(Log)


 


























































Sex: Female


Day(s) Relative to Start Date



G1


0 mg/kg/day



G2


111 mg/kg/day



G3


333 mg/kg/day



G4


1000 mg/kg/day



Pituitary (%)



103b [a]



Mean


SD


N


%Diff



0.0065  


0.0010  


10  


.  



0.0066  


0.0012  


10  


1.4967  



0.0063  


0.0015  


10   -2.2692  



0.0060  


0.0010  


10   -6.7863  



Spleen (%)



91 [a]



Mean


SD


N


%Diff



0.2309  


0.0303  


10  


.  



0.2368  


0.0249  


10  


2.5798  



0.2504  


0.0246  


10  


8.4454  



0.2293  


0.0365  


10   -0.6766  



Thymus (%)



91 [a1]



Mean


SD


N


%Diff



0.1210  


0.0110  


10  


.  



0.1186  


0.0258  


10   -2.0196  



0.1383  


0.0247  


10  


14.2483  



0.1201  


0.0221  


10   -0.8066  



Thyroid with parathyroids


(%)



103b [a]



Mean


SD


N


%Diff



0.0108  


0.0021  


10  


.  



0.0105  


0.0013  


10   -2.9900  



0.0112  


0.0022  


10  


3.9089  



0.0094  


0.0014  


10   -12.6217  



Uterus with cervix


(%)



91 [a2]



Mean


SD


N


%Diff



0.2639  


0.0630  


10  


.  



0.2439  


0.0379  


10   -7.6065  



0.3615  


0.1556  


10  


36.9683  



0.2584  


0.1003  


10   -2.0991  



Anova & Dunnett;           


Anova & Dunnett(Log)


Anova & Dunnett(Rank)


Appendix 15, TABLE 25. Summary of Brain Weights and Organ to Brain Weight Ratios on Day 91 –  Females











































































Sex: Female


Day(s) Relative to Start Date



G1


0 mg/kg/day



G2


111 mg/kg/day



G3


333 mg/kg/day



G4


1000 mg/kg/day



Brain (g)



91 [a]



Mean


SD


N


%Diff



2.0429  


0.0704  


10  


-  



2.0222  


0.0982  


10   -1.0123  



2.0281  


0.0700  


10   -0.7274  



2.0674  


0.0795  


10  


1.2002  



Adrenals (%)



91 [a1]



Mean


SD


N


%Diff



4.3415  


0.3593  


10  


.  



4.4316  


0.8093  


10  


2.0750  



4.6008  


0.2698  


10  


5.9727  



4.5661  


0.5515  


10  


5.1736  



Heart (%)



91 [a]



Mean


SD


N


%Diff



48.2463  


2.5304  


10  


.  



51.0142  


4.5978  


10  


5.7370  



49.4542  


4.2601  


10  


2.5037  



48.8776  


3.7949  


10  


1.3085  



Kidneys (%)



91 [a]



Mean


SD


N


%Diff



84.2497  


7.0691  


10  


.  



85.6302  


7.8237  


10  


1.6386  



82.1495  


7.3198  


10   -2.4928  



87.0850  


7.7178  


10  


3.3654  



Liver (%)



91 [a]



Mean


SD


N


%Diff



367.3662  


36.9055  


10  


.  



395.4801  


42.1633  


10  


7.6528  



364.7693  


32.3434  


10   -0.7069  



371.0654  


22.4820  


10  


1.0070  



Ovaries (%)



91 [a]



Mean


SD


N


%Diff



5.4819  


0.7512  


10  


.  



6.2745  


0.6765  


10  


14.4589  



5.5378  


0.8738  


10  


1.0205  



5.3669  


0.7279  


10   -2.0970  



Pituitary (%)



103b [a]



Mean


SD


N


%Diff



0.8686  


0.1242  


10  


.  



0.9153  


0.1566  


10  


5.3783  



0.8523  


0.1979  


10   -1.8750  



0.8362  


0.1354  


10   -3.7309  



[a] - Anova & Dunnett;            b: Weighed after fixation


[a1] Anova & Dunnett(Rank)


 

















































Sex: Female


Day(s) Relative to Start Date



G1


0 mg/kg/day



G2


111 mg/kg/day



G3


333 mg/kg/day



G4


1000 mg/kg/day



Spleen (%)



91 [a]



Mean


SD


N


%Diff



31.1921  


4.8330  


10  


.  



33.1079  


2.6272  


10  


6.1419  



33.9355  


4.4713  


10  


8.7951  



31.7195  


3.6721  


10  


1.6908  



Thymus (%)



91 [a1]



Mean


SD


N


%Diff



16.3847  


2.2777  


10  


.  



16.5824  


3.4157  


10  


1.2068  



18.6606  


3.3939  


10  


13.8905  



16.6820  


2.6937  


10  


1.8145  



Thyroid with parathyroids


(%)



103b [a]



Mean


SD


N


%Diff



1.4555  


0.2912  


10  


.  



1.4653  


0.1703  


10  


0.6801  



1.5131  


0.2947  


10  


3.9596  



1.3079  


0.1598  


10   -10.1370  



Uterus with cervix


(%)



91 [a2]



Mean


SD


N


%Diff



35.5778  


8.2525  


10  


.  



33.9622  


3.5255  


10   -4.5412  



49.0565  


21.7419  


10  


37.8850  



35.6692  


12.8037  


10  


0.2569  



[a] - Anova & Dunnett;            [a1] - Anova & Dunnett(Log)


[a2] Anova & Dunnett(Rank)


Appendix 15, TABLE 26. Summary of Terminal Fasting Body Weights and Organ Weights on Day 119 – Females



























































Sex: Female


Day(s) Relative to Start Date



G1R


0 mg/kg/day



G4R


1000 mg/kg/day



Terminal


Fasting BW


(g)



Day 119 [p]



Mean


SD


N


%Diff



293.36  


16.57  


5  


-  



308.97  


25.53  


5  


5.32  



Adrenals (g)



Day 119 [p1]



Mean


SD


N


%Diff



0.0917  


0.0158  


5  


-  



0.1034  


0.0173  


5  


12.8300  



Brain (g)



Day 119 [p2]



Mean


SD


N


%Diff



2.0873  


0.0470  


5  


-  



2.1181  


0.1572  


5  


1.4756  



Heart (g)



Day 119 [p2]



Mean


SD


N


%Diff



1.0674  


0.1027  


5  


-  



1.0941  


0.1462  


5  


2.4976  



Kidneys


(g)



Day 119 [p2]



Mean


SD


N


%Diff



1.8137  


0.2827  


5  


-  



1.9450  


0.2069  


5  


7.2439  



Liver (g)



Day 119 [p]



Mean


SD


N


%Diff



8.3472  


1.1153  


5  


-  



8.3153  


1.2175  


5  


-0.3829  



Ovaries (g)



Day 119 [p]



Mean


SD


N


%Diff



0.1200  


0.0160  


5  


-  



0.0875 * 0.0257  


5  


-27.1121  



[p] - Wilcoxon(Rank): [G1RvsG4R]


  {Ovaries} G1RvsG4R: *: Significantly different from vehicle control group at p < 0.05


[p1] - T-Test(Log): [G1RvsG4R]


[p2] T-Test: [G1RvsG4R]


 














































Sex: Female


Day(s) Relative to Start Date



G1R


0 mg/kg/day



G4R


1000 mg/kg/day



Pituitary (g)



Day 124b [p]



Mean


SD


N


%Diff



0.0177  


0.0025  


5  


-  



0.0188  


0.0044  


5  


6.2288  



Spleen (g)



Day 119 [p]



Mean


SD


N


%Diff



0.6260  


0.1032  


5  


-  



0.6587  


0.1059  


5  


5.2169  



Thymus


(g)



Day 119 [p1]



Mean


SD


N


%Diff



0.3458  


0.0447  


5  


.  



0.3144  


0.0458  


5  


-9.0967  



Thyroid with parathyroids


(g)



Day 124b [p1]



Mean


SD


N


%Diff



0.0276  


0.0019  


5  


-  



0.0278  


0.0042  


5  


0.5069  



Uterus with cervix


(g)



Day 119 [p1]



Mean


SD


N


%Diff



0.9721  


0.2480  


5  


.  



1.0763  


0.1814  


5  


10.7255  



[p] - Wilcoxon(Rank): [G1RvsG4R] ;            b: Weighed after fixation


[p1] T-Test: [G1RvsG4R]


Appendix 15, TABLE 27. Summary of Terminal Fasting Body Weights and Organ to Body Weight  Ratios on Day 119 – Females



























































Sex: Female


Day(s) Relative to Start Date



G1R


0 mg/kg/day



G4R


1000 mg/kg/day



Terminal


Fasting BW


(g)



Day 119 [p]



Mean


SD


N


%Diff



293.36  


16.57  


5  


-  



308.97  


25.53  


5  


5.32  



Adrenals (%)



Day 119 [p1]



Mean


SD


N


%Diff



0.0311  


0.0036  


5  


.  



0.0333  


0.0030  


5  


7.1958  



Brain (%)



Day 119 [p1]



Mean


SD


N


%Diff



0.7131  


0.0377  


5  


.  



0.6887  


0.0709  


5  


-3.4179  



Heart (%)



Day 119 [p1]



Mean


SD


N


%Diff



0.3635  


0.0208  


5  


.  



0.3543  


0.0403  


5  


-2.5080  



Kidneys (%)



Day 119 [p1]



Mean


SD


N


%Diff



0.6159  


0.0670  


5  


.  



0.6294  


0.0398  


5  


2.1967  



Liver (%)



Day 119 [p1]



Mean


SD


N


%Diff



2.8368  


0.2202  


5  


.  



2.6833  


0.2081  


5  


-5.4091  



Ovaries (%)



Day 119 [p1]



Mean


SD


N


%Diff



0.0409  


0.0053  


5  


.  



0.0283 * 0.0086  


5  


-30.7594  



[p] - Wilcoxon(Rank): [G1RvsG4R]


[p1] - T-Test: [G1RvsG4R]


  {Ovaries} G1RvsG4R: *: Significantly different from vehicle control group at p < 0.05


 














































Sex: Female


Day(s) Relative to Start Date



G1R


0 mg/kg/day



G4R


1000 mg/kg/day



Pituitary (%)



Day 124b [p]



Mean


SD


N


%Diff



0.0060  


0.0007  


5  


.  



0.0061  


0.0018  


5  


2.2410  



Spleen (%)



Day 119 [p]



Mean


SD


N


%Diff



0.2125  


0.0232  


5  


.  



0.2125  


0.0204  


5  


-0.0015  



Thymus (%)



Day 119 [p]



Mean


SD


N


%Diff



0.1178  


0.0129  


5  


.  



0.1020  


0.0151  


5  


-13.4214  



Thyroid with parathyroids


(%)



Day 124b [p]



Mean


SD


N


%Diff



0.0094  


0.0007  


5  


.  



0.0090  


0.0010  


5  


-4.8501  



Uterus with cervix


(%)



Day 119 [p]



Mean


SD


N


%Diff



0.3343  


0.0992  


5  


.  



0.3481  


0.0506  


5  


4.1434  



[p] - T-Test: [G1RvsG4R] ;            b: Weighed after fixation


Appendix 15, TABLE 28. Summary of Brain Weights and Organ to Brain Weight Ratios on Day 119 –  Females



























































Sex: Female

Day(s) Relative to Start Date


G1R

0 mg/kg/day


G4R

1000 mg/kg/day


Brain (g)


Day 119 [p]


Mean

SD

N

%Diff


2.0873  

0.0470  

5  

-  


2.1181  

0.1572  

5  

1.4756  


Adrenals (%)


Day 119 [p]


Mean

SD

N

%Diff


4.3925  

0.7559  

5  

.  


4.9093  

0.9212  

5  

11.7651  


Heart (%)


Day 119 [p]


Mean

SD

N

%Diff


51.1143  

4.4546  

5  

.  


51.8664  

7.8725  

5  

1.4714  


Kidneys (%)


Day 119 [p1]


Mean

SD

N

%Diff


86.8344  

12.9778  

5  

.  


92.4427  

13.7068  

5  

6.4586  


Liver (%)


Day 119 [p1]


Mean

SD

N

%Diff


400.0200  

53.7943  

5  

.  


393.6943  

59.5292  

5  

-1.5813  


Ovaries (%)


Day 119 [p]


Mean

SD

N

%Diff


5.7514  

0.7765  

5  

.  


4.0846 * 1.0524  

5  

-28.9809  


Pituitary (%)


Day 124b [p2]


Mean

SD

N

%Diff


0.8457  

0.1125  

5  

.  


0.8995  

0.2815  

5  

6.3657  


[p] - T-Test: [G1RvsG4R] ;            b: Weighed after fixation


  {Ovaries} G1RvsG4R: *: Significantly different from vehicle control group at p < 0.05


[p1] - Wilcoxon(Rank): [G1RvsG4R]


[p2] - T-Test(Log): [G1RvsG4R]


 







































Sex: Female


Day(s) Relative to Start Date



G1R


0 mg/kg/day



G4R


1000 mg/kg/day



Spleen (%)



Day 119 [p]



Mean


SD


N


%Diff



29.9853  


4.8725  


5  


.  



31.2922  


5.8740  


5  


4.3584  



Thymus (%)



Day 119 [p1]



Mean


SD


N


%Diff



16.5505  


1.9143  


5  


.  



14.8833  


2.1876  


5  


-10.0731  



Thyroid with parathyroids


(%)



Day 124b [p1]



Mean


SD


N


%Diff



1.3240  


0.0954  


5  


.  



1.3145  


0.2039  


5  


-0.7193  



Uterus with cervix


(%)



Day 119 [p1]



Mean


SD


N


%Diff



46.6818  


12.6254  


5  


.  



50.7888  


7.4906  


5  


8.7980  



[p] - Wilcoxon(Rank): [G1RvsG4R] ;            b: Weighed after fixation [p1] - T-Test: [G1RvsG4R]


Appendix 15, TABLE 29. Summary of Sperm Parameters



























































































































































































































Group No.


Dose


(mg/kg/day)



 



Sperm Motility



Sperm Morphology



Cauda Epididymal Sperm Counts



Percentage of progressive motile sperms



Percentage of motile sperms



Percentage of normal sperms



Percentage of abnormal sperms



Cauda epididymis weight (g)



No. of sperms per cauda epididymis   (x106)



No. of sperms per gram of cauda epididymis (x106)



G1



Mean



64.40



86.40



99.05



0.95



0.32



250.78



793.73



0



SD



8.13



6.70



1.01



1.01



0.02



17.00



84.79



 



n



10



10



10



10



10



10



10



G2



Mean



66.00



87.70



 



 



 



 



 



111



SD



6.86



5.06



NA



NA



NA



NA



NA



 



n



10



10



 



 



 



 



 



G3



Mean



64.60



85.20



 



 



 



 



 



333



SD



10.24



7.74



NA



NA



NA



NA



NA



 



n



10



10



 



 



 



 



 



G4



Mean



62.60



85.10



97.25



2.75



0.30



235.30



774.63



1000



SD



8.57



6.66



2.62



2.62



0.02



26.41



51.20



 



n



10



10



10



10



10



10



10



G1R



Mean



61.00



85.20



 



 



 



 



 



0



SD



15.36



7.98



NA



NA



NA



NA



NA



 



n



5



5



 



 



 



 



 



G4R



Mean



61.40



82.20



 



 



 



 



 



1000



SD



7.47



6.83



NA



NA



NA



NA



NA



 



n



5



5



 



 



 



 



 



Appendix 15, TABLE 30. Summary of Gross Pathology Findings 

----------------------- MALES ----------------------- ---------------------- FEMALES ----------------------


Removal Reason: TERMINAL SACRIFICE                  


 


                      Group No.:                       G1       G2       G3       G4      G1R      G4R       G1       G2       G3       G4      G1R      G4R   


                      Dose (mg/kg/day):                0       111      333      1000      0       1000      0       111      333      1000      0       1000  


                      Number of Animals on Study :     10       10       10       10        5        5       10       10       10       10        5        5                       Number of Animals Completed:    (10)     (10)     (10)     (10)      (5)      (5)     (10)     (10)     (10)     (10)      (5)      (5)                                                    


 


CECUM;


  Submitted.......................................    (10)     (10)     (10)     (10)      (5)      (5)     (10)     (10)     (10)     (10)      (5)      (5)   No Visible Lesions..............................     10        3        0        0        5        5       10        3        0        0        5        5   Contents orange ................................      0        7       10       10        0        0        0        7       10       10        0        0


 


COLON;


  Submitted.......................................    (10)     (10)     (10)     (10)      (5)      (5)     (10)     (10)     (10)     (10)      (5)      (5)   No Visible Lesions..............................     10        8        4        6        5        5       10        9        4        4        5        5   Contents orange ................................      0        2        6        4        0        0        0        1        6        6        0        0


 


ILEUM;


  Submitted.......................................    (10)     (10)     (10)     (10)      (5)      (5)     (10)     (10)     (10)     (10)      (5)      (5)   No Visible Lesions..............................     10        8        9        9        5        5       10       10        9        9        5        5   Contents orange ................................      0        2        1        1        0        0        0        0        1        1        0        0


 


RECTUM;


  Submitted.......................................    (10)     (10)     (10)     (10)      (5)      (5)     (10)     (10)     (10)     (10)      (5)      (5)


  No Visible Lesions..............................     10       10        6        6        5        5       10        9        5        5        5        5   Contents orange ................................      0        0        4        4        0        0        0        1        5        5        0        0


 


TESTES;


  Submitted.......................................    (10)     (10)     (10)     (10)      (5)      (5)      (-)      (-)      (-)      (-)      (-)      (-)   No Visible Lesions..............................     10       10        8        9        5        5        -        -        -        -        -        -


  Flaccid; bilateral .............................      0        0        1        1        0        0        -        -        -        -        -        -


  Flaccid; unilateral ............................      0        0        1        0        0        0        -        -        -        -        -        -


                                                    ----------------------- MALES ----------------------- ---------------------- FEMALES ----------------------


Removal Reason: TERMINAL SACRIFICE                


                      Group No.:                       G1       G2       G3       G4      G1R      G4R       G1       G2       G3       G4      G1R      G4R   


                      Dose (mg/kg/day):                0       111      333      1000      0       1000      0       111      333      1000      0       1000  


                      Number of Animals on Study :     10       10       10       10        5        5       10       10       10       10        5        5                       Number of Animals Completed:    (10)     (10)     (10)     (10)      (5)      (5)     (10)     (10)     (10)     (10)      (5)      (5)                                                    


UTERUS;


  Submitted.......................................     (-)      (-)      (-)      (-)      (-)      (-)     (10)     (10)     (10)     (10)      (5)      (5)   No Visible Lesions..............................      -        -        -        -        -        -       10       10        8        9        4        3   Dilated ........................................      -        -        -        -        -        -        0        0        2        1        1        2


Note:  All the tissues were examined grossly and no gross abnormalities were observed for all the animals.


Appendix 15, TABLE 31. Summary of Histopathology Findings 

Dose (mg/kg/day):                0       111      333      1000      0       1000      0       111      333      1000      0       1000  


                      Number of Animals on Study :     10       10       10       10        5        5       10       10       10       10        5        5                       Number of Animals Completed:    (10)     (10)     (10)     (10)      (5)      (5)     (10)     (10)     (10)     (10)      (5)      (5)                                                    


 


GLANDS, ADRENAL;


  Examined........................................    (10)      (0)      (0)     (10)      (0)      (0)     (10)      (0)      (0)     (10)      (0)      (0)   Within Normal Limits............................      8        0        0        8        0        0       10        0        0       10        0        0   Vacuolation; cortical; bilateral ...............     (2)      (0)      (0)      (2)      (0)      (0)      (0)      (0)      (0)      (0)      (0)      (0)     minimal ......................................      2        0        0        2        0        0        0        0        0        0        0        0


 


AORTA;


  Examined........................................    (10)      (0)      (0)     (10)      (0)      (0)     (10)      (0)      (0)     (10)      (0)      (0)


  Within Normal Limits............................     10        0        0       10        0        0       10        0        0       10        0        0


 


BONE MARROW SMEAR;


  Examined........................................    (10)      (0)      (0)     (10)      (0)      (0)     (10)      (0)      (0)     (10)      (0)      (0)


  Within Normal Limits............................     10        0        0       10        0        0       10        0        0       10        0        0 


BRAIN CEREBRUM;


  Examined........................................    (10)      (0)      (0)     (10)      (0)      (0)     (10)      (0)      (0)     (10)      (0)      (0)


  Within Normal Limits............................     10        0        0       10        0        0       10        0        0       10        0        0


 


BRAIN CEREBELLUM;


  Examined........................................    (10)      (0)      (0)     (10)      (0)      (0)     (10)      (0)      (0)     (10)      (0)      (0)


  Within Normal Limits............................     10        0        0       10        0        0       10        0        0       10        0        0


 


BRAIN-MEDULLA OBLONGATA;


  Examined........................................    (10)      (0)      (0)     (10)      (0)      (0)     (10)      (0)      (0)     (10)      (0)      (0)


  Within Normal Limits............................     10        0        0       10        0        0       10        0        0       10        0        0


Observations: Non Neo-Plastic                       ----------------------- MALES ----------------------- ---------------------- FEMALES ----------------------


Removal Reason: TERMINAL SACRIFICE                  


 


                      Group No.:                       G1       G2       G3       G4      G1R      G4R       G1       G2       G3       G4      G1R      G4R   


                      Dose (mg/kg/day):                0       111      333      1000      0       1000      0       111      333      1000      0       1000                         Number of Animals on Study :     10       10       10       10        5        5       10       10       10       10        5        5                       Number of Animals Completed:    (10)     (10)     (10)     (10)      (5)      (5)     (10)     (10)     (10)     (10)      (5)      (5)                                                    


 


BRAIN-PONS;


  Examined........................................     (9)      (0)      (0)     (10)      (0)      (0)     (10)      (0)      (0)     (10)      (0)      (0)   Within Normal Limits............................      9        0        0       10        0        0       10        0        0       10        0        0   Not Examined: NOT PRESENT ......................      1        0        0        0        0        0        0        0        0        0        0        0


 


CECUM;


  Examined........................................    (10)      (0)      (0)     (10)      (0)      (0)     (10)      (0)      (0)     (10)      (0)      (0)


  Within Normal Limits............................     10        0        0       10        0        0       10        0        0       10        0        0


 


CERVIX;


  Examined........................................     (-)      (-)      (-)      (-)      (-)      (-)     (10)      (0)      (0)     (10)      (0)      (0)


  Within Normal Limits............................      -        -        -        -        -        -       10        0        0       10        0        0


 


COLON;


  Examined........................................    (10)      (0)      (0)     (10)      (0)      (0)     (10)      (0)      (0)     (10)      (0)      (0)


  Within Normal Limits............................     10        0        0       10        0        0       10        0        0       10        0        0


 


COAGULATING GLAND;


  Examined........................................    (10)      (0)      (0)     (10)      (0)      (0)      (-)      (-)      (-)      (-)      (-)      (-)


  Within Normal Limits............................     10        0        0       10        0        0        -        -        -        -        -        -


 


DUODENUM;


  Examined........................................    (10)      (0)      (0)     (10)      (0)      (0)     (10)      (0)      (0)     (10)      (0)      (0)


  Within Normal Limits............................     10        0        0       10        0        0       10        0        0       10        0        0


 


Observations: Non Neo-Plastic                       ----------------------- MALES ----------------------- ---------------------- FEMALES ----------------------


Removal Reason: TERMINAL SACRIFICE                  


 


                      Group No.:                       G1       G2       G3       G4      G1R      G4R       G1       G2       G3       G4      G1R      G4R   


                      Dose (mg/kg/day):                0       111      333      1000      0       1000      0       111      333      1000      0       1000  


                      Number of Animals on Study :     10       10       10       10        5        5       10       10       10       10        5        5                       Number of Animals Completed:    (10)     (10)     (10)     (10)      (5)      (5)     (10)     (10)     (10)     (10)      (5)      (5)                                                    


 


ESOPHAGUS;


  Examined........................................    (10)      (0)      (0)     (10)      (0)      (0)     (10)      (0)      (0)     (10)      (0)      (0)


  Within Normal Limits............................     10        0        0       10        0        0       10        0        0       10        0        0


 


EYES;


  Examined........................................    (10)      (0)      (0)     (10)      (0)      (0)     (10)      (0)      (0)     (10)      (0)      (0)


  Within Normal Limits............................     10        0        0       10        0        0       10        0        0       10        0        0


 


BONE, FEMUR/JOINT, FEMOROTIBIAL;


  Examined........................................    (10)      (0)      (0)     (10)      (0)      (0)     (10)      (0)      (0)     (10)      (0)      (0)


  Within Normal Limits............................     10        0        0       10        0        0       10        0        0       10        0        0 


FEMORAL MUSCLE;


  Examined........................................    (10)      (0)      (0)     (10)      (0)      (0)     (10)      (0)      (0)     (10)      (0)      (0)


  Within Normal Limits............................     10        0        0       10        0        0       10        0        0       10        0        0


 


GUT ASSOCIATED LYMPHOID TISSUE;


  Examined........................................    (10)      (0)      (0)      (9)      (0)      (0)     (10)      (0)      (0)     (10)      (0)      (0)   Within Normal Limits............................     10        0        0        9        0        0       10        0        0       10        0        0   Not Examined: NOT PRESENT ......................      0        0        0        1        0        0        0        0        0        0        0        0


 


HEART;


  Examined........................................    (10)      (0)      (0)     (10)      (0)      (0)     (10)      (0)      (0)     (10)      (0)      (0)   Within Normal Limits............................      8        0        0        7        0        0       10        0        0       10        0        0   Infiltration; mononuclear cell .................     (2)      (0)      (0)      (3)      (0)      (0)      (0)      (0)      (0)      (0)      (0)      (0)     minimal ......................................      2        0        0        3        0        0        0        0        0        0        0        0


 


Observations: Non Neo-Plastic                       ----------------------- MALES ----------------------- ---------------------- FEMALES ----------------------


Removal Reason: TERMINAL SACRIFICE                  


 


                      Group No.:                       G1       G2       G3       G4      G1R      G4R       G1       G2       G3       G4      G1R      G4R   


                      Dose (mg/kg/day):                0       111      333      1000      0       1000      0       111      333      1000      0       1000  


                      Number of Animals on Study :     10       10       10       10        5        5       10       10       10       10        5        5                       Number of Animals Completed:    (10)     (10)     (10)     (10)      (5)      (5)     (10)     (10)     (10)     (10)      (5)      (5)                                                    


 


ILEUM;


  Examined........................................    (10)      (0)      (0)     (10)      (0)      (0)     (10)      (0)      (0)     (10)      (0)      (0)


  Within Normal Limits............................     10        0        0       10        0        0       10        0        0       10        0        0


 


JEJUNUM;


  Examined........................................    (10)      (0)      (0)     (10)      (0)      (0)     (10)      (0)      (0)     (10)      (0)      (0)


  Within Normal Limits............................     10        0        0       10        0        0       10        0        0       10        0        0


 


KIDNEYS;


  Examined........................................    (10)      (0)      (0)     (10)      (0)      (0)     (10)      (0)      (0)     (10)      (0)      (0)   Within Normal Limits............................     10        0        0        9        0        0        8        0        0        9        0        0   Mineralization; corticomedullary junction;


        unilateral ...............................     (0)      (0)      (0)      (0)      (0)      (0)      (2)      (0)      (0)      (1)      (0)      (0)     minimal ......................................      0        0        0        0        0        0        2        0        0        1        0        0   Casts; tubular; bilateral ......................     (0)      (0)      (0)      (1)      (0)      (0)      (0)      (0)      (0)      (0)      (0)      (0)     minimal ......................................      0        0        0        1        0        0        0        0        0        0        0        0


 


LARYNX;


  Examined........................................    (10)      (0)      (0)      (9)      (0)      (0)     (10)      (0)      (0)     (10)      (0)      (0)


  Within Normal Limits............................     10        0        0        9        0        0       10        0        0       10        0        0


  Not Examined: NOT PRESENT ......................      0        0        0        1        0        0        0        0        0        0        0        0


 


Observations: Non Neo-Plastic                       ----------------------- MALES ----------------------- ---------------------- FEMALES ----------------------


Removal Reason: TERMINAL SACRIFICE                  


 


                      Group No.:                       G1       G2       G3       G4      G1R      G4R       G1       G2       G3       G4      G1R      G4R   


                      Dose (mg/kg/day):                0       111      333      1000      0       1000      0       111      333      1000      0       1000  


                      Number of Animals on Study :     10       10       10       10        5        5       10       10       10       10        5        5                       Number of Animals Completed:    (10)     (10)     (10)     (10)      (5)      (5)     (10)     (10)     (10)     (10)      (5)      (5)                                                    


 


LEFT EPIDIDYMIS;


  Examined........................................    (10)      (0)      (2)     (10)      (0)      (0)      (-)      (-)      (-)      (-)      (-)      (-)   Within Normal Limits............................     10        0        1        9        0        0        -        -        -        -        -        -   Reduced sperm; luminal .........................     (0)      (0)      (1)      (0)      (0)      (0)      (-)      (-)      (-)      (-)      (-)      (-)     marked .......................................      0        0        1        0        0        0        -        -        -        -        -        -   Cell debris; luminal ...........................     (0)      (0)      (1)      (1)      (0)      (0)      (-)      (-)      (-)      (-)      (-)      (-)     minimal ......................................      0        0        1        1        0        0        -        -        -        -        -        -


 


LIVER;


  Examined........................................    (10)      (0)      (0)     (10)      (0)      (0)     (10)      (0)      (0)     (10)      (0)      (0)


  Within Normal Limits............................      4        0        0        2        0        0       10        0        0        9        0        0   Vacuolation; hepatocyte ........................     (6)      (0)      (0)      (7)      (0)      (0)      (0)      (0)      (0)      (1)      (0)      (0)     minimal ......................................      4        0        0        6        0        0        0        0        0        1        0        0     mild .........................................      2        0        0        1        0        0        0        0        0        0        0        0   Infiltration; mononuclear cell .................     (0)      (0)      (0)      (1)      (0)      (0)      (0)      (0)      (0)      (0)      (0)      (0)     minimal ......................................      0        0        0        1        0        0        0        0        0        0        0        0


 


LUNGS (WITH BRONCHI AND BRONCHIOLES);


  Examined........................................    (10)      (0)      (0)     (10)      (0)      (0)     (10)      (0)      (0)     (10)      (0)      (0)   Within Normal Limits............................      9        0        0        8        0        0       10        0        0        9        0        0   Mineralization; blood vessels ..................     (1)      (0)      (0)      (2)      (0)      (0)      (0)      (0)      (0)      (1)      (0)      (0)     minimal ......................................      1        0        0        2        0        0        0        0        0        1        0        0 


MAMMARY GLANDS;


  Examined........................................    (10)      (0)      (0)     (10)      (0)      (0)     (10)      (0)      (0)     (10)      (0)      (0)


  Within Normal Limits............................     10        0        0       10        0        0       10        0        0       10        0        0


 


Observations: Non Neo-Plastic                       ----------------------- MALES ----------------------- ---------------------- FEMALES ----------------------


Removal Reason: TERMINAL SACRIFICE                 


 


                      Group No.:                       G1       G2       G3       G4      G1R      G4R       G1       G2       G3       G4      G1R      G4R   


                      Dose (mg/kg/day):                0       111      333      1000      0       1000      0       111      333      1000      0       1000  


                      Number of Animals on Study :     10       10       10       10        5        5       10       10       10       10        5        5                       Number of Animals Completed:    (10)     (10)     (10)     (10)      (5)      (5)     (10)     (10)     (10)     (10)      (5)      (5)                                                    


 


LYMPH NODES, MESENTERIC;


  Examined........................................    (10)      (0)      (0)     (10)      (0)      (0)     (10)      (0)      (0)     (10)      (0)      (0)


  Within Normal Limits............................     10        0        0       10        0        0       10        0        0       10        0        0


 


LYMPH NODES, MANDIBULAR;


  Examined........................................    (10)      (0)      (0)     (10)      (0)      (0)     (10)      (0)      (0)     (10)      (0)      (0)


  Within Normal Limits............................     10        0        0       10        0        0       10        0        0       10        0        0 


NERVE, OPTIC;


  Examined........................................    (10)      (0)      (0)     (10)      (0)      (0)     (10)      (0)      (0)     (10)      (0)      (0)


  Within Normal Limits............................     10        0        0       10        0        0       10        0        0       10        0        0


 


OVARIES;


  Examined........................................     (-)      (-)      (-)      (-)      (-)      (-)     (10)      (0)      (0)     (10)      (0)      (0)


  Within Normal Limits............................      -        -        -        -        -        -       10        0        0       10        0        0


 


OVIDUCTS;


  Examined........................................     (-)      (-)      (-)      (-)      (-)      (-)     (10)      (0)      (0)     (10)      (0)      (0)


  Within Normal Limits............................      -        -        -        -        -        -       10        0        0       10        0        0


 


PANCREAS;


  Examined........................................    (10)      (0)      (0)     (10)      (0)      (0)     (10)      (0)      (0)     (10)      (0)      (0)


  Within Normal Limits............................     10        0        0       10        0        0       10        0        0       10        0        0


 


PARATHYROID GLAND;


  Examined........................................    (10)      (0)      (0)     (10)      (0)      (0)     (10)      (0)      (0)     (10)      (0)      (0)


  Within Normal Limits............................     10        0        0       10        0        0       10        0        0       10        0        0


 


Observations: Non Neo-Plastic                       ----------------------- MALES ----------------------- ---------------------- FEMALES ----------------------


Removal Reason: TERMINAL SACRIFICE                  


 


                      Group No.:                       G1       G2       G3       G4      G1R      G4R       G1       G2       G3       G4      G1R      G4R   


                      Dose (mg/kg/day):                0       111      333      1000      0       1000      0       111      333      1000      0       1000  


                      Number of Animals on Study :     10       10       10       10        5        5       10       10       10       10        5        5                       Number of Animals Completed:    (10)     (10)     (10)     (10)      (5)      (5)     (10)     (10)     (10)     (10)      (5)      (5)                                                    


 


PHARYNX;


  Examined........................................    (10)      (0)      (0)      (9)      (0)      (0)     (10)      (0)      (0)     (10)      (0)      (0)


  Within Normal Limits............................     10        0        0        9        0        0       10        0        0       10        0        0


  Not Examined: NOT PRESENT ......................      0        0        0        1        0        0        0        0        0        0        0        0


 


PITUITARY GLAND;


  Examined........................................    (10)      (0)      (0)     (10)      (0)      (0)     (10)      (0)      (0)     (10)      (0)      (0)   Within Normal Limits............................      8        0        0        9        0        0       10        0        0       10        0        0   Cyst; pars distalis ............................     (2)      (0)      (0)      (1)      (0)      (0)      (0)      (0)      (0)      (0)      (0)      (0)     present ......................................      2        0        0        1        0        0        0        0        0        0        0        0


 


PROSTATE;


  Examined........................................    (10)      (0)      (0)     (10)      (0)      (0)      (-)      (-)      (-)      (-)      (-)      (-)   Within Normal Limits............................      8        0        0        9        0        0        -        -        -        -        -        -   Infiltration; mononuclear cell .................     (2)      (0)      (0)      (1)      (0)      (0)      (-)      (-)      (-)      (-)      (-)      (-)     minimal ......................................      2        0        0        1        0        0        -        -        -        -        -        -


 


RECTUM;


  Examined........................................    (10)      (0)      (0)     (10)      (0)      (0)     (10)      (0)      (0)     (10)      (0)      (0)


  Within Normal Limits............................     10        0        0       10        0        0       10        0        0       10        0        0


GLANDS, SALIVARY;


  Examined........................................    (10)      (0)      (0)     (10)      (0)      (0)     (10)      (0)      (0)     (10)      (0)      (0)   Within Normal Limits............................     10        0        0       10        0        0       10        0        0       10        0        0                                                   


Observations: Non Neo-Plastic                       ----------------------- MALES ----------------------- ---------------------- FEMALES ----------------------


Removal Reason: TERMINAL SACRIFICE                  


 


                      Group No.:                       G1       G2       G3       G4      G1R      G4R       G1       G2       G3       G4      G1R      G4R   


                      Dose (mg/kg/day):                0       111      333      1000      0       1000      0       111      333      1000      0       1000  


                      Number of Animals on Study :     10       10       10       10        5        5       10       10       10       10        5        5                       Number of Animals Completed:    (10)     (10)     (10)     (10)      (5)      (5)     (10)     (10)     (10)     (10)      (5)      (5)                                                    


 


NERVE, SCIATIC;


  Examined........................................    (10)      (0)      (0)     (10)      (0)      (0)     (10)      (0)      (0)     (10)      (0)      (0)


  Within Normal Limits............................     10        0        0       10        0        0       10        0        0       10        0        0


 


SEMINAL VESICLE;


  Examined........................................    (10)      (0)      (0)     (10)      (0)      (0)      (-)      (-)      (-)      (-)      (-)      (-)


  Within Normal Limits............................     10        0        0       10        0        0        -        -        -        -        -        -


 


SKIN;


  Examined........................................    (10)      (0)      (0)     (10)      (0)      (0)     (10)      (0)      (0)     (10)      (0)      (0)


  Within Normal Limits............................     10        0        0       10        0        0       10        0        0       10        0        0 


SPINAL CORD;


  Examined........................................    (10)      (0)      (0)     (10)      (0)      (0)     (10)      (0)      (0)     (10)      (0)      (0)


  Within Normal Limits............................     10        0        0       10        0        0       10        0        0       10        0        0


SPLEEN;


  Examined........................................    (10)      (0)      (0)     (10)      (0)      (0)     (10)      (0)      (0)     (10)      (0)      (0)   Within Normal Limits............................      9        0        0        8        0        0        9        0        0        9        0        0   Extramedullary hematopoiesis; increased ........     (1)      (0)      (0)      (2)      (0)      (0)      (1)      (0)      (0)      (1)      (0)      (0)     minimal ......................................      1        0        0        2        0        0        1        0        0        1        0        0


 


STERNUM WITH MARROW;


  Examined........................................    (10)      (0)      (0)     (10)      (0)      (0)     (10)      (0)      (0)     (10)      (0)      (0)


  Within Normal Limits............................     10        0        0       10        0        0       10        0        0       10        0        0


Observations: Non Neo-Plastic                       ----------------------- MALES ----------------------- ---------------------- FEMALES ----------------------


Removal Reason: TERMINAL SACRIFICE                 


 


                      Group No.:                       G1       G2       G3       G4      G1R      G4R       G1       G2       G3       G4      G1R      G4R   


                      Dose (mg/kg/day):                0       111      333      1000      0       1000      0       111      333      1000      0       1000  


                      Number of Animals on Study :     10       10       10       10        5        5       10       10       10       10        5        5                       Number of Animals Completed:    (10)     (10)     (10)     (10)      (5)      (5)     (10)     (10)     (10)     (10)      (5)      (5)                                                    


 


STOMACH;


  Examined........................................    (10)      (0)      (0)     (10)      (0)      (0)     (10)      (0)      (0)     (10)      (0)      (0)


  Within Normal Limits............................     10        0        0       10        0        0       10        0        0       10        0        0


 


TESTES;


  Examined........................................    (10)      (0)      (2)     (10)      (0)      (0)      (-)      (-)      (-)      (-)      (-)      (-)


  Within Normal Limits............................      0        0        0        0        0        0        -        -        -        -        -        -   Atrophy; seminiferous tubules; unilateral ......     (0)      (0)      (1)      (1)      (0)      (0)      (-)      (-)      (-)      (-)      (-)      (-)     minimal ......................................      0        0        0        1        0        0        -        -        -        -        -        -     severe .......................................      0        0        1        0        0        0        -        -        -        -        -        -   Dilatation; seminiferous tubules; bilateral ....     (0)      (0)      (1)      (1)      (0)      (0)      (-)      (-)      (-)      (-)      (-)      (-)     minimal ......................................      0        0        1        1        0        0        -        -        -        -        -        -   Sperm granuloma;


unilateral ....................     (0)      (0)      (1)      (0)      (0)      (0)      (-)      (-)      (-)      (-)      (-)      (-)     minimal ......................................      0        0        1        0        0        0        -        -        -        -        -        -   All stages of spermatogenesis normal ...........     10        0        2       10        0        0        -        -        -        -        -        -


 


THYMUS;


  Examined........................................    (10)      (0)      (0)     (10)      (0)      (0)     (10)      (0)      (0)     (10)      (0)      (0)   Within Normal Limits............................     10        0        0       10        0        0        9        0        0        8        0        0   Cyst(s); epithelial ............................     (0)      (0)      (0)      (0)      (0)      (0)      (1)      (0)      (0)      (2)      (0)      (0)     present ......................................      0        0        0        0        0        0        1        0        0        2        0        0 


THYROID GLAND;


  Examined........................................    (10)      (0)      (0)     (10)      (0)      (0)     (10)      (0)      (0)     (10)      (0)      (0)


  Within Normal Limits............................     10        0        0       10        0        0       10        0        0       10        0        0


Observations: Non Neo-Plastic                       ----------------------- MALES ----------------------- ---------------------- FEMALES ----------------------


Removal Reason: TERMINAL SACRIFICE                 


 


                      Group No.:                       G1       G2       G3       G4      G1R      G4R       G1       G2       G3       G4      G1R      G4R   


                      Dose (mg/kg/day):                0       111      333      1000      0       1000      0       111      333      1000      0       1000  


                      Number of Animals on Study :     10       10       10       10        5        5       10       10       10       10        5        5                       Number of Animals Completed:    (10)     (10)     (10)     (10)      (5)      (5)     (10)     (10)     (10)     (10)      (5)      (5)                                                


 


TONGUE;


  Examined........................................    (10)      (0)      (0)     (10)      (0)      (0)     (10)      (0)      (0)     (10)      (0)      (0)


  Within Normal Limits............................     10        0        0       10        0        0       10        0        0       10        0        0


 


TRACHEA;


  Examined........................................    (10)      (0)      (0)     (10)      (0)      (0)     (10)      (0)      (0)     (10)      (0)      (0)


  Within Normal Limits............................     10        0        0       10        0        0       10        0        0       10        0        0


URINARY BLADDER;


  Examined........................................    (10)      (0)      (0)     (10)      (0)      (0)     (10)      (0)      (0)     (10)      (0)      (0)


  Within Normal Limits............................     10        0        0       10        0        0       10        0        0       10        0        0


 


UTERUS;


  Examined........................................     (-)      (-)      (-)      (-)      (-)      (-)     (10)      (0)      (2)     (10)      (1)      (2)


  Within Normal Limits............................      -        -        -        -        -        -        9        0        0        9        0        0   Dilatation; luminal ............................     (-)      (-)      (-)      (-)      (-)      (-)      (1)      (0)      (2)      (1)      (1)      (2)     minimal ......................................      -        -        -        -        -        -        1        0        0        0        0        0     mild .........................................      -        -        -        -        -        -        0        0        0        0        0        1     moderate .....................................      -        -        -        -        -        -        0        0        2        1        1        1


 


VAGINA;


  Examined........................................     (-)      (-)      (-)      (-)      (-)      (-)     (10)      (0)      (0)     (10)      (0)      (0)


  Within Normal Limits............................      -        -        -        -        -        -       10        0        0       10        0        0


 

Conclusions:
As there were no treatment-related adverse effects observed up to the highest dose the No Observed Adverse Effect Level (NOAEL) for systemic toxicity of the test item C.I. Pigment Orange 36 is considered to be 1000 mg/kg/day under the test conditions and doses employed
Executive summary:

 


The purpose of this repeated dose toxicity study was to evaluate the systemic toxicity profile of the test item, C.I. Pigment Orange 36 in Wistar rats when administered orally by gavage for a period of 90 consecutive daysand to assess the reversibilityof any effects during a subsequent 28days recovery period. This study was also intended to provide the information on major toxic effects, target organs and an estimation of a No Observed Adverse Effect Level (NOAEL). 


The test item was weighed and suspended in vehicle,i.e.,0.5% Carboxymethylcellulose Sodium salt (medium viscosity) in Milli-Q®Water and administered to rats at the graduated dose levels of 111, 333 and 1000 mg/kg/day for low dose (G2), mid dose (G3) and high dose (G4 )/ high dose recovery (G4R) group rats, respectively. The rats in the vehicle control group (G1)/vehicle control recovery (G1R) groups received vehicle Carboxymethylcellulose alone. The dose volume administered was 10 mL/kg body weight. Each main group in the experiment was comprised of 10 male and 10 female rats and recovery groups comprised of 5 male and 5 female rats.


The identity of the test item was provided by the Sponsor by a Certificate of Analysis (CoA). The authenticity of the test item was not determined at the test facility. The stability of the test item in the vehicle was established separately under Eurofins Advinus Study No. G19462 at 1 and 100 mg/mL. Based on the results, the test item was found to be stable and homogeneous in the vehicle up to 24 hours when stored at room temperature.


During the conduct of this study, the prepared dose formulations and vehicle (Carboxy methylcellulose Sodium salt (medium viscosity) were analyzed for homogeneity and active ingredient (a.i.) concentration on Day 1 and during 2ndmonth (Day 34) and 3rdmonth (Day 63) of the treatment.The results indicated thatthe percent agreement of the analyzed concentrations were in the range, 85% to 115% of the claimed concentrations and the overall % RSD from six replicates at each dose level was<10.0%. This indicates that the prepared dose formulation met the acceptance criteria for concentration and % RSD.


Each rat in the experiment was observed for clinical signs, mortality and morbidity. Ophthalmological examination was carried out for all the rats prior to start of treatment, at the end of treatment for main groups and at the end of recovery period for recovery groups. The body weights and food consumption were measured during in-life phase of the experiment. Neurological examinations were conducted towards the end of treatment (Day 84) for main groups and towards the end of recovery period (Day 117) for recovery groups.The clinical laboratory investigations such as haematology, coagulation, clinical chemistry, hormone analysis and urine analysis were performed at termination. Vaginal smear was examined in the female rats and the stage ofoestrous cycle was recordedprior to necropsy.


All rats in the experiment were subjected to detailed necropsy and the organ weights and their ratios were derived as percent fasting body weights and brain weight. Histopathological examination was carried out on the preserved organs of the vehicle control (G1) and high dose(G4) group animals. Histopathological examination of the testes included a qualitative assessment of stages of spermatogenesis.In addition, gross lesions from all the animals were examined microscopically.There were no test item-related histopathological changes observed in any organ/tissue in high dose group (G4); hence, histopathological evaluation was not carried out in thelower dose (G2and G3) and recovery groups (G1R and G4R).


Under the experimental conditions employed, the following results were obtained:


·        Clinical Signs and Mortality:Orangecolour faecal matter(light to dark) were observed at all the tested doses in both sexes. This could be due to physical nature of the test item. There were no mortality observed at any of the doses tested in both sexes.


·        Ophthalmological Examination:Ophthalmological examination did not reveal any ocular abnormalities.


·        Neurological Findings:No treatment-related neurological abnormalities /dysfunctions were observed at all the doses tested.


·        Body Weights:Treatment did not affect body weight at all the tested doses in either sex.


·        Food Consumption:Treatment did not affect food consumption at all the tested doses in either sex.


·        Haematology, Coagulation, Clinical Chemistry and urine parameters:There were no test item related alterations observed at any of the tested dose levels in either sex.


·        Thyroid Hormone Profile:Thyroid hormone profile (TSH, T4 and T3) was not affected in both sexes across the treated groups when compared to the concurrent vehicle control group.


·        Terminal Fasting Body Weights and Organ Weights:No significant changes in terminal fasting body weights and organ weights attributed to test item were observedat any of the tested dose levels in either sex.


·        Sperm Parameter:There were no test item-related changes in any of the sperm parameters.


·        Gross pathology:There were no test item-related gross pathological changes observed in both sexes. Orange colouration of intestinal contents (ileum, cecum, colon and rectum) observed in both sexes at all doses at the end of treatment period was attributed to the test item colour.


·        Histopathology:There were no test item-related microscopic lesions in any evaluated organs or tissues of male and female rats at the end of treatment period at tested dose levels.


No Observed Adverse Effect Level (NOAEL):


As there were no treatment-related adverse effects observed up to the highest dose the No Observed Adverse Effect Level (NOAEL)for systemic toxicityof the test item C.I. Pigment Orange 36 is considered to be 1000 mg/kg/day under the test conditions and doses employed.

Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study (OECD 422, GLP-compliant)
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EPA OPPTS 870.3650 (Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Tocixity Screening Test)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Animals: Rat, RccHanTM: WIST(SPF)
- Rationale: Recognized by international guidelines as a recommended test system.
- Source: Harlan Laboratories, Inc., Maasheseweg 87c, 5800 AN Vernay / Netherlands
- Number of Animals: 44 males (11 per group) and 44 females (11 per group)
- Age (at Start of Treatment): 11 weeks
- Body Weight Range (at Start of Treatment): 301 to 362 g (males), 216 to 247 g (females)
- Identification: Parent animals had cage card and individual animal number (ear tattoo), pups were individually tattooed with Indian ink on day 1 post partum
- Randomization: Performed after at least three days of acclimatization using a computer-generated random algorithm. Body weights (recorded on the day of allocation) were taken into consideration in order to ensure similar mean body weights in all groups.
- Accommodation: In groups of five in Makrolon type-4 cages with wire mesh tops up to the day of randomization and afterwards individually in Makrolon type-3 cages with wire mesh tops and sterilized standard softwood bedding (‘Lignocel’ J. Rettenmaier & Söhne GmbH & CoKG, 73494 Rosenberg / Germany, imported by Provimi Kliba SA, 4303 Kaiseraugst / Switzerland) with paper enrichment (ISO-BLOX from Harlan Laboratories B.V., Netherlands), batch/lot nos. 02105111001, 02105111201, 02105120301 and 6960C.CS-100099). During the pre-pairing period, cages with males were interspersed amongst those holding females to promote the development of regular estrus cycles.
- Diet: Pelleted standard Harlan Teklad 2018C (batch no. 80/11) rodent maintenance diet (Provimi Kliba SA, 4303 Kaiseraugst / Switzerland) was available ad libitum.
- Water: Community tap-water from Füllinsdorf was available ad libitum in water bottles.
- Acclimatization: Under test conditions after health examination. Only animals without any visible signs of illness were used for the study.

ENVIRONMENTAL CONDITIONS
Standard laboratory conditions, continuously monitored.
- Temperature (°C): 22 +/- 3
- Humidity (%): 30 to 70
- Air changes (per hr): . Air-conditioned with 10 - 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12/12 (with at least eight hours music during the light period)
Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
DOSE FORMULATIONS
The dose formulations were prepared weekly using the test item as supplied by the Sponsor.
The test item was weighed into a glass beaker on a tared precision balance and approximately 80% of the vehicle was added (w/v). Using a magnetic stirrer, a homogeneous suspension was prepared. Having obtained a homogeneous mixture, the remaining vehicle was added. Separate formulations were prepared for each concentration.
Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.

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

TREATMENT
- Method: Oral, by gavage
- Rationale for Method: Administration by gavage is a common and accepted route of exposure for this type of studies.
- Frequency of Administration: Once daily
- Target Dose Levels: 0 mg/kg/day (control group), 100 mg/kg/day (group 2), 300 mg/kg/day (group 3) and 1000 mg/kg/day (group 4)
- Rationale for Dose Level Selection: The dose levels were selected based on a previous non-GLP dose range-finding toxicity study in Han Wistar rats, Harlan Laboratories Study D33711, using dose levels of 0, 100, 300 and 1000 mg/kg/ day, where no adverse effects were observed up to and including the highest dose level.
- Dose Volume: 10 mL/kg body weight
- Dose Concentrations: 0 mg/mL/day (control group), 10 mg/mL/day (group 2), 30 mg/mL/day (group 3) and 100 mg/mL/day (group 4).
- Duration of Acclimatization Period: 7 days.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
METHOD
On the first treatment day samples from the control group as well as three samples (top, middle and bottom) of about 0.5 g of each concentration were taken prior to dosing for analysis of concentration and homogeneity. Samples of about 0.5 g of each concentration were taken from the middle only to confirm stability (8 days). During the last week of the treatment, samples were taken from the middle to confirm concentration. The aliquots for analysis of dose formulations were frozen (-20 +/- 5 °C) and delivered on dry ice to the responsible for formulation analysis (Harlan Laboratories Ltd., Itingen / Switzerland) and stored there at -20 +/- 5 °C until analysis.
The samples were analyzed by UV-VIS spectroscopy following an analytical procedure developed at Harlan Laboratories. The test item was used as the analytical standard.

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

In conclusion, the results indicate the accurate use of the test item and highly purified water as vehicle during this study. Application formulations were found to be homogeneously prepared and stable over a storage period of eight days (20 +/- 5 °C).
Duration of treatment / exposure:
MALES: 40 days
FEMALES: Approximately 7 weeks
Frequency of treatment:
Once daily
Remarks:
Doses / Concentrations:
0, 100, 300, 1000 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
11

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

FEMALES
- Acclimatization: 7 days
- First Test Item Administration: Day 1 of pre-pairing
- Pre-Pairing: 14 days
- Pairing: 14 days maximum
- Gestation: Approximately 21 days
- Treatment Ends: On day 4 post partum
- Blood Sampling: Day 5 post partum
- Necropsy: On day 5 post partum (pups on day 4 post partum)
Positive control:
Not required
Observations and examinations performed and frequency:
VIABILITY/MORTALITY: Twice daily

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

HISTOPATHOLOGY
Slides of all organs and tissues listed collected at terminal sacrifice from the animals of the control and high-dose groups were examined by the study pathologist. The same applied to all occurring gross lesions.
Special emphasis was made on the stages of spermatogenesis and histopathology of interstitial cell structure.
Histological examination of ovaries was carried out on any females that did not give birth. In addition, microscopic examination of the reproductive organs of all infertile males was made, where necessary.
A histopathology peer review was performed. A histopathology phase report was provided by the principal investigator which was included in the report.
Other examinations:
MATING, GESTATION, LACTATION
During the pairing period, females were housed with sexually mature males (1:1) until evidence of copulation was observed. The females were removed and housed individually if the daily vaginal smear was sperm positive, or a copulation plug was observed. The day on which a positive mating was determined (copulation plug or sperm) was designated day 0 post coitum. For a female which did not mate during the 14-day pairing period, a second pairing of this female with a male in the same group, which had already mated successfully, was performed. All dams were allowed to give birth and rear their litters (F1 pups) up to day 4 post partum. Day 0 was designated as the day on which a female had delivered all her pups.

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

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

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

Statistics:
The following statistical methods were used to analyze food consumption, body weights and reproduction data:
- Means and standard deviations of various data were calculated.
- The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.
- Fisher's exact-test was applied when the variables could be dichotomized without loss of information.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Red stained faeces were noted in all males and females in dose groups; this was due to the staining properties of the test item
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Red stained faeces were noted in all males and females in dose groups; this was due to the staining properties of the test item
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
In males at the dose level of 1000 mg/kg bw/day, lower body weight gain during the pre-pairing period
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Locomotor activity was not affected and functional observational battery gave no indication of a test item-related effect.
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Details on results:
1. IN-LIFE DATA OF PARENTAL ANIMALS
VIABILITY / MORTALITY
All animals survived scheduled study period.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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


Key result
Dose descriptor:
NOAEL
Remarks:
P (for general toxicity)
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed up to 1000 mg/kg bw/day, the highest dose level tested.
Dose descriptor:
NOAEL
Remarks:
P (for reproduction)
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed up to 1000 mg/kg bw/day, the highest dose level tested.
Dose descriptor:
NOAEL
Remarks:
F1 (for development)
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed up to 1000 mg/kg bw/day, the highest dose level tested.
Critical effects observed:
not specified

1. REPRODUCTION, BREEDING AND PUP DATA

SUMMARY OF PERFORMANCE

P Animals Breeding for F1 Litters

Group
(mg/kg/day)

1
(0)

2
(100)

3
(300)

4
(100)

Female numbers

45-55

56-66

67-77

78-88

Number of females paired

11

11

11

11

Number of females mated

11

11

11

11

Number of non pregnant females (A)

3

1

3

0

Numbers of pregnant females,
which did not deliver any pups (B)

0

0

0

1

Number of females which reared their pups until day 4 post partum

8

10

8

10

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

(B)  Female No. 85 had implantations only.

 

MATING PERFORMANCE AND FERTILITY

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

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

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

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

 

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

 

CORPORA LUTEA COUNT

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

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

 

DURATION OF GESTATION

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

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

 

IMPLANTATION RATE AND POST-IMPLANTATION LOSS

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

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

 

LITTER SIZE AT FIRST LITTER CHECK

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

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

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

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

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

 

POSTNATAL LOSS DAYS 0 - 4 POST PARTUM

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

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

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

 

EXTERNAL EXAMINATION AT FIRST LITTER CHECK AND DURING LACTATION

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

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

 

SEX RATIOS

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

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

 

 BODY WEIGHTS TO DAY 4 POST PARTUM

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

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

 

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

 

MACROSCOPICAL FINDINGS

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

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

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

 

Conclusions:
This study is a valid investigation of the toxicological effects resulting from repeated oral-gavage administration of the test item to rats (according to OECD 422, GLP compliant). The test item was administered in vehicle (highly purified water) at dosages of 0, 100, 300, and 1000 mg/kg body weight/day, animals in control groups received the vehicle only. Test item was administered to male rats for 28 days and to female rats for 14 days prior to pairing, through the pairing and gestation periods until the F1 generation reached day 4 post partum.
Under the conditions of this study, no adverse effects were found in males or females up to the highest dose level of 1000 mg/kg bw/day.

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

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

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

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

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


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

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

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

 

The test item was administered to male rats for 39 days and to female rats for 14 days prior to pairing, through the pairing and gestation periods until the F1 generation reached day 4 post partum.

 

The following dose levels were applied:

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

Group 2:                    100 mg/kg body weight/day

Group 3:                    300 mg/kg body weight/day

Group 4:                   1000 mg/kg body weight/day

 

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

 

The following results were obtained:

 MORTALITY AND GENERAL TOLERABILITY OF PARENTAL ANIMALS

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

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

 

FUNCTIONAL OBSERVATIONAL BATTERY IN PARENTAL ANIMALS

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

 

FOOD CONSUMPTION OF PARENTAL ANIMALS

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

 

BODY WEIGHTS OF PARENTAL ANIMALS

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

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

 

CLINICAL LABORATORY INVESTIGATIONS IN PARENTAL ANIMALS

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

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

 

REPRODUCTION AND BREEDING DATA

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

 

SEMINOLOGY AND SPERMATID COUNT IN PARENTAL MALES

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

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

These findings were shown to be artefacts due to processing of the cells rather than effects of the test material in a separate study described hereafter (see chapter 7.8.1 Key_Sperm-motility)

ORGAN WEIGHTS OF PARENTAL ANIMALS

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

 

MACROSCOPICAL FINDINGS AND HISTOPATHOLOGICAL EXAMINATIONS OF PARENTAL ANIMALS

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

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

 

FINDINGS IN PUPS AT FIRST LITTER CHECK AND DURING LACTATION

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

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

 

PUP WEIGHTS TO DAY 4 POST PARTUM

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

 

MACROSCOPICAL FINDINGS OF PUPS

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

 

CONCLUSION

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

Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study (OECD 422, GLP-compliant)
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EPA OPPTS 870.3650 (Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Tocixity Screening Test)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Animals: Rat, RccHanTM: WIST(SPF)
- Rationale: Recognized by international guidelines as a recommended test system.
- Source: Harlan Laboratories, Inc., Maasheseweg 87c, 5800 AN Vernay / Netherlands
- Number of Animals: 44 males (11 per group) and 44 females (11 per group)
- Age (at Start of Treatment): 11 weeks
- Body Weight Range (at Start of Treatment): 301 to 362 g (males), 216 to 247 g (females)
- Identification: Parent animals had cage card and individual animal number (ear tattoo), pups were individually tattooed with Indian ink on day 1 post partum
- Randomization: Performed after at least three days of acclimatization using a computer-generated random algorithm. Body weights (recorded on the day of allocation) were taken into consideration in order to ensure similar mean body weights in all groups.
- Accommodation: In groups of five in Makrolon type-4 cages with wire mesh tops up to the day of randomization and afterwards individually in Makrolon type-3 cages with wire mesh tops and sterilized standard softwood bedding (‘Lignocel’ J. Rettenmaier & Söhne GmbH & CoKG, 73494 Rosenberg / Germany, imported by Provimi Kliba SA, 4303 Kaiseraugst / Switzerland) with paper enrichment (ISO-BLOX from Harlan Laboratories B.V., Netherlands), batch/lot nos. 02105111001, 02105111201, 02105120301 and 6960C.CS-100099). During the pre-pairing period, cages with males were interspersed amongst those holding females to promote the development of regular estrus cycles.
- Diet: Pelleted standard Harlan Teklad 2018C (batch no. 80/11) rodent maintenance diet (Provimi Kliba SA, 4303 Kaiseraugst / Switzerland) was available ad libitum.
- Water: Community tap-water from Füllinsdorf was available ad libitum in water bottles.
- Acclimatization: Under test conditions after health examination. Only animals without any visible signs of illness were used for the study.

ENVIRONMENTAL CONDITIONS
Standard laboratory conditions, continuously monitored.
- Temperature (°C): 22 +/- 3
- Humidity (%): 30 to 70
- Air changes (per hr): . Air-conditioned with 10 - 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12/12 (with at least eight hours music during the light period)
Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
DOSE FORMULATIONS
The dose formulations were prepared weekly using the test item as supplied by the Sponsor.
The test item was weighed into a glass beaker on a tared precision balance and approximately 80% of the vehicle was added (w/v). Using a magnetic stirrer, a homogeneous suspension was prepared. Having obtained a homogeneous mixture, the remaining vehicle was added. Separate formulations were prepared for each concentration.
Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.

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

TREATMENT
- Method: Oral, by gavage
- Rationale for Method: Administration by gavage is a common and accepted route of exposure for this type of studies.
- Frequency of Administration: Once daily
- Target Dose Levels: 0 mg/kg/day (control group), 100 mg/kg/day (group 2), 300 mg/kg/day (group 3) and 1000 mg/kg/day (group 4)
- Rationale for Dose Level Selection: The dose levels were selected based on a previous non-GLP dose range-finding toxicity study in Han Wistar rats, Harlan Laboratories Study D33711, using dose levels of 0, 100, 300 and 1000 mg/kg/ day, where no adverse effects were observed up to and including the highest dose level.
- Dose Volume: 10 mL/kg body weight
- Dose Concentrations: 0 mg/mL/day (control group), 10 mg/mL/day (group 2), 30 mg/mL/day (group 3) and 100 mg/mL/day (group 4).
- Duration of Acclimatization Period: 7 days.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
METHOD
On the first treatment day samples from the control group as well as three samples (top, middle and bottom) of about 0.5 g of each concentration were taken prior to dosing for analysis of concentration and homogeneity. Samples of about 0.5 g of each concentration were taken from the middle only to confirm stability (8 days). During the last week of the treatment, samples were taken from the middle to confirm concentration. The aliquots for analysis of dose formulations were frozen (-20 +/- 5 °C) and delivered on dry ice to the responsible for formulation analysis (Harlan Laboratories Ltd., Itingen / Switzerland) and stored there at -20 +/- 5 °C until analysis.
The samples were analyzed by UV-VIS spectroscopy following an analytical procedure developed at Harlan Laboratories. The test item was used as the analytical standard.

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

In conclusion, the results indicate the accurate use of the test item and highly purified water as vehicle during this study. Application formulations were found to be homogeneously prepared and stable over a storage period of eight days (20 +/- 5 °C).
Duration of treatment / exposure:
MALES: 40 days
FEMALES: Approximately 7 weeks
Frequency of treatment:
Once daily
Remarks:
Doses / Concentrations:
0, 100, 300, 1000 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
11

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

FEMALES
- Acclimatization: 7 days
- First Test Item Administration: Day 1 of pre-pairing
- Pre-Pairing: 14 days
- Pairing: 14 days maximum
- Gestation: Approximately 21 days
- Treatment Ends: On day 4 post partum
- Blood Sampling: Day 5 post partum
- Necropsy: On day 5 post partum (pups on day 4 post partum)
Positive control:
Not required
Observations and examinations performed and frequency:
VIABILITY/MORTALITY: Twice daily

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

HISTOPATHOLOGY
Slides of all organs and tissues listed collected at terminal sacrifice from the animals of the control and high-dose groups were examined by the study pathologist. The same applied to all occurring gross lesions.
Special emphasis was made on the stages of spermatogenesis and histopathology of interstitial cell structure.
Histological examination of ovaries was carried out on any females that did not give birth. In addition, microscopic examination of the reproductive organs of all infertile males was made, where necessary.
A histopathology peer review was performed. A histopathology phase report was provided by the principal investigator which was included in the report.
Other examinations:
MATING, GESTATION, LACTATION
During the pairing period, females were housed with sexually mature males (1:1) until evidence of copulation was observed. The females were removed and housed individually if the daily vaginal smear was sperm positive, or a copulation plug was observed. The day on which a positive mating was determined (copulation plug or sperm) was designated day 0 post coitum. For a female which did not mate during the 14-day pairing period, a second pairing of this female with a male in the same group, which had already mated successfully, was performed. All dams were allowed to give birth and rear their litters (F1 pups) up to day 4 post partum. Day 0 was designated as the day on which a female had delivered all her pups.

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

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

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

Statistics:
The following statistical methods were used to analyze food consumption, body weights and reproduction data:
- Means and standard deviations of various data were calculated.
- The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.
- Fisher's exact-test was applied when the variables could be dichotomized without loss of information.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Red stained faeces were noted in all males and females in dose groups; this was due to the staining properties of the test item
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Red stained faeces were noted in all males and females in dose groups; this was due to the staining properties of the test item
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
In males at the dose level of 1000 mg/kg bw/day, lower body weight gain during the pre-pairing period
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Locomotor activity was not affected and functional observational battery gave no indication of a test item-related effect.
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Details on results:
1. IN-LIFE DATA OF PARENTAL ANIMALS
VIABILITY / MORTALITY
All animals survived scheduled study period.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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


Key result
Dose descriptor:
NOAEL
Remarks:
P (for general toxicity)
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed up to 1000 mg/kg bw/day, the highest dose level tested.
Dose descriptor:
NOAEL
Remarks:
P (for reproduction)
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed up to 1000 mg/kg bw/day, the highest dose level tested.
Dose descriptor:
NOAEL
Remarks:
F1 (for development)
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed up to 1000 mg/kg bw/day, the highest dose level tested.
Critical effects observed:
not specified

1. REPRODUCTION, BREEDING AND PUP DATA

SUMMARY OF PERFORMANCE

P Animals Breeding for F1 Litters

Group
(mg/kg/day)

1
(0)

2
(100)

3
(300)

4
(100)

Female numbers

45-55

56-66

67-77

78-88

Number of females paired

11

11

11

11

Number of females mated

11

11

11

11

Number of non pregnant females (A)

3

1

3

0

Numbers of pregnant females,
which did not deliver any pups (B)

0

0

0

1

Number of females which reared their pups until day 4 post partum

8

10

8

10

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

(B)  Female No. 85 had implantations only.

 

MATING PERFORMANCE AND FERTILITY

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

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

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

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

 

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

 

CORPORA LUTEA COUNT

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

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

 

DURATION OF GESTATION

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

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

 

IMPLANTATION RATE AND POST-IMPLANTATION LOSS

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

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

 

LITTER SIZE AT FIRST LITTER CHECK

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

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

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

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

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

 

POSTNATAL LOSS DAYS 0 - 4 POST PARTUM

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

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

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

 

EXTERNAL EXAMINATION AT FIRST LITTER CHECK AND DURING LACTATION

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

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

 

SEX RATIOS

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

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

 

 BODY WEIGHTS TO DAY 4 POST PARTUM

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

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

 

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

 

MACROSCOPICAL FINDINGS

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

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

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

 

Conclusions:
This study is a valid investigation of the toxicological effects resulting from repeated oral-gavage administration of the test item to rats (according to OECD 422, GLP compliant). The test item was administered in vehicle (highly purified water) at dosages of 0, 100, 300, and 1000 mg/kg body weight/day, animals in control groups received the vehicle only. Test item was administered to male rats for 28 days and to female rats for 14 days prior to pairing, through the pairing and gestation periods until the F1 generation reached day 4 post partum.
Under the conditions of this study, no adverse effects were found in males or females up to the highest dose level of 1000 mg/kg bw/day.

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

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

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

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

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


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

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

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

 

The test item was administered to male rats for 39 days and to female rats for 14 days prior to pairing, through the pairing and gestation periods until the F1 generation reached day 4 post partum.

 

The following dose levels were applied:

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

Group 2:                    100 mg/kg body weight/day

Group 3:                    300 mg/kg body weight/day

Group 4:                   1000 mg/kg body weight/day

 

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

 

The following results were obtained:

 MORTALITY AND GENERAL TOLERABILITY OF PARENTAL ANIMALS

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

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

 

FUNCTIONAL OBSERVATIONAL BATTERY IN PARENTAL ANIMALS

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

 

FOOD CONSUMPTION OF PARENTAL ANIMALS

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

 

BODY WEIGHTS OF PARENTAL ANIMALS

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

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

 

CLINICAL LABORATORY INVESTIGATIONS IN PARENTAL ANIMALS

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

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

 

REPRODUCTION AND BREEDING DATA

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

 

SEMINOLOGY AND SPERMATID COUNT IN PARENTAL MALES

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

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

These findings were shown to be artefacts due to processing of the cells rather than effects of the test material in a separate study described hereafter (see chapter 7.8.1 Key_Sperm-motility)

ORGAN WEIGHTS OF PARENTAL ANIMALS

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

 

MACROSCOPICAL FINDINGS AND HISTOPATHOLOGICAL EXAMINATIONS OF PARENTAL ANIMALS

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

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

 

FINDINGS IN PUPS AT FIRST LITTER CHECK AND DURING LACTATION

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

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

 

PUP WEIGHTS TO DAY 4 POST PARTUM

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

 

MACROSCOPICAL FINDINGS OF PUPS

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

 

CONCLUSION

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

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
see Rationale and Justification for the Analogue Read-Across Approach – Nanoforms and Bulk Forms (Chapter 13)
Reason / purpose for cross-reference:
read-across source
Key result
Dose descriptor:
NOAEL
Remarks:
P; general toxicity
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no effects were observed
Key result
Critical effects observed:
no
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Valid: Guideline study
System:
other: no effects

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14 May 2021 to 29 November 2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
28-day (Sub-acute) inhalation toxicity study” adopted on 25 June 2018.
Qualifier:
according to guideline
Guideline:
OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
Version / remarks:
28-day (Sub-acute) inhalation toxicity study” adopted on 25 June 2018.
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
Rat is the preferred laboratory rodent species for inhalation toxicity assessment and is also recommended by various regulatory guidelines.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: In-House
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 7 - 8 weeks on the day of randomization
- Weight at study initiation: Males: 140.14 g to 153.11 g
Females: 120.06 g to 137.46 g
- Fasting period before study: No
- Housing: Maximum of three animals
- Diet (e.g. ad libitum): yes
- Water (e.g. ad libitum):yes
- Acclimation period:7 days

DETAILS OF FOOD AND WATER QUALITY:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.0°C to 22.7°C
- Humidity (%): 46% to 65%
- Air changes (per hr): 12-15
- Photoperiod (hrs dark / hrs light): 12 hours fluorescent light and 12 hours dark cycle

IN-LIFE DATES: From: To: 17 May 2021 to 16 August 2021
Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Vehicle:
clean air
Mass median aerodynamic diameter (MMAD):
>= 1 - < 2 µm
Geometric standard deviation (GSD):
3
Remarks on MMAD:
1.82 to 1.97
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Inhalation exposure was conducted using a flow-past, nose-only dynamic inhalation exposure system (Refer Annexure 5) supplied by CH Technologies, USA, with a provision of at least 12 air changes per hour.
- Method of holding animals in test chamber: restraining tubes
- Source and rate of air: Compressor air and 20 L/min
- Method of conditioning air: Filtered air
- System of generating particulates/aerosols: The Rotating Brush Generator (Palas RBG 1000 - supplied by Palas GmbH) was used to generate the dust particles (aerosols) into an airborne state.
- Temperature, humidity, pressure in air chamber: 23.2, 57.6, 60psi
- Air flow rate: 20 L/min
- Air change rate: 12 air cahnges per hour
- Method of particle size determination: gravimetric
- Treatment of exhaust air: NaoH

TEST ATMOSPHERE
- Samples taken from breathing zone: yes

Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
6 Hours
Frequency of treatment:
Weekly 5 days
Dose / conc.:
0.003 mg/L air
Remarks:
Low dose
Dose / conc.:
0.01 mg/L air
Remarks:
Mid dose
Dose / conc.:
0.03 mg/L air
Remarks:
High dose
No. of animals per sex per dose:
5 males + 5 females
Details on study design:
- Dose selection rationale: Inhalation is one of the most possible routes of exposure to human. Primarily used to derive regulatory concentrations for assessing worker risk in occupational settings, also used to identify and assess human residential, consumer, transportation, and environmental risk.
- Rationale for selecting satellite groups: persistence of toxic effects.
- Post-exposure recovery period in satellite groups: Following the 28 days exposure period, the animals in the recovery groups (G1R and G4R) were not be given any treatment and was maintained for 56 days post treatment period and observed for reversibility or persistence of toxic effects.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes / No / Not specified
- Time schedule: twice daily
- Cage side observations checked in table [yes] were included.
Table 1

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly once

BODY WEIGHT: Yes
- Time schedule for examinations: weekly once

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: before exposure and week 4 and week 12
- Dose groups that were examined: control and high dose

HAEMATOLOGY: Yes
- Time schedule for collection of blood: day 27 and 85
- Anaesthetic used for blood collection: Yes
- Animals fasted: Yes
- How many animals: 60
- Parameters checked in table [yes] were examined.
table 10

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: day 27 and 85
- Animals fasted: Yes
- How many animals: 60
- Parameters checked in table [yes] were examined.
Table 12

URINALYSIS: Yes
- Time schedule for collection of urine: 27
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked in table [yes] were examined.
Table 13

NEUROBEHAVIOURAL EXAMINATION: Yes / No / Not specified
- Time schedule for examinations:
- Dose groups that were examined:
- Battery of functions tested: sensory activity / grip strength / motor activity / other:

IMMUNOLOGY: Yes
- Time schedule for examinations: week 4 and 12
- How many animals: 40
- Dose groups that were examined: control and high dose
- Parameters checked in table [yes] were examined.
Tables 5, 6, 7 & 8

OTHER:
BALF Fluid
Table 11
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
Statistics:
The raw data were subjected to statistical analysis. The computer printout of the data (in the form of the appendix) was verified with the original raw data. After verification, the data were subjected to statistical analysis using SPSS Software version 22. Body weight, percent change in body weight with respect to Day 1, feed consumption, FOB, organ weight ratios, hematology, and clinical chemistry estimations, urine volume, pH, and specific gravity were subjected to statistical analysis. One-way ANOVA followed by Dunnett’s post-test was done for different treatment groups comparing with the control group data. All analyses and comparisons were evaluated at the 95% level of confidence (P<0.05). Statistically significant changes obtained from the aforementioned tests were designated by the superscripts in the summary table throughout the study report, as stated below:
*: Statistically significant (p<0.05).
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
All animals survived to scheduled sacrifice, and there were no clinical signs attributable to the test item. Post-exposure, all animals were normal throughout the experimental period. No clinical signs were noted during the recovery period.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
No statistically significant treatment-related variations were observed in all the tested dose groups when compared with control group.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
No statistically significant treatment-related variations were observed in all the tested dose groups when compared with control group.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
No ocular abnormalities were observed during ophthalmoscopic examination
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No toxicologically significant treatment related changes in haematology parameters were noted. However, the following statistically significant variations were noted.
In main group males, increase in RBC, haematocrit, MPV, PT in G4 and decrease in MCHC in G3 was noted. In females increase in APTT in G4 was noted.
In recovery males, decrease absolute and relative reticulocyte count (G4R) was noted. In recovery females decrease in percent lymphocytes, absolute and relative reticulocyte count and increase in percent neutrophils (G4R) was noted.
All the noted variations in main group are considered incidental in the absence of dose responsiveness. Further, in the absence of any microscopic variations in the marrow (sternum with marrow), the noted changes are not considered to be adverse. Variations noted at the end of recovery period are considered incidental as similar changes were not noted at the end of treatment period.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
No toxicologically significant changes were observed in clinical chemistry parameters when compared to the control group. However, the following statistically significant variations were noted.
In main group males, increase in AST and calcium in G3 group and sodium in G4 was noted; in females increase in glucose, urea and BUN in G4 group was noted.
In recovery group females, increase in potassium (G4R) was noted.
The variations noted are considered incidental due to lack of dose responsiveness and/or could be due to random biological variation.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
No significant changes were observed in main group and recovery when compared with control group.
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
There were no adverse effects during neurological/functional examinations in main group (G1 and G4) and recovery group (G1R and G4R). However statistically significant decrease in rearing in G4 males, increase in movement counts in G4R females was noted. The noted variations are considered incidental in the absence of any changes in other neurological parameters. Also, all the animals were normal during the experimental period.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
The following statistically significant variations were noted in organ weights and its ratios.
Absolute and relative to fasting body weight
In main group females, decrease in absolute and relative weight of liver (G4), thymus (G2 and G4) was noted.
In recovery group females, decrease in absolute weight of spleen, uterus and relative weight of spleen, uterus (G4R) was noted.

Organ weight changes (relative to brain weight)
In main group females, decrease in relative weight of liver (G4) and thymus (G2 and G4) was noted; in males increase in relative weight of testis (G4M) was noted.
In recovery group females, decrease in relative weight of spleen and uterus (G4R) was noted.
All the observed changes are considered incidental in the absence of any associated gross and histopathological changes in the high dose main group animals.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no gross pathological findings in the study.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Microscopically, test item-related changes were observed in lungs and are summarized as below.
Sex Males
Group G1 G2 G3 G4 G1R G4R
Number of Animals Examined 5 5 5 5 5 5
Lungs
Pigment, golden brown - 5 5 5 - 5

Sex Females
Group G1 G2 G3 G4 G1R G4R
Number of Animals Examined 5 5 5 5 5 5
Lungs
Pigment, golden brown - 5 5 5 - 5
In lungs, minimal to moderate, multifocal, variably sized, pigmented (golden brown) granular material was observed throughout the parenchyma of lung at low, mid, high and high dose recovery groups of rats. This pigment was distributed both in alveolar and bronchiolar spaces with no accompanying inflammation or tissue destruction to the lung parenchyma. This pigment was suggestive of inhaled test item particles. In the absence of cellular changes to lung parenchyma, mere presence of pigment in lungs could be considered as non-adverse effect up to the nominal target dose level of 0.03 mg/L. (Nikula KJ et.al., 2014).
Few microscopic findings observed in this study such as cyst/s in kidneys, dilatation of uterus and all other findings were considered incidental as they occurred randomly across the dose groups including concurrent controls and/or were expected for laboratory rats.
Histopathological findings: neoplastic:
effects observed, non-treatment-related
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
13.6.2 BALF Fluid
The bronchio-alveolar lavage fluid measured at termination revealed the following statistically significant changes:
In main group females, decrease in percent lymphocytes and increase in percent neutrophils in G4 group was noted; increase in WBC and absolute lymphocytes in G3 group was noted.
In recovery group, males increase in LDH and decrease in percent basophils (G4R) was noted. The basophils (%) in recovery males appear decreased because of higher values in the control recovery group and hence the significance noted is of no toxicological relevance. Increase in LDH in recovery males is because of higher value in one animal, and hence considered incidental.
Few variations in differential counts could be secondary to accumulation of test item in the lungs. All other changes are considered incidental as they lack dose responsiveness and/or were noted in only one sex.
Details on results:
Based on the observed results, it is concluded that the No Observed Adverse Effect Concentration (NOAEC) of test item was found to be 0.03 mg/L when exposed for 6 hours/day, 5 days/week, for 4 weeks by flow-past nose-only inhalation route to Sprague Dawley rats. The nominal dose of 0.03 mg/L (6 hrs/day, 5 days/week) is the actual exposure concentration in males and females.

Key result
Dose descriptor:
NOAEC
Effect level:
> 30 mg/m³ air
Based on:
test mat.
Sex:
male/female
Basis for effect level:
behaviour (functional findings)
body weight and weight gain
clinical biochemistry
clinical signs
food consumption and compound intake
gross pathology
haematology
histopathology: non-neoplastic
mortality
organ weights and organ / body weight ratios
serum/plasma biochemistry
urinalysis
Key result
Critical effects observed:
no

TABLE 1.   SUMMARY OF CLINICAL SIGNS AND MORTALITY RECORD

                                                                                                                        Refer Appendix 1

Group, Sex & Dose

(mg/L)

No. of

Animals

Clinical Signs of Toxicity

Mortality                               (No. of Incidences/

No. of Animals)

G1, M & 0

5

N

0/5

G2, M & 0.003

5

N

0/5

G3, M & 0.01

5

N

0/5

G4, M & 0.03

5

N

0/5

G1R, M & 0

5

N

0/5

G4R, M & 0.03

5

N

0/5

M: Male; N: None; R: Recovery.


TABLE 1 (Contd…). SUMMARY OF CLINICAL SIGNS AND MORTALITY RECORD

                                                                                                                              Refer Appendix 1

Group, Sex & Dose

(mg/L)

No. of

Animals

Clinical Signs of Toxicity

Mortality                               (No. of Incidence/

No. of Animals)

G1, F & 0

5

N

0/5

G2, F & 0.003

5

N

0/5

G3, F & 0.01

5

N

0/5

G4, F & 0.03

5

N

0/5

G1R, F & 0

5

N

0/5

G4R, F & 0.03

5

N

0/5

F: Female; N: None; R: Recovery.

 

                            


TABLE 2.  SUMMARY OF BODY WEIGHTS (g)RECORD

 Refer Appendix 2

Group, Sex & Dose (mg/L)

Body Weight (g) on Days

1

5

8

12

15

22

26

G1, M & 0

Mean

166.51

171.75

178.32

185.81

197.59

214.19

225.14

±SD

9.48

10.36

10.11

10.38

12.00

12.39

13.10

n

5

5

5

5

5

5

5

G2, M & 0.003

Mean

170.65

174.98

180.83

188.27

204.29

219.31

229.19

±SD

14.03

13.78

13.91

13.41

13.53

13.31

11.58

n

5

5

5

5

5

5

5

G3, M & 0.01

Mean

163.66

168.12

174.43

181.48

198.20

213.03

224.77

±SD

9.42

9.17

9.25

9.10

10.10

9.87

9.39

n

5

5

5

5

5

5

5

G4, M & 0.03

Mean

163.99

168.87

175.43

182.93

198.64

214.21

225.74

±SD

9.27

9.25

8.92

8.36

8.87

8.00

8.84

n

5

5

5

5

5

5

5

G1, F & 0

Mean

148.51

152.03

155.66

160.28

172.22

183.16

191.20

±SD

11.18

11.98

11.73

11.04

11.11

10.71

11.54

n

5

5

5

5

5

5

5

G2, F & 0.003

Mean

150.58

154.23

158.35

163.93

177.05

187.21

195.08

±SD

9.04

8.92

8.43

8.83

8.94

8.94

9.12

n

5

5

5

5

5

5

5

G3, F & 0.01

Mean

147.71

151.43

155.06

160.47

173.49

182.83

191.88

±SD

9.33

9.83

9.54

9.60

9.58

9.25

9.64

n

5

5

5

5

5

5

5

G4, F & 0.03

Mean

147.30

150.33

154.63

159.60

172.10

182.59

191.08

±SD

8.92

9.02

9.36

8.45

6.81

6.49

6.69

n

5

5

5

5

5

5

5

 M: Male; F: Female; SD: Standard Deviation; n: Number of animals


TABLE 2 (Contd...). SUMMARY OF BODY WEIGHTS (g) RECORD

 Refer Appendix 2

Group, Sex & Dose (mg/L)

Body Weight (g) on Days

1

5

8

12

15

22

29

36

43

50

57

64

71

78

84

G1R, M & 0

Mean

166.20

171.02

177.66

185.62

200.07

214.49

232.63

253.26

274.79

299.32

325.27

349.03

372.79

397.94

413.98

±SD

11.24

11.18

10.45

10.60

12.29

11.09

10.88

10.72

10.05

10.33

9.74

8.98

8.83

7.95

5.30

n

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

G4R, M & 0.03

Mean

164.61

169.11

175.81

183.14

199.36

215.18

234.98

256.10

278.55

303.45

328.88

353.30

374.05

398.71

415.74

±SD

5.82

6.41

6.91

6.52

8.72

7.25

7.24

8.04

7.24

7.94

8.28

7.95

4.25

4.86

3.34

n

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

G1R, F & 0

Mean

148.78

151.72

155.52

160.51

173.51

184.31

198.22

210.64

225.19

239.40

255.46

270.87

285.04

301.89

317.03

±SD

6.32

5.93

5.77

5.65

6.78

6.41

6.86

8.13

7.78

7.89

7.61

8.09

9.30

8.70

8.48

n

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

G4R, F & 0.03

Mean

146.83

150.42

154.40

158.83

170.78

181.28

196.23

210.43

224.38

239.40

255.44

270.86

283.41

300.91

314.61

±SD

5.66

5.28

5.62

5.57

5.71

4.93

4.84

5.04

5.63

4.95

4.82

4.24

7.37

7.36

5.82

n

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

 M: Male; F: Female; R: Recovery; -: Not applicable; SD: Standard Deviation; n: Number of animals


TABLE 3.  SUMMARY OF PERCENT CHANGE IN BODY WEIGHT WITH RESPECT TO DAY 1RECORD

Refer Appendix 3

Group, Sex & Dose (mg/L)

Percent Change in Body Weight (%) during Days

1 to 5

1 to 8

1 to 12

1 to 15

1 to 22

1 to 26

G1, M & 0

Mean

3.13

7.09

11.59

18.65

28.64

35.21

±SD

0.42

0.29

0.62

1.61

1.60

1.89

n

5

5

5

5

5

5

G2, M & 0.003

Mean

2.56

6.00

10.41

19.85

28.72

34.60

±SD

0.57

0.62

1.30

2.90

3.74

4.82

n

5

5

5

5

5

5

G3, M & 0.01

Mean

2.74

6.61

10.93

21.15

30.24

37.45

±SD

0.74

0.86

1.34

1.71

2.10

2.63

n

5

5

5

5

5

5

G4, M & 0.03

Mean

2.98

7.00

11.60

21.19

30.73

37.77

±SD

0.25

0.90

1.19

1.65

2.41

3.00

n

5

5

5

5

5

5

G1, F & 0

Mean

2.35

4.81

7.96

16.04

23.45

28.86

±SD

0.73

0.44

0.97

1.64

2.43

2.48

n

5

5

5

5

5

5

G2, F & 0.003

Mean

2.44

5.19

8.90

17.64

24.40

29.64

±SD

0.57

0.96

0.74

1.44

1.75

1.82

n

5

5

5

5

5

5

G3, F & 0.01

Mean

2.51

4.99

8.66

17.51

23.86

29.99

±SD

0.84

0.80

0.54

1.62

1.82

1.86

n

5

5

5

5

5

5

G4, F & 0.03

Mean

2.06

4.98

8.39

16.96

24.11

29.88

±SD

0.24

0.39

1.16

2.98

3.66

3.41

n

5

5

5

5

5

5

M: Male; F: Female; R: Recovery; SD: Standard Deviation; n: Number of animals

 

 

 

 

 

 

 

 


TABLE 3 (Contd...). SUMMARY OF PERCENT CHANGE IN BODY WEIGHT WITH RESPECT TO DAY 1 RECORD

Refer Appendix 3

Group, Sex & Dose (mg/L)

Percent Change in Body Weight (%) during Days

1 to 5

1 to 8

1 to 12

1 to 15

1 to 22

1 to 29

1 to 36

1 to 43

1 to 50

1 to 57

1 to 64

1 to 71

1 to 78

1 to 84

G1R, M & 0

Mean

2.91

6.95

11.75