Registration Dossier
Registration Dossier
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EC number: 235-462-4 | CAS number: 12236-62-3
Endpoint summary
Administrative data
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
- 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
- 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
- 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
- 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
Referenceopen allclose all
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 | 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 | 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: FemaleDay(s) Relative to Start Date | G1R0 mg/kg/day | G4R1000 mg/kg/day | ||
Brain (g) | Day 119 [p] | MeanSDN%Diff | 2.08730.04705- | 2.11810.157251.4756 |
Adrenals (%) | Day 119 [p] | MeanSDN%Diff | 4.39250.75595. | 4.90930.9212511.7651 |
Heart (%) | Day 119 [p] | MeanSDN%Diff | 51.11434.45465. | 51.86647.872551.4714 |
Kidneys (%) | Day 119 [p1] | MeanSDN%Diff | 86.834412.97785. | 92.442713.706856.4586 |
Liver (%) | Day 119 [p1] | MeanSDN%Diff | 400.020053.79435. | 393.694359.52925-1.5813 |
Ovaries (%) | Day 119 [p] | MeanSDN%Diff | 5.75140.77655. | 4.0846 * 1.05245-28.9809 |
Pituitary (%) | Day 124b [p2] | MeanSDN%Diff | 0.84570.11255. | 0.89950.281556.3657 |
[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
1. REPRODUCTION, BREEDING AND PUP DATA
SUMMARY OF PERFORMANCE
P Animals Breeding for F1 Litters
Group |
1 |
2 |
3 |
4 |
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, |
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.
1. REPRODUCTION, BREEDING AND PUP DATA
SUMMARY OF PERFORMANCE
P Animals Breeding for F1 Litters
Group |
1 |
2 |
3 |
4 |
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, |
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.
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
- 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
Reference
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 | ||||||||||