Registration Dossier

Diss Factsheets

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

Referenceopen allclose all

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

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


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


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


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


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


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


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


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


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


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


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


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


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


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


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


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


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


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


No Observed Adverse Effect Level (NOAEL):


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

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Details on species / strain selection:
Rat is the standard laboratory rodent species used for toxicity assessment and recommended by various regulatory authorities.
The Wistar rat was selected due to the large amount of background data available for this strain.
Sex:
male/female
Details on test animals or test system and environmental conditions:
Hylasco Biotechnology Pvt. Ltd., Plot 4B, AKP, Turkapally Village, Shameerpet Mandal, RR Dist, Telangana 500078
- Females (if applicable) nulliparous and non-pregnant: [yes]
- Age at study initiation: 6 weeks
Body weight range at the start of treatment: Males: 189.68 to 249.91 g & Females : 156.56 to 192.12 g
At the commencement of the treatment, the weight variation of rats used did not exceed ± 20 % of the mean body weight in each sex and group.
Conditions: Rats were housed in an environment controlled room. The temperature maintained during the experiment was between 20 to 24°C and relative humidity was between 49 to 68%. The photoperiod was 12 hours light and 12 hours dark cycle. Adequate fresh air supply of 12-15 air changes/hour was maintained in the experimental room. The maximum and minimum temperature in the experimental room was recorded once daily. The relative humidity in the experimental room was calculated daily from dry and wet bulb temperature recordings.
Housing: Two rats of same sex were housed per cage in sterilized standard polysulfone cages (Size: L 425 x B 266 x H 185 mm), with stainless steel top grill having facilities for pelleted food and drinking water in polycarbonate bottles with stainless steel sipper tubes. The last animal in recovery group of each sex was housed individually. Polycarbonate rat huts were provided to the animals as environmental enrichment objects and changed along with cage at least once a week. During the experimental period, animals were housed in a single experimental room of barrier area.
Bedding: Steam sterilized corn cob was used as bedding and changed along with the cage atleast twice a week.
Diet: Altromin Rat/Mice Maintenance diets manufactured by Altromin Spezialfutter GmbH & Co. KG, Im Seelenkamp 20, 32791 Lage, Germany, was provided ad libitum.
Water: Deep bore-well water passed through activated charcoal filter and exposed to UV rays in Aquaguard on-line water filter-cum-purifier manufactured by Eureka Forbes Ltd., Mumbai 400 001,India, was provided ad libitum to rats in polycarbonate bottles with stainless steel sipper tubes.
Route of administration:
oral: gavage
Details on route of administration:
The dose formulations were administered orally by gavage to specific group of rats once daily at approximately the same time (± 3 hours) each day for a period of 90 consecutive days. Similarly, the vehicle was administered to rats in vehicle control/vehicle control recovery group once daily orally for 90 consecutive days.
The vehicle or the dose formulations were not administered to recovery groups for 28 days following the 90-day treatment period. The dose formulation and the vehicle were administered at an equivolume of 10 mL/kg/day. The dose volume was calculated for individual animals on the first day of treatment and was adjusted according to the most recent body weights recorded during the treatment period
Vehicle:
CMC (carboxymethyl cellulose)
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
For homogeneity and active ingredient (a.i.) concentration analysis, prepared formulation samples were sampled in duplicate sets on Day 1 and during 2nd (Day 34) and 3rd (Day 63) month of the treatment period and analysed in-house. For each set, duplicate samples from top, middle and bottom layers were drawn from each preparation and in case of control, duplicate samples were drawn from middle layer.
The analysis was done as per the method validated under Eurofins Advinus Study No.: G19462. One set of samples was analyzed for concentration. The back up samples were discarded as analysis results of the first set of samples were within the acceptable limits. Formulations were considered acceptable as overall mean results of all the layers and mean of each layer were within ± 15.0 % of the claimed concentration and relative standard deviation (% RSD) was less than 10.0 %.
Duration of treatment / exposure:
90 Days
Frequency of treatment:
Daily
Dose / conc.:
111 mg/kg bw/day (nominal)
Dose / conc.:
333 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10
Control animals:
yes
Details on study design:
No. of groups : 6
Vehicle control (G1)
Low dose (G2)
Mid dose (G3)
High dose (G4)
Vehicle control recovery (G1R)
High dose recovery (G4R)
No. of rats/group: Main groups: 10 males + 10 females
Recovery groups: 5 males + 5 females
Total = 100 (50 males + 50 females)
Observations and examinations performed and frequency:
Observations and examinations performed and frequency
Morbidity and Mortality: All rats were observed for morbidity and mortalities twice daily i.e., once in the morning and once in the afternoon except during holidays wherein the observation was done once daily as there were no clinical signs observed. Clinical Signs: Each rat was observed for checking general clinical signs twice once daily during treatment period once daily during the recovery period. On the days of scheduled detailed clinical examination, clinical signs were included as a part of detailed clinical observations except on Day 1 wherein detailed clinical examination was done prior to the treatment and observations for general clinical signs was done after dosing the animals.
Detailed Clinical Examination: Detailed clinical examination was done prior to the test item administration on Day 1 and at weekly intervals thereafter (± 2 days) during treatment period. During detailed clinical examination, all rats were observed for changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern), changes in gait, posture and response to handling as well as the presen ce of clonic or tonic movements, stereotypies (e.g., excessive grooming, repetitive circling or bizarre behaviour (e.g. self-mutilation, walking backwards). On the days of detailed clinical examination, observation for general clinical signs (first post-dose) was not performed except on Day 1. Ophthalmological Examination: Ophthalmological examination of all animals was performed with an ophthalmoscope prior to start of the treatment, at the end of the treatment period for main groups (Day 84) and at the end of recovery period (Day 117)for recovery groups. Before examination, my driasis was induced using a 1 % solution of Tropicamide.
Functional Observation Battery Tests (FOB)
The following neurological examination was performed during the 12th week (Day 84) of treatment period for main groups and towards the end of recovery period (Day 117) for recovery groups.
Home Cage Observations: Each rat was observed in the home cage for posture and for presence or absence of abnormal vocalizations, tremors and convulsions.
Observations during Removal of Animal from Home Cage and Handling: The objective of this phase of neurological examination was to observe the subject’s response to handling and to conduct other procedures of the FOB that can best be performed when the rat is being held. Each rat was observed for the following examinations:
ease of removal from home cage
handling reactivity
palpebral closure
eye examination
piloerection
lacrimation
salivation
skin/fur examination
perineum wetness
respiration
muscle tone and
extensor thrust response
The observations were recorded using scores/ranks.
Open Field Observation: Rat was placed (one at a time) in an open arena, on a flat surface with a clea
n absorbent paper and observed for at least 2 minutes. Absorbent paper was replaced for each group.
During this observation period, rat was evaluated as it moves about freely/unperturbed and the fol
lowing observations were made and observations were recorded using score/ranks:
gait
posture
tremors
mobility score
arousal level
clonic or tonic movements
stereotypic behaviour
bizarre behaviour
urination
defecation
rearing
abnormal vocalizations
Functional Tests: Functional testing includes motor activity, sensory evaluation, landing hindlimbs footsplay and measurement of grip performance.
Motor Activity: The motor activity of rats was measured using an automated animal activity measuring system (Make: Columbus Instruments) equipped with a computer analyzer. Each rat was individually placed in the activity cages of the instrument. The rats were monitored for 30 minutes. During this motor activity measurement session, parameters viz., the stereotypic time (small movements) in seconds, the ambulatory time (large ambulatory movement) in seconds, horizontal counts, a mbulatory counts were monitored. The Opto-Varimex 4 motor activity measurement system provided the data at 1 minute interval and the data was analyzed in blocks of 10 minutes interval and the same was reported.
Sensory Reactivity Measurements: After the 2 minutes (approximately) observation period, while the rat was in the open field arena, the following tests were conducted. The rat was allowed to move freely in the open field box for these tests but positioned in the box by the observer in order to administer stimulus. During sensory reactivity measurements, rats were observed for following and the observations were recorded using scores/ranks.
approach response
touch response
click response
tail-pinch response
pupil response
aerial righting reflex
Landing Hindlimbs Footsplay: The landing hind limbs foot splay was performed by dropping the rat onto a horizontal surface of the table top from a short height and measuring the distance between the hind feet upon landing. The hind feet of the rat were gently pressed to an ink pad just prior to testing. The rat was suspended in a prone position and then dropped from a height of approximately 30 cm on to a SOP format, which contains the details such as Study no., Animal no, Group and Sex. A clean recording SOP format was used for each rat. A total of 3 readings were recorded for each rat and average of 3 footsplay values is presented in the report along with the individual footsplay values.
Grip Performance: Hindlimbs and forelimbs grip performance was tested using computerized dual grip strength meter (Model: Columbus Instruments). Three trials were conducted for each rat i.e., three trials each for forelimb and hind limbs. Averages of three trials for both forelimb and hindlimbs are calculated and presented in the report along with the individual grip strength values.
Physiological Observations: Body temperature (rectal temperature) was measured in degree Celsius (°C) using digital thermometer. At the end of the functional test, body weight of each rat was measured.
Body Weight: Individual body weights (g) was recorded prior to test item administration on Day 1 and weekly thereafter (± 2 day) for all groups of rats during treatment and recovery period. Fasting body weight was recorded prior to sacrifice for all animals.
Food Consumption: The food consumption was measured at weekly intervals (± 2 days) during treatment and recovery period. The cage wise average food consumption (g/rat/day) was calculated and presented in the report.
Oestrous Cycle Evaluation: Vaginal smear was examined in the female rats and the stage of oestrous cycle was recorded prior to necropsy.
Clinical Pathology Investigations
Blood Collection: At the end of the treatment and recovery periods, all rats were fasted overnight (water allowed) and approximately 4.0 mL of blood was collected under isoflurane anaesthesia, with a fine capillary tube, by retro-orbital sinus puncture: After analysis and data review by the analyst, the residual samples were disposed. Haematology, Coagulation, Clinical Chemistry, Hormone Analysis & Urinalysis Parameters were done as study plan.
Sacrifice and pathology:
All rats from toxicity groups at the end of the scheduled period (Day 91 and 119) were subjected to detailed necropsy (examination of external surfaces of the body, all orifices; cranial, thoracic and abdominal cavities and their contents) and findings were recorded. Terminal fasting body weights were recorded for all animals immediately prior to terminal sacrifice and used in calculation of relative organ weights. All rats sacrificed at term were fasted overnight (water allowed), euthanized with isoflurane (as per the random numbers generated for the study), exsanguinated and subjected for gross examination.
At sacrifice, sperm motility was evaluated using the sperm samples collected from the right vas deferens, immediately after necropsy using Hamilton-Thorne TOX-IVOS sperm analyzer for all rats. For morphological evaluation of sperms, smears were made using semen samples collected from right vas deferens of all rats immediately after necropsy and fixed with acetone for evaluation by manual method. Initially, sperm morphology was assessed for control and high dose rats. The right epididymis was collected and frozen for enumeration of cauda epididymal sperm reserves. As the high dose group did not show any test item-related effect, the analysis was not extended to low, mid dose and recovery rats. Unused frozen samples were discarded at the time of final report preparation On completion of gross pathology examination, the tissues/organs noted in following table were collected from all rats. The below listed organs were weighed from all terminally sacrificed animals. The organ weight ratios (organ to body weight and brain weight) as percentage of fasting body weight
were determined and presented in the report. Paired organs were weighed together, and combined weights were presented.
Histopathological examination was carried out on the preserved organs of vehicle control (G1) and high dose group animals (G4). Examination of the testes also included a qualitative assessment of stages of spermatogenesis. In addition, all gross lesions from all the animals were examined microscopically. In the absence of test item-related changes, the tissues from low (G2), mid dose (G3) and recovery (G1R and G4R) groups were not evaluated.
The tissues were processed for routine paraffin embedding and 4-5-micron sections were stained with Haematoxylin and Eosin stain. In addition, testes were sectioned at 3-4 μm and stained with PAS reagent and haematoxylin to aid in qualitative assessment of spermatogenesis. Unused tissues were archived
Statistics:
For comparative statistics, data 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:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study (OECD 422, GLP-compliant)
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EPA OPPTS 870.3650 (Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Tocixity Screening Test)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Animals: Rat, RccHanTM: WIST(SPF)
- Rationale: Recognized by international guidelines as a recommended test system.
- Source: Harlan Laboratories, Inc., Maasheseweg 87c, 5800 AN Vernay / Netherlands
- Number of Animals: 44 males (11 per group) and 44 females (11 per group)
- Age (at Start of Treatment): 11 weeks
- Body Weight Range (at Start of Treatment): 301 to 362 g (males), 216 to 247 g (females)
- Identification: Parent animals had cage card and individual animal number (ear tattoo), pups were individually tattooed with Indian ink on day 1 post partum
- Randomization: Performed after at least three days of acclimatization using a computer-generated random algorithm. Body weights (recorded on the day of allocation) were taken into consideration in order to ensure similar mean body weights in all groups.
- Accommodation: In groups of five in Makrolon type-4 cages with wire mesh tops up to the day of randomization and afterwards individually in Makrolon type-3 cages with wire mesh tops and sterilized standard softwood bedding (‘Lignocel’ J. Rettenmaier & Söhne GmbH & CoKG, 73494 Rosenberg / Germany, imported by Provimi Kliba SA, 4303 Kaiseraugst / Switzerland) with paper enrichment (ISO-BLOX from Harlan Laboratories B.V., Netherlands), batch/lot nos. 02105111001, 02105111201, 02105120301 and 6960C.CS-100099). During the pre-pairing period, cages with males were interspersed amongst those holding females to promote the development of regular estrus cycles.
- Diet: Pelleted standard Harlan Teklad 2018C (batch no. 80/11) rodent maintenance diet (Provimi Kliba SA, 4303 Kaiseraugst / Switzerland) was available ad libitum.
- Water: Community tap-water from Füllinsdorf was available ad libitum in water bottles.
- Acclimatization: Under test conditions after health examination. Only animals without any visible signs of illness were used for the study.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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


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

1. REPRODUCTION, BREEDING AND PUP DATA

SUMMARY OF PERFORMANCE

P Animals Breeding for F1 Litters

Group
(mg/kg/day)

1
(0)

2
(100)

3
(300)

4
(100)

Female numbers

45-55

56-66

67-77

78-88

Number of females paired

11

11

11

11

Number of females mated

11

11

11

11

Number of non pregnant females (A)

3

1

3

0

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

0

0

0

1

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

8

10

8

10

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

(B)  Female No. 85 had implantations only.

 

MATING PERFORMANCE AND FERTILITY

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

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

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

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

 

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

 

CORPORA LUTEA COUNT

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

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

 

DURATION OF GESTATION

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

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

 

IMPLANTATION RATE AND POST-IMPLANTATION LOSS

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

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

 

LITTER SIZE AT FIRST LITTER CHECK

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

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

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

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

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

 

POSTNATAL LOSS DAYS 0 - 4 POST PARTUM

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

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

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

 

EXTERNAL EXAMINATION AT FIRST LITTER CHECK AND DURING LACTATION

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

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

 

SEX RATIOS

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

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

 

 BODY WEIGHTS TO DAY 4 POST PARTUM

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

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

 

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

 

MACROSCOPICAL FINDINGS

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

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

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

 

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

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

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

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

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

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


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

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

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

 

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

 

The following dose levels were applied:

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

Group 2:                    100 mg/kg body weight/day

Group 3:                    300 mg/kg body weight/day

Group 4:                   1000 mg/kg body weight/day

 

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

 

The following results were obtained:

 MORTALITY AND GENERAL TOLERABILITY OF PARENTAL ANIMALS

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

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

 

FUNCTIONAL OBSERVATIONAL BATTERY IN PARENTAL ANIMALS

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

 

FOOD CONSUMPTION OF PARENTAL ANIMALS

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

 

BODY WEIGHTS OF PARENTAL ANIMALS

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

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

 

CLINICAL LABORATORY INVESTIGATIONS IN PARENTAL ANIMALS

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

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

 

REPRODUCTION AND BREEDING DATA

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

 

SEMINOLOGY AND SPERMATID COUNT IN PARENTAL MALES

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

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

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

ORGAN WEIGHTS OF PARENTAL ANIMALS

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

 

MACROSCOPICAL FINDINGS AND HISTOPATHOLOGICAL EXAMINATIONS OF PARENTAL ANIMALS

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

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

 

FINDINGS IN PUPS AT FIRST LITTER CHECK AND DURING LACTATION

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

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

 

PUP WEIGHTS TO DAY 4 POST PARTUM

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

 

MACROSCOPICAL FINDINGS OF PUPS

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

 

CONCLUSION

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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


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

1. REPRODUCTION, BREEDING AND PUP DATA

SUMMARY OF PERFORMANCE

P Animals Breeding for F1 Litters

Group
(mg/kg/day)

1
(0)

2
(100)

3
(300)

4
(100)

Female numbers

45-55

56-66

67-77

78-88

Number of females paired

11

11

11

11

Number of females mated

11

11

11

11

Number of non pregnant females (A)

3

1

3

0

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

0

0

0

1

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

8

10

8

10

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

(B)  Female No. 85 had implantations only.

 

MATING PERFORMANCE AND FERTILITY

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

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

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

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

 

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

 

CORPORA LUTEA COUNT

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

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

 

DURATION OF GESTATION

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

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

 

IMPLANTATION RATE AND POST-IMPLANTATION LOSS

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

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

 

LITTER SIZE AT FIRST LITTER CHECK

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

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

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

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

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

 

POSTNATAL LOSS DAYS 0 - 4 POST PARTUM

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

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

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

 

EXTERNAL EXAMINATION AT FIRST LITTER CHECK AND DURING LACTATION

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

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

 

SEX RATIOS

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

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

 

 BODY WEIGHTS TO DAY 4 POST PARTUM

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

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

 

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

 

MACROSCOPICAL FINDINGS

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

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

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

 

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

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

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

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

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

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


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

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

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

 

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

 

The following dose levels were applied:

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

Group 2:                    100 mg/kg body weight/day

Group 3:                    300 mg/kg body weight/day

Group 4:                   1000 mg/kg body weight/day

 

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

 

The following results were obtained:

 MORTALITY AND GENERAL TOLERABILITY OF PARENTAL ANIMALS

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

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

 

FUNCTIONAL OBSERVATIONAL BATTERY IN PARENTAL ANIMALS

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

 

FOOD CONSUMPTION OF PARENTAL ANIMALS

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

 

BODY WEIGHTS OF PARENTAL ANIMALS

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

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

 

CLINICAL LABORATORY INVESTIGATIONS IN PARENTAL ANIMALS

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

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

 

REPRODUCTION AND BREEDING DATA

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

 

SEMINOLOGY AND SPERMATID COUNT IN PARENTAL MALES

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

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

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

ORGAN WEIGHTS OF PARENTAL ANIMALS

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

 

MACROSCOPICAL FINDINGS AND HISTOPATHOLOGICAL EXAMINATIONS OF PARENTAL ANIMALS

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

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

 

FINDINGS IN PUPS AT FIRST LITTER CHECK AND DURING LACTATION

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

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

 

PUP WEIGHTS TO DAY 4 POST PARTUM

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

 

MACROSCOPICAL FINDINGS OF PUPS

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

 

CONCLUSION

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

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

Repeated dose toxicity: inhalation - systemic effects

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

DETAILS OF FOOD AND WATER QUALITY:

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

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

TEST ATMOSPHERE
- Samples taken from breathing zone: yes

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

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly once

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

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

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

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

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

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

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

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

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

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

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

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

TABLE 1.   SUMMARY OF CLINICAL SIGNS AND MORTALITY RECORD

                                                                                                                        Refer Appendix 1

Group, Sex & Dose

(mg/L)

No. of

Animals

Clinical Signs of Toxicity

Mortality                               (No. of Incidences/

No. of Animals)

G1, M & 0

5

N

0/5

G2, M & 0.003

5

N

0/5

G3, M & 0.01

5

N

0/5

G4, M & 0.03

5

N

0/5

G1R, M & 0

5

N

0/5

G4R, M & 0.03

5

N

0/5

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


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

                                                                                                                              Refer Appendix 1

Group, Sex & Dose

(mg/L)

No. of

Animals

Clinical Signs of Toxicity

Mortality                               (No. of Incidence/

No. of Animals)

G1, F & 0

5

N

0/5

G2, F & 0.003

5

N

0/5

G3, F & 0.01

5

N

0/5

G4, F & 0.03

5

N

0/5

G1R, F & 0

5

N

0/5

G4R, F & 0.03

5

N

0/5

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

 

                            


TABLE 2.  SUMMARY OF BODY WEIGHTS (g)RECORD

 Refer Appendix 2

Group, Sex & Dose (mg/L)

Body Weight (g) on Days

1

5

8

12

15

22

26

G1, M & 0

Mean

166.51

171.75

178.32

185.81

197.59

214.19

225.14

±SD

9.48

10.36

10.11

10.38

12.00

12.39

13.10

n

5

5

5

5

5

5

5

G2, M & 0.003

Mean

170.65

174.98

180.83

188.27

204.29

219.31

229.19

±SD

14.03

13.78

13.91

13.41

13.53

13.31

11.58

n

5

5

5

5

5

5

5

G3, M & 0.01

Mean

163.66

168.12

174.43

181.48

198.20

213.03

224.77

±SD

9.42

9.17

9.25

9.10

10.10

9.87

9.39

n

5

5

5

5

5

5

5

G4, M & 0.03

Mean

163.99

168.87

175.43

182.93

198.64

214.21

225.74

±SD

9.27

9.25

8.92

8.36

8.87

8.00

8.84

n

5

5

5

5

5

5

5

G1, F & 0

Mean

148.51

152.03

155.66

160.28

172.22

183.16

191.20

±SD

11.18

11.98

11.73

11.04

11.11

10.71

11.54

n

5

5

5

5

5

5

5

G2, F & 0.003

Mean

150.58

154.23

158.35

163.93

177.05

187.21

195.08

±SD

9.04

8.92

8.43

8.83

8.94

8.94

9.12

n

5

5

5

5

5

5

5

G3, F & 0.01

Mean

147.71

151.43

155.06

160.47

173.49

182.83

191.88

±SD

9.33

9.83

9.54

9.60

9.58

9.25

9.64

n

5

5

5

5

5

5

5

G4, F & 0.03

Mean

147.30

150.33

154.63

159.60

172.10

182.59

191.08

±SD

8.92

9.02

9.36

8.45

6.81

6.49

6.69

n

5

5

5

5

5

5

5

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


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

 Refer Appendix 2

Group, Sex & Dose (mg/L)

Body Weight (g) on Days

1

5

8

12

15

22

29

36

43

50

57

64

71

78

84

G1R, M & 0

Mean

166.20

171.02

177.66

185.62

200.07

214.49

232.63

253.26

274.79

299.32

325.27

349.03

372.79

397.94

413.98

±SD

11.24

11.18

10.45

10.60

12.29

11.09

10.88

10.72

10.05

10.33

9.74

8.98

8.83

7.95

5.30

n

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

G4R, M & 0.03

Mean

164.61

169.11

175.81

183.14

199.36

215.18

234.98

256.10

278.55

303.45

328.88

353.30

374.05

398.71

415.74

±SD

5.82

6.41

6.91

6.52

8.72

7.25

7.24

8.04

7.24

7.94

8.28

7.95

4.25

4.86

3.34

n

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

G1R, F & 0

Mean

148.78

151.72

155.52

160.51

173.51

184.31

198.22

210.64

225.19

239.40

255.46

270.87

285.04

301.89

317.03

±SD

6.32

5.93

5.77

5.65

6.78

6.41

6.86

8.13

7.78

7.89

7.61

8.09

9.30

8.70

8.48

n

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

G4R, F & 0.03

Mean

146.83

150.42

154.40

158.83

170.78

181.28

196.23

210.43

224.38

239.40

255.44

270.86

283.41

300.91

314.61

±SD

5.66

5.28

5.62

5.57

5.71

4.93

4.84

5.04

5.63

4.95

4.82

4.24

7.37

7.36

5.82

n

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

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


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

Refer Appendix 3

Group, Sex & Dose (mg/L)

Percent Change in Body Weight (%) during Days

1 to 5

1 to 8

1 to 12

1 to 15

1 to 22

1 to 26

G1, M & 0

Mean

3.13

7.09

11.59

18.65

28.64

35.21

±SD

0.42

0.29

0.62

1.61

1.60

1.89

n

5

5

5

5

5

5

G2, M & 0.003

Mean

2.56

6.00

10.41

19.85

28.72

34.60

±SD

0.57

0.62

1.30

2.90

3.74

4.82

n

5

5

5

5

5

5

G3, M & 0.01

Mean

2.74

6.61

10.93

21.15

30.24

37.45

±SD

0.74

0.86

1.34

1.71

2.10

2.63

n

5

5

5

5

5

5

G4, M & 0.03

Mean

2.98

7.00

11.60

21.19

30.73

37.77

±SD

0.25

0.90

1.19

1.65

2.41

3.00

n

5

5

5

5

5

5

G1, F & 0

Mean

2.35

4.81

7.96

16.04

23.45

28.86

±SD

0.73

0.44

0.97

1.64

2.43

2.48

n

5

5

5

5

5

5

G2, F & 0.003

Mean

2.44

5.19

8.90

17.64

24.40

29.64

±SD

0.57

0.96

0.74

1.44

1.75

1.82

n

5

5

5

5

5

5

G3, F & 0.01

Mean

2.51

4.99

8.66

17.51

23.86

29.99

±SD

0.84

0.80

0.54

1.62

1.82

1.86

n

5

5

5

5

5

5

G4, F & 0.03

Mean

2.06

4.98

8.39

16.96

24.11

29.88

±SD

0.24

0.39

1.16

2.98

3.66

3.41

n

5

5

5

5

5

5

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

 

 

 

 

 

 

 

 


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

Refer Appendix 3

Group, Sex & Dose (mg/L)

Percent Change in Body Weight (%) during Days

1 to 5

1 to 8

1 to 12

1 to 15

1 to 22

1 to 29

1 to 36

1 to 43

1 to 50

1 to 57

1 to 64

1 to 71

1 to 78

1 to 84

G1R, M & 0

Mean

2.91

6.95

11.75

20.43

29.17

40.12

52.59

65.61

80.42

96.11

110.48

124.83

140.04

149.84

±SD

0.67

1.58

1.96

1.83

2.75

3.14

4.02

5.24

6.27

7.78

8.81

10.07

11.39

14.09

n

5

5

5

5

5

5

5

5

5

5

5

5

5

5

G4R, M & 0.03

Mean

2.72

6.79

11.26

21.08

30.74

42.77

55.60

69.26

84.40

99.85

114.71

127.39

142.38

152.75

±SD

0.52

0.60

0.54

1.10

1.63

1.00

1.14

1.90

2.29

2.19

2.89

5.59

5.82

6.84

n

5

5

5

5

5

5

5

5

5

5

5

5

5

5

G1R, F & 0

Mean

1.99

4.55

7.91

16.63

23.91

33.27

41.61

51.41

60.97

71.80

82.16

91.70

103.06

113.25

±SD

0.66

0.67

1.07

0.62

1.05

1.18

1.88

2.74

2.57

3.42

3.53

5.57

6.21

6.31

n

5

5

5

5

5

5

5

5

5

5

5

5

5

5

G4R, F & 0.03

Mean

2.45

5.16

8.18

16.34

23.52

33.72

43.39

52.91

63.15

74.09

84.61

93.13

105.07

114.44

±SD

0.52

0.95

0.96

2.29

2.66

3.11

3.00

3.85

3.92

4.45

4.61

5.07

5.26

6.20

n

5

5

5

5

5

5

5

5

5

5

5

5

5

5

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


TABLE 4.  SUMMARY OF AVERAGE FEED CONSUMPTION (g/rat/day)RECORD

 Refer Appendix 4

Group, Sex & Dose (mg/L)

Week 1

Week 2

Week 3

Week 4

G1, M & 0

Mean

18.67

18.63

18.49

28.45

±SD

2.73

2.83

2.81

3.70

n

2

2

2

2

G2, M & 0.003

Mean

18.61

18.66

18.53

28.03

±SD

3.03

2.77

2.62

3.35

n

2

2

2

2

G3, M & 0.01

Mean

18.78

18.78

18.54

28.01

±SD

3.04

2.87

2.77

3.54

n

2

2

2

2

G4, M & 0.03

Mean

18.53

18.76

18.56

28.05

±SD

2.85

3.06

3.00

3.38

n

2

2

2

2

G1, F & 0

Mean

14.63

14.73

14.61

21.69

±SD

1.59

1.62

1.48

1.31

n

2

2

2

2

G2, F & 0.003

Mean

14.65

14.80

14.64

21.44

±SD

1.41

1.46

1.40

1.43

n

2

2

2

2

G3, F & 0.01

Mean

14.55

14.80

14.68

20.95

±SD

1.40

1.41

1.48

1.22

n

2

2

2

2

G4, F & 0.03

Mean

14.49

14.66

14.53

20.60

±SD

1.37

1.38

1.43

1.13

n

2

2

2

2

 M: Male; F: Female; R: Recovery; SD: Standard Deviation; n: number of cages.


TABLE 4 (Contd...). SUMMARY OF AVERAGE FEED CONSUMPTION (g/rat/day) RECORD

 Refer Appendix 4

Group, Sex & Dose (mg/L)

Week 1

Week 2

Week 3

Week 4

Week 5

Week 6

Week 7

Week 8

Week 9

Week 10

Week 11

Week 12

G1R, M & 0

Mean

18.68

18.77

18.50

18.66

18.72

18.78

18.92

19.23

19.43

19.62

19.84

23.11

±SD

3.04

3.04

2.90

2.93

3.02

3.02

3.00

3.27

3.32

3.32

3.44

4.06

n

2

2

2

2

2

2

2

2

2

2

2

2

G4R, M & 0.03

Mean

18.69

18.76

18.59

18.72

18.73

18.80

18.96

19.35

19.41

19.59

19.76

23.06

±SD

2.60

2.87

2.78

2.91

3.06

3.03

2.98

3.21

3.25

3.37

3.40

4.08

n

2

2

2

2

2

2

2

2

2

2

2

2

G1R, F & 0

Mean

14.53

14.76

14.64

14.85

14.99

14.99

15.18

15.56

15.67

15.83

16.03

18.64

±SD

1.39

1.47

1.34

1.74

1.77

1.68

1.78

1.90

1.91

1.91

2.12

2.44

n

2

2

2

2

2

2

2

2

2

2

2

2

G4R, F & 0.03

Mean

14.38

14.61

14.59

14.85

14.94

14.96

15.21

15.41

15.59

15.74

15.87

18.39

±SD

1.31

1.38

1.25

1.52

1.58

1.52

1.60

1.59

1.65

1.76

1.71

2.03

n

2

2

2

2

2

2

2

2

2

2

2

2

 M: Male; F: Female; R: Recovery; SD: Standard Deviation; n: number of cages.


TABLE 5.  SUMMARY OF NEUROLOGICAL/FUNCTIONAL OBSERVATION BATTERY (FOB)

Refer Appendix 5

Week 4

Parameters↓

Group & Sex

G1 & M

G4 & M

G1 & F

G4 & F

Dose (mg/L)

0

0.03

0

0.03

Number of Animals

5

5

5

5

Home cage observations

Home Cage posture

1

1

1

1

Respiratory pattern

1

1

1

1

Clonic involuntary movements

1

1

1

1

Tonic involuntary movements

1

1

1

1

Vocalization

1

1

1

1

Palpebral Closure

1

1

1

1

Handling observation

 

 

Ease of removal from the cage

2

2

2

2

Ease of handling animal in hand

2

2

2

2

Red or crusty deposits

Eyes

1

1

1

1

Nose

1

1

1

1

Mouth

1

1

1

1

Lacrimation

1

1

1

1

Salivation

1

1

1

1

Fur Appearance

1

1

1

1

Piloerection

1

1

1

1

Eye Prominence

1

1

1

1

Muscle Tone

1

1

1

1

Home cage observations: Home cage posture- 1=Normal,Respiratory Pattern -1=Normal,Clonic involuntary movements -1=None/Normal,Tonic involuntary movements -1=None/Normal,Vocalization -1=Absent (Normal),Palpebral closure -1=Eyelids wide open (Normal),Handling observation : Ease of removal from the cage -2=Normal,Ease of handling animal in hand -2=Normal,Red/Crusty deposits -1=Absent,Lacrimation- 1=None,Salivation-1=Absent (Normal),Fur Appearance -1=Normal,Piloerection -1=None, Eye Prominence -1=Normal,Muscle tone -1=Normal

M: Male; F: Female

TABLE 5 (Contd...). SUMMARY OF NEUROLOGICAL/FUNCTIONAL OBSERVATION BATTERY (FOB) RECORD

Refer Appendix 5

Week 4

Parameters↓

Group & Sex

G1 & M

G4 & M

G1 & F

G4 & F

Dose (mg/L)

0

0.03

0

0.03

Number of Animals

5

5

5

5

Open field Observation

Mobility

1

1

1

1

Gait

1

1

1

1

Arousal

3

3

3

3

Number of Rearing

Mean

8.0

5.4*

8.8

8.6

±SD

1.0

1.7

1.1

1.1

Numbers of Urination

Mean

1.2

1.2

1.4

1.4

±SD

0.4

0.4

0.5

0.9

Number of Defecation

Mean

0.8

0.8

0.8

1.2

±SD

0.4

0.4

0.4

0.8

Clonic involuntary movement

1

1

1

1

Tonic involuntary movement

1

1

1

1

Stereotype Behaviour

1

1

1

1

Excessive Grooming

Mean

4.8

5.4

5.2

7.2

±SD

0.8

1.1

0.8

1.8

Sensory Observations

 

Approach Response

2

2

2

2

Auditory Response

2

2

2

2

Touch Response

2

2

2

2

Pupil Reflex

2

2

2

2

Tail Pinch Response

2

2

2

2

Righting Reflex

1

1

1

1

Physiological observation

 

Body temperature (°F)

Mean

96.7

97.1

98.5

98.5

±SD

0.8

1.1

0.1

0.1

Open field Observation: Mobility -1=Normal,Gait -1=Normal,Arousal -3=Normal,Clonic involuntary movements -1=None/Normal,Tonic involuntary movements -1=None/Normal,Stereotype Behaviour -1=Absent, Sensory Observations: Approach response -2=Normal, Auditory Response -2=Normal,Touch Response -2=Normal,Pupil Reflex -2=Normal,Tail Pinch Response -2=Normal,Righting Reflex -1=Present

*: Statistically significant (p<0.05)


TABLE 5 (Contd...). SUMMARY OF NEUROLOGICAL/FUNCTIONAL OBSERVATION BATTERY (FOB) RECORD

Refer Appendix 5

Week 12

Parameters↓

Group & Sex

G1R & M

G4R & M

G1R & F

G4R & F

Dose (mg/L)

0

0.03

0

0.03

Number of Animals

5

5

5

5

Home cage observations

Home Cage posture

1

1

1

1

Respiratory pattern

1

1

1

1

Clonic involuntary movements

1

1

1

1

Tonic involuntary movements

1

1

1

1

Vocalization

1

1

1

1

Palpebral Closure

1

1

1

1

Handling observation

 

 

Ease of removal from the cage

2

2

2

2

Ease of handling animal in hand

2

2

2

2

Red or crusty deposits

Eyes

1

1

1

1

Nose

1

1

1

1

Mouth

1

1

1

1

Lacrimation

1

1

1

1

Salivation

1

1

1

1

Fur Appearance

1

1

1

1

Piloerection

1

1

1

1

Eye Prominence

1

1

1

1

Muscle Tone

1

1

1

1

Home cage observations: Home cage posture- 1=Normal,Respiratory Pattern -1=Normal,Clonic involuntary movements -1=None/Normal,Tonic involuntary movements -1=None/Normal,Vocalization -1=Absent (Normal),Palpebral closure -1=Eyelids wide open (Normal),Handling observation : Ease of removal from the cage -2=Normal,Ease of handling animal in hand -2=Normal,Red/Crusty deposits -1=Absent,Lacrimation- 1=None,Salivation-1=Absent (Normal),Fur Appearance -1=Normal,Piloerection -1=None, Eye Prominence -1=Normal,Muscle tone -1=Normal

M: Male; F: Female; R: Recovery

 


TABLE 5 (Contd...). SUMMARY OF NEUROLOGICAL/FUNCTIONAL OBSERVATION BATTERY (FOB) RECORD

                                                                                                     Refer Appendix 5

Week 12

Parameters↓

Group & Sex

G1R & M

G4R & M

G1R & F

G4R & F

Dose (mg/L)

0

0.03

0

0.03

Number of Animals

5

5

5

5

Open field Observation

Mobility

1

1

1

1

Gait

1

1

1

1

Arousal

3

3

3

3

Number of Rearing

Mean

6.8

5.6

8.8

8.0

±SD

1.5

1.3

0.8

2.0

Numbers of Urination

Mean

1.0

1.2

1.2

1.2

±SD

0.0

0.4

0.4

0.4

Number of Defecation

Mean

1.0

1.2

1.0

1.2

±SD

0.7

0.8

0.7

0.8

Clonic involuntary movement

1

1

1

1

Tonic involuntary movement

1

1

1

1

Stereotype Behaviour

1

1

1

1

Excessive Grooming

Mean

5.0

5.0

5.6

5.8

±SD

0.7

0.7

1.3

0.8

Sensory Observations

 

Approach Response

2

2

2

2

Auditory Response

2

2

2

2

Touch Response

2

2

2

2

Pupil Reflex

2

2

2

2

Tail Pinch Response

2

2

2

2

Righting Reflex

1

1

1

1

Physiological observation

 

Body temperature (°F)

Mean

96.9

96.6

98.4

98.6

±SD

0.2

0.4

0.2

0.2

Open field Observation: Mobility -1=Normal,Gait -1=Normal,Arousal -3=Normal,Clonic involuntary movements -1=None/Normal,Tonic involuntary movements -1=None/Normal,Stereotype Behaviour -1=Absent, Sensory Observations: Approach response -2=Normal, Auditory Response -2=Normal,Touch Response -2=Normal,Pupil Reflex -2=Normal,Tail Pinch Response -2=Normal,Righting Reflex -1=Present

M: Male; F: Female; R: Recovery


TABLE 6.  SUMMARY OF HIND LIMB FOOT SPLAY (cm)RECORD

                                                                                                                                                       Refer Appendix 6

# Week 4 & 12

Group, Sex & Dose (mg/L)

Hind Limb Foot Splay (cm)

G1, M & 0

Mean

6.2

±SD

0.5

n

5

G4, M & 0.03

Mean

6.1

±SD

0.4

n

5

G1, F & 0

Mean

5.7

±SD

0.5

n

5

G4, F & 0.03

Mean

5.6

±SD

0.4

n

5

G1R, M & 0

Mean

8.7

±SD

0.5

n

5

G4R, M & 0.03

Mean

8.1

±SD

0.5

n

5

G1R, F & 0

Mean

5.5

±SD

0.2

n

5

G4R, F & 0.03

Mean

5.5

±SD

0.1

n

5

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

# Week 4 for main group animals and week 12 for recovery group animals.

 


TABLE 7.SUMMARY OF ACTIMETER READING RECORD

                                                                                                                                                       Refer Appendix 7

# Week 4 & 12

Group, Sex & Dose (mg/L)

Movement Counts (no.)

G1, M & 0

Mean

2171.8

±SD

70.6

n

5

G4, M & 0.03

Mean

2202.0

±SD

53.8

n

5

G1, F & 0

Mean

2407.8

±SD

28.4

n

5

G4, F & 0.03

Mean

2395.8

±SD

65.2

n

5

G1R, M & 0

Mean

2239.6

±SD

33.0

n

5

G4R, M & 0.03

Mean

2260.8

±SD

57.3

n

5

G1R, F & 0

Mean

2341.8

±SD

39.5

n

5

G4R, F & 0.03

Mean

2420.6*

±SD

35.9

n

5

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

# Week 4 for main group animals and week 12 for recovery group animals; *: Statistically significant (p<0.05).

 

 


TABLE 8.  SUMMARY OF GRIP STRENGTH (kgf) MEASUREMENT RECORD

Refer Appendix 8

# Week 4 & 12

Group, Sex & Dose (mg/L)

Fore limb

Hind limb

G1, M & 0

Mean

1.505

0.534

±SD

0.051

0.087

n

5

5

G4, M & 0.03

Mean

1.486

0.472

±SD

0.049

0.022

n

5

5

G1, F & 0

Mean

1.432

0.407

±SD

0.039

0.048

n

5

5

G4, F & 0.03

Mean

1.410

0.375

±SD

0.022

0.058

n

5

5

G1R, M & 0

Mean

1.507

0.533

±SD

0.052

0.085

n

5

5

G4R, M & 0.03

Mean

1.489

0.478

±SD

0.056

0.021

n

5

5

G1R, F & 0

Mean

1.432

0.407

±SD

0.041

0.048

n

5

5

G4R, F & 0.03

Mean

1.416

0.381

±SD

0.024

0.062

n

5

5

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

# Week 4 for main group animals and week 12 for recovery group animals.

 

 

 

 

 

 

 

 

 

 

 

 


TABLE 9.     SUMMARY OF OPHTHALMOSCOPIC EXAMINATION RECORD

Week 4 and 12                                                                                                                               Refer Appendix 9

Week 4

Week 12

Group, Sex & Dose

(mg/L)

G1, M & 0

G4, M & 0.03

G1R, M & 0

G4R, M & 0.03

Number of Animals

05

05

05

05

Observations

Eye Parameters ↓

Eye

L

R

L

R

L

R

L

R

Eye Lids

N

N

N

N

N

N

N

N

Cornea

N

N

N

N

N

N

N

N

Iris

N

N

N

N

N

N

N

N

Aqueous Humour

N

N

N

N

N

N

N

N

Lens

N

N

N

N

N

N

N

N

Vitreous Humour

N

N

N

N

N

N

N

N

Retina/Optic disc

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N: No Abnormality Detected; L: Left; R: Right; M: Male; F: Female.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 


TABLE 9 (Contd…).  SUMMARY OF OPHTHALMOSCOPIC EXAMINATION RECORD

Week 4 and 12                                                                                                                              Refer Appendix 9 

Week 4

Week 12

Group, Sex & Dose

(mg/L)

G1, F & 0

G4, F & 0.03

G1R, F & 0

G4R, F & 0.03

Number of Animals

05

05

05

05

Observations

Eye Parameters ↓

Eye

L

R

L

R

L

R

L

R

Eye Lids

N

N

N

N

N

N

N

N

Cornea

N

N

N

N

N

N

N

N

Iris

N

N

N

N

N

N

N

N

Aqueous Humour

N

N

N

N

N

N

N

N

Lens

N

N

N

N

N

N

N

N

Vitreous Humour

N

N

N

N

N

N

N

N

Retina/Optic disc

N

N

N

N

N

N

N

N

N: No Abnormality Detected; L: Left; R: Right; F: Female.


TABLE 10. SUMMARY OF HAEMATOLOGY RECORD

                                        Refer Appendix 10

Group, Sex & Dose (mg/L)

Total Leucocyte Count

Total Erythrocyte Count

Hemoglobin

Haematocrit

Mean Corpuscular Volume

Mean Corpuscular Hemoglobin

Mean Corpuscular Hemoglobin Concentration

Platelet Count

Mean Platelet Volume

(WBC)

(RBC)

(HGB)

(HCT)

(MCV)

(MCH)

(MCHC)

(PLT)

(MPV)

(103cells/µL)

(106cells/µL)

(g/dL)

(%)

(fL)

(pg)

(g/dL)

(103cells/µL)

(fL)

G1, M & 0

Mean

7.63

7.81

15.52

44.20

56.60

19.94

35.26

759.00

6.18

±SD

2.12

0.60

0.98

3.50

0.52

1.32

2.33

344.07

0.48

n

5

5

5

5

5

5

5

5

5

G2, M & 0.003

Mean

9.19

8.42

15.68

46.98

55.86

18.66

33.40

1063.80

6.14

±SD

2.75

0.57

0.69

1.92

1.81

0.85

0.61

178.32

0.23

n

5

5

5

5

5

5

5

5

5

G3, M & 0.01

Mean

9.38

8.17

15.58

47.12

57.68

19.06

33.06*

1081.40

6.44

±SD

3.42

0.34

1.16

3.13

1.71

0.78

0.48

214.16

0.36

n

5

5

5

5

5

5

5

5

5

G4, M & 0.03

Mean

8.11

8.92*

16.50

49.08*

55.04

18.52

33.64

1039.80

6.80*

±SD

0.95

0.66

1.00

2.92

1.46

0.64

0.39

149.35

0.40

n

5

5

5

5

5

5

5

5

5

G1R, M & 0

Mean

9.48

9.58

16.80

51.10

53.34

17.54

32.86

783.60

6.42

±SD

1.23

0.14

0.52

1.08

0.70

0.44

0.43

122.98

0.19

n

5

5

5

5

5

5

5

5

5

G4R, M & 0.03

Mean

10.11

9.07

16.88

48.48

53.58

18.76

34.96

930.60

6.38

±SD

1.65

0.86

0.15

3.15

1.88

1.94

2.50

172.00

0.33

n

5

5

5

5

5

5

5

5

5

      M: Male; R: Recovery; SD: Standard Deviation; n: number of animals; *: Statistically significant (p<0.05).

 

 

 

 

 

 

 

 


TABLE 10 (Contd…). SUMMARY OF HAEMATOLOGY RECORD

                                                                                      Refer Appendix 10

Group, Sex & Dose (mg/L)

Reticulocyte Count

Neutrophils

Lymphocytes

Monocytes

Eosinophils

Basophils

Absolute Reticulocyte Count

(Retic)

(Neut)

(Lymph)

(Mono)

(Eos)

(Baso)

(Retic)

(%)

(%)

(%)

(%)

(%)

(%)

(109cells/L)

G1, M & 0

Mean

2.94

23.88

70.18

3.50

1.16

0.32

229.14

±SD

0.85

6.93

6.12

0.89

0.33

0.08

68.43

n

5

5

5

5

5

5

5

G2, M & 0.003

Mean

2.63

18.86

76.02

3.10

0.82

0.34

221.26

±SD

0.96

2.70

2.61

0.92

0.11

0.09

76.02

n

5

5

5

5

5

5

5

G3, M & 0.01

Mean

2.77

23.30

71.54

2.48

1.30

0.38

225.82

±SD

0.70

3.45

2.77

0.82

0.81

0.11

54.77

n

5

5

5

5

5

5

5

G4, M & 0.03

Mean

2.49

23.78

71.38

2.70

1.00

0.36

217.74

±SD

0.95

7.63

8.14

0.83

0.34

0.11

67.75

n

5

5

5

5

5

5

5

G1R, M & 0

Mean

1.81

25.66

69.22

2.68

1.32

0.54

173.32

±SD

0.22

7.18

7.42

0.83

0.40

0.24

21.10

n

5

5

5

5

5

5

5

G4R, M & 0.03

Mean

1.43*

26.90

68.00

2.80

1.08

0.46

129.64*

±SD

0.11

2.90

3.09

0.88

0.29

0.11

18.15

n

5

5

5

5

5

5

5

 M: Male; R: Recovery; SD: Standard Deviation; n: Number of animals; *: Statistically significant (p<0.05)

 

                                       

 

 

 

 

TABLE 10 (Contd…). SUMMARY OF HAEMATOLOGY RECORD

   Refer Appendix 10

Group, Sex & Dose (mg/L)

Absolute Neutrophils

Absolute Lymphocytes

Absolute Monocytes

Absolute Eosinophils

Absolute Basophils

Prothrombin Time

Activated Prothrombin Time

(Neut)

(Lymph)

(Mono)

(Eos)

(Baso)

(PT)

(APTT)

(103cells/µL)

(103cells/µL)

(103cells/µL)

(103cells/µL)

(103cells/µL)

(Seconds)

(Seconds)

G1, M & 0

Mean

1.82

5.35

0.27

0.09

0.02

14.42

16.94

±SD

0.78

1.55

0.11

0.04

0.01

0.68

3.96

n

5

5

5

5

5

5

5

G2, M & 0.003

Mean

1.78

6.94

0.28

0.07

0.03

15.82

19.02

±SD

0.72

1.90

0.11

0.02

0.02

1.30

5.09

n

5

5

5

5

5

5

5

G3, M & 0.01

Mean

2.10

6.77

0.24

0.13

0.04

15.34

23.22

±SD

0.53

2.67

0.15

0.12

0.02

1.20

9.17

n

5

5

5

5

5

5

5

G4, M & 0.03

Mean

1.90

5.82

0.22

0.08

0.03

17.42*

24.40

±SD

0.53

1.17

0.06

0.02

0.01

1.59

2.02

n

5

5

5

5

5

5

5

G1R, M & 0

Mean

2.48

6.51

0.26

0.12

0.05

16.14

28.36

±SD

0.97

0.54

0.11

0.04

0.02

1.85

11.80

n

5

5

5

5

5

5

5

G4R, M & 0.03

Mean

2.70

6.88

0.29

0.12

0.05

15.66

27.48

±SD

0.45

1.21

0.12

0.05

0.01

1.19

4.01

n

5

5

5

5

5

5

5

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

 

                                                                                                                                  


TABLE 10 (Contd…). SUMMARY OF HAEMATOLOGY RECORD

       Refer Appendix 10

Group, Sex & Dose (mg/L)

Total Leucocyte Count

Total Erythrocyte Count

Hemoglobin

Haematocrit

Mean Corpuscular Volume

Mean Corpuscular Hemoglobin

Mean Corpuscular Hemoglobin Concentration

Platelet Count

Mean Platelet Volume

(WBC)

(RBC)

(HGB)

(HCT)

(MCV)

(MCH)

(MCHC)

(PLT)

(MPV)

(103cells/µL)

(106cells/µL)

(g/dL)

(%)

(fL)

(pg)

(g/dL)

(103cells/µL)

(fL)

G1, F & 0

Mean

9.14

8.51

16.12

48.02

56.32

18.92

33.66

906.20

6.96

±SD

2.24

0.30

0.64

2.65

1.48

0.71

1.88

313.91

0.23

n

5

5

5

5

5

5

5

5

5

G2, F & 0.003

Mean

8.33

8.39

16.02

48.42

57.76

19.08

33.08

782.40

6.90

±SD

1.71

0.45

0.82

2.72

1.55

0.48

0.60

256.78

0.22

n

5

5

5

5

5

5

5

5

5

G3, F & 0.01

Mean

8.87

9.00

16.52

51.16

56.86

18.36

32.28

1092.80

6.92

±SD

1.95

0.49

0.67

2.35

2.08

0.73

0.25

89.68

0.22

n

5

5

5

5

5

5

5

5

5

G4, F & 0.03

Mean

8.54

8.95

16.42

50.80

56.78

18.34

32.32

977.40

6.90

±SD

1.49

0.28

0.52

1.16

1.14

0.40

0.41

117.58

0.19

n

5

5

5

5

5

5

5

5

5

G1R, F & 0

Mean

9.67

8.51

16.04

47.36

55.70

18.82

33.80

918.40

6.62

±SD

2.17

0.44

0.68

1.43

2.09

0.65

0.66

130.17

0.33

n

5

5

5

5

5

5

5

5

5

G4R, F & 0.03

Mean

7.58

9.06

16.56

48.78

53.90

18.32

33.94

597.80

6.98

±SD

2.19

0.33

0.72

2.11

1.12

0.53

0.40

308.99

1.65

n

5

5

5

5

5

5

5

5

5

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

  

 

 


TABLE 10 (Contd…). SUMMARY OF HAEMATOLOGY RECORD

       Refer Appendix 10

Group, Sex & Dose (mg/L)

Reticulocyte Count

Neutrophils

Lymphocytes

Monocytes

Eosinophils

Basophils

Absolute Reticulocyte Count

(Retic)

(Neut)

(Lymph)

(Mono)

(Eos)

(Baso)

(Retic)

(%)

(%)

(%)

(%)

(%)

(%)

(109cells/L)

G1, F & 0

Mean

2.55

24.28

69.98

3.34

1.34

0.26

217.08

±SD

0.59

5.92

6.34

0.91

0.82

0.09

48.26

n

5

5

5

5

5

5

5

G2, F & 0.003

Mean

2.36

24.52

70.00

3.38

0.78

0.32

198.08

±SD

0.37

5.33

5.42

1.13

0.36

0.08

31.99

n

5

5

5

5

5

5

5

G3, F & 0.01

Mean

2.64

25.72

68.92

3.36

0.82

0.36

236.94

±SD

0.48

7.95

7.60

0.68

0.42

0.11

38.20

n

5

5

5

5

5

5

5

G4, F & 0.03

Mean

2.35

22.20

72.14

3.44

0.80

0.34

209.38

±SD

0.69

5.59

6.30

0.80

0.33

0.05

55.93

n

5

5

5

5

5

5

5

G1R, F & 0

Mean

2.43

24.74

69.50

3.04

1.16

0.54

205.22

±SD

0.83

1.48

1.73

0.70

0.42

0.25

62.92

n

5

5

5

5

5

5

5

G4R, F & 0.03

Mean

1.31*

30.22*

64.16*

2.96

1.50

0.56

118.78*

±SD

0.16

3.47

3.51

0.94

0.72

0.30

18.19

n

5

5

5

5

5

5

5

      F: Female; R: Recovery; SD: Standard Deviation; n: Number of animals; *: Statistically significant (p<0.05)

 

      

 

 

 


TABLE 10 (Contd…). SUMMARY OF HAEMATOLOGY RECORD

     Refer Appendix 10

Group, Sex & Dose (mg/L)

Absolute Neutrophils

Absolute Lymphocytes

Absolute Monocytes

Absolute Eosinophils

Absolute Basophils

Prothrombin Time

Activated Prothrombin Time

(Neut)

(Lymph)

(Mono)

(Eos)

(Baso)

(PT)

(APTT)

(103cells/µL)

(103cells/µL)

(103cells/µL)

(103cells/µL)

(103cells/µL)

(Seconds)

(Seconds)

G1, F & 0

Mean

2.16

6.46

0.31

0.11

0.03

16.36

16.84

±SD

0.45

1.93

0.12

0.05

0.02

1.87

2.96

n

5

5

5

5

5

5

5

G2, F & 0.003

Mean

2.08

5.80

0.28

0.07

0.02

17.10

21.68

±SD

0.83

1.02

0.11

0.04

0.01

2.14

4.63

n

5

5

5

5

5

5

5

G3, F & 0.01

Mean

2.32

6.09

0.29

0.07

0.04

13.74

19.86

±SD

0.98

1.38

0.05

0.03

0.02

1.90

2.10

n

5

5

5

5

5

5

5

G4, F & 0.03

Mean

1.91

6.13

0.30

0.07

0.03

16.46

27.30*

±SD

0.71

0.97

0.12

0.04

0.00

1.62

3.79

n

5

5

5

5

5

5

5

G1R, F & 0

Mean

2.38

6.74

0.30

0.11

0.05

16.92

33.88

±SD

0.46

1.64

0.10

0.03

0.03

3.47

3.09

n

5

5

5

5

5

5

5

G4R, F & 0.03

Mean

2.25

4.88

0.22

0.12

0.05

15.44

33.02

±SD

0.58

1.45

0.12

0.08

0.03

2.09

7.57

n

5

5

5

5

5

5

5

      F: Female; R: Recovery; SD: Standard Deviation; n: Number of animals; *: Statistically significant (p<0.05)

      

 

      


TABLE 11.SUMMARY OF BALF RECORD

                                                                                                                                                                                            Refer Appendix 11

Group, Sex & Dose (mg/L)

Total Leucocyte Count

Neutrophils

Lymphocytes

Monocytes

Eosinophils

Basophils

Absolute Neutrophils

(WBC)

(Neut)

(Lymph)

(Mono)

(Eos)

(Baso)

(Neut)

(103cells/µL)

(%)

(%)

(%)

(%)

(%)

(103cells/µL)

G1, M & 0

 

Mean

1.19

29.60

54.24

3.00

0.86

1.44

0.58

±SD

1.45

20.44

18.64

2.65

1.06

0.52

1.05

n

5

5

5

5

5

5

5

G2, M & 0.003

Mean

0.77

32.70

56.88

2.08

0.80

2.60

0.23

±SD

0.51

9.76

10.60

1.55

0.63

1.89

0.17

n

5

5

5

5

5

5

5

G3, M & 0.01

Mean

0.32

30.42

59.32

1.92

0.88

1.84

0.10

±SD

0.16

5.85

5.34

1.31

0.73

0.90

0.05

n

5

5

5

5

5

5

5

G4, M & 0.03

Mean

0.72

27.22

66.56

2.18

1.58

2.18

0.16

±SD

0.85

10.58

10.94

0.64

1.06

0.39

0.13

n

5

5

5

5

5

5

5

G1R, M & 0

Mean

0.12

16.92

68.74

1.64

12.34

10.54

0.01

±SD

0.12

13.35

32.50

2.05

20.46

6.17

0.01

n

5

5

5

5

5

5

5

G4R, M & 0.03

Mean

0.03

27.54

63.42

3.88

4.22

2.94*

0.01

±SD

0.01

24.10

29.51

5.23

3.22

3.20

0.01

n

5

5

5

5

5

5

5

      M: Male; R: Recovery; SD: Standard Deviation; n: Number of animals; *: Statistically significant (p<0.05)

 

       


TABLE 11 (Contd…). SUMMARY OF BALF RECORD

       Refer Appendix 11

Group, Sex & Dose (mg/L)

Absolute Lymphocytes

Absolute Monocytes

Absolute Eosinophils

Absolute Basophils

Lactate Dehydrogenase

Total Protein

(Lymph)

(Mono)

(Eos)

(Baso)

(LDH)

(TPR)

(103cells/µL)

(103cells/µL)

(103cells/µL)

(103cells/µL)

(u/L)

(g/dL)

G1, M & 0

Mean

0.44

0.07

0.01

0.01

135.52

349.19

±SD

0.25

0.12

0.01

0.02

130.92

127.80

n

5

5

5

5

5

5

G2, M & 0.003

Mean

0.43

0.02

0.00

0.01

82.20

321.13

±SD

0.31

0.02

0.01

0.00

39.77

171.66

n

5

5

5

5

5

5

G3, M & 0.01

Mean

0.20

0.01

0.00

0.01

56.22

325.64

±SD

0.11

0.01

0.00

0.01

19.47

107.69

n

5

5

5

5

5

5

G4, M & 0.03

Mean

0.51

0.01

0.01

0.01

80.04

299.91

±SD

0.66

0.02

0.01

0.02

51.42

111.84

n

5

5

5

5

5

5

G1R, M & 0

Mean

0.10

0.00

0.01

0.01

56.16

296.06

±SD

0.12

0.00

0.01

0.02

24.94

116.35

n

5

5

5

5

5

5

G4R, M & 0.03

Mean

0.02

0.00

0.00

0.00

140.88*

185.63

±SD

0.02

0.00

0.00

0.00

56.13

31.78

n

5

5

5

5

5

5

      M: Male; R: Recovery; SD: Standard Deviation; n: Number of animals; *: Statistically significant (p<0.05)


TABLE 11 (Contd…). SUMMARY OF BALF RECORD

       Refer Appendix 11

Group, Sex & Dose (mg/L)

Total Leucocyte Count

Neutrophils

Lymphocytes

Monocytes

Eosinophils

Basophils

Absolute Neutrophils

(WBC)

(Neut)

(Lymph)

(Mono)

(Eos)

(Baso)

(Neut)

(103cells/µL)

(%)

(%)

(%)

(%)

(%)

(103cells/µL)

G1, F & 0

Mean

0.32

21.94

65.62

1.72

0.32

5.42

0.07

±SD

0.07

2.42

5.73

0.60

0.18

4.49

0.01

n

5

5

5

5

5

5

5

G2, F & 0.003

Mean

0.43

27.32

52.80

1.66

0.30

1.68

0.12

±SD

0.17

5.99

4.72

0.73

0.22

0.73

0.06

n

5

5

5

5

5

5

5

G3, F & 0.01

Mean

0.87*

27.86

59.40

1.76

3.56

2.12

0.23

±SD

0.51

6.20

7.70

0.55

6.22

1.10

0.10

n

5

5

5

5

5

5

5

G4, F & 0.03

Mean

0.46

51.66*

42.10*

1.66

2.00

2.78

0.23

±SD

0.28

17.30

17.59

1.73

1.17

4.35

0.16

n

5

5

5

5

5

5

5

G1R, F & 0

Mean

0.27

19.08

69.36

2.72

4.80

11.52

0.06

±SD

0.17

6.77

8.10

1.29

2.53

6.91

0.05

n

5

5

5

5

5

5

5

G4R, F & 0.03

Mean

1.89

22.94

66.34

4.62

3.28

9.90

1.08

±SD

3.60

24.01

30.37

7.23

2.26

7.57

2.30

n

5

5

5

5

5

5

5

      F: Female; R: Recovery; SD: Standard Deviation; n: Number of animals; *: Statistically significant (p<0.05)

 

 


TABLE 11 (Contd…). SUMMARY OF BALF RECORD

       Refer Appendix 11

Group, Sex & Dose (mg/L)

Absolute Lymphocytes

Absolute Monocytes

Absolute Eosinophils

Absolute Basophils

Lactate Dehydrogenase

Total Protein

(Lymph)

(Mono)

(Eos)

(Baso)

(LDH)

(TPR)

(103cells/µL)

(103cells/µL)

(103cells/µL)

(103cells/µL)

(u/L)

(g/dL)

G1, F & 0

Mean

0.21

0.00

0.00

0.02

47.56

322.15

±SD

0.06

0.01

0.00

0.01

6.02

135.05

n

5

5

5

5

5

5

G2, F & 0.003

Mean

0.23

0.01

0.00

0.01

41.26

326.31

±SD

0.09

0.00

0.00

0.00

18.91

87.24

n

5

5

5

5

5

5

G3, F & 0.01

Mean

0.50*

0.02

0.05

0.02

102.52

369.37

±SD

0.26

0.02

0.12

0.01

39.86

120.36

n

5

5

5

5

5

5

G4, F & 0.03

Mean

0.20

0.01

0.01

0.02

146.44

371.40

±SD

0.13

0.02

0.01

0.03

188.18

300.11

n

5

5

5

5

5

5

G1R, F & 0

Mean

0.18

0.00

0.01

0.03

49.50

193.58

±SD

0.12

0.01

0.01

0.02

30.42

55.52

n

5

5

5

5

5

5

G4R, F & 0.03

Mean

0.46

0.29

0.04

0.09

140.56

229.77

±SD

0.59

0.65

0.08

0.15

175.38

44.27

n

5

5

5

5

5

5

      F: Female; R: Recovery; SD: Standard Deviation; n: Number of animals; *: Statistically significant (p<0.05)


TABLE 12.  SUMMARY OF CLINICAL CHEMISTRYRECORD

                                                                                                                                                                                                                                                                 Refer Appendix 12

Group, Sex & Dose (mg/L)

Glucose

Urea

Creatinine

Total Cholesterol

Triglycerides

Total Protein

Albumin

Alanine aminotransferase

Aspartate aminotransferase

(GLU)

(CRE)

(CHO)

(TRI)

(TPR)

(ALB)

(ALT)

(AST)

(mg/dL)

(mg/dL)

(mg/dL)

(mg/dL)

(mg/dL)

(g/dL)

(g/dL)

(U/L)

(U/L)

G1, M & 0

Mean

96.80

39.10

0.57

53.20

39.20

7.22

3.57

41.20

92.00

±SD

4.44

2.85

0.06

2.95

7.85

0.33

0.13

5.07

7.11

n

5

5

5

5

5

5

5

5

5

G2, M & 0.003

Mean

103.80

39.82

0.57

46.80

37.20

7.42

3.44

43.20

103.80

±SD

26.07

6.93

0.05

14.24

15.30

0.33

0.27

6.61

13.01

n

5

5

5

5

5

5

5

5

5

G3, M & 0.01

Mean

90.20

41.28

0.54

54.60

40.80

7.58

3.39

54.00

112.80*

±SD

8.67

4.69

0.03

12.97

15.02

0.22

0.14

15.44

13.05

n

5

5

5

5

5

5

5

5

5

G4, M & 0.03

Mean

95.60

39.16

0.54

42.80

37.00

7.70

3.52

39.40

101.00

±SD

4.83

6.64

0.04

13.07

3.94

0.67

0.23

7.54

9.67

n

5

5

5

5

5

5

5

5

5

G1R, M & 0

Mean

107.00

29.42

0.49

48.80

40.00

7.14

3.66

67.60

129.40

±SD

18.19

6.19

0.02

7.01

12.83

0.30

0.09

37.04

90.48

n

5

5

5

5

5

5

5

5

5

G4R, M & 0.03

Mean

101.00

28.38

0.46

56.60

53.00

7.10

3.78

56.40

88.60

±SD

10.37

3.04

0.02

14.66

6.12

0.48

0.25

5.37

10.74

n

5

5

5

5

5

5

5

5

5

              M: Male; R: Recovery; SD: Standard Deviation; n: number of animals; *: Statistically significant (p<0.05)

 

 

TABLE 12 (Contd…). SUMMARY OF CLINICAL CHEMISTRY RECORD

Refer Appendix 12

Group, Sex & Dose (mg/L)

Alkaline phosphatase

Total Bilirubin

Calcium

Phosphorous

Globulin

Blood Urea Nitrogen

Sodium

Potassium

Chloride

(ALP)

(BIT)

(CAL)

(PHO)

(GLO)

(BUN)

(Na)

(K)

(CLO)

(U/L)

(mg/dL)

(mg/dL)

(mg/dL)

(g/dL)

(mg/dL)

(mmol/L)

(mmol/L)

(mmol/L)

G1, M & 0

Mean

135.20

0.01

9.88

6.22

3.65

18.25

141.42

3.50

110.18

±SD

34.04

0.01

0.26

0.36

0.33

1.33

0.68

0.18

0.86

n

5

5

5

5

5

5

5

5

5

G2, M & 0.003

Mean

116.80

0.02

10.06

6.86

3.98

18.58

142.36

4.02

110.62

±SD

29.58

0.02

0.15

0.47

0.25

3.23

0.53

0.59

1.82

n

5

5

5

5

5

5

5

5

5

G3, M & 0.01

Mean

142.60

0.03

10.26*

6.86

4.19

19.26

143.06

3.92

110.80

±SD

39.84

0.03

0.09

1.00

0.20

2.19

1.21

0.31

2.59

n

5

5

5

5

5

5

5

5

5

G4, M & 0.03

Mean

105.80

0.02

9.68

6.36

4.18

18.28

143.46*

3.66

110.12

±SD

11.95

0.01

0.20

0.45

0.60

3.10

1.78

0.25

2.37

n

5

5

5

5

5

5

5

5

5

G1R, M & 0

Mean

143.00

0.01

9.44

6.46

3.48

13.73

142.42

3.74

106.80

±SD

49.28

0.01

0.30

0.34

0.29

2.89

0.71

0.45

0.70

n

5

5

5

5

5

5

5

5

5

G4R, M & 0.03

Mean

161.60

0.01

9.76

6.66

3.32

13.25

142.08

3.57

105.20

±SD

94.73

0.01

0.23

0.55

0.28

1.42

0.73

0.16

1.51

n

5

5

5

5

5

5

5

5

5

M: Male; R: Recovery; SD: Standard Deviation; n: Number of animals; *: Statistically significant (p<0.05)

 

 

 

TABLE 12 (Contd…). SUMMARY OF CLINICAL CHEMISTRY

Refer Appendix 12

Group, Sex & Dose (mg/L)

Glucose

Urea

Creatinine

Total Cholesterol

Triglycerides

Total Protein

Albumin

Alanine aminotransferase

Aspartate aminotransferase

(GLU)

(CRE)

(CHO)

(TRI)

(TPR)

(ALB)

(ALT)

(AST)

(mg/dL)

(mg/dL)

(mg/dL)

(mg/dL)

(mg/dL)

(g/dL)

(g/dL)

(U/L)

(U/L)

G1, F & 0

Mean

79.80

39.56

0.52

42.80

34.40

7.00

3.41

52.20

108.60

±SD

8.56

4.16

0.07

3.96

4.67

0.31

0.11

7.79

16.70

n

5

5

5

5

5

5

5

5

5

G2, F & 0.003

Mean

87.80

37.04

0.51

48.80

55.00

7.20

3.41

60.40

123.40

±SD

11.56

4.05

0.05

8.58

30.10

0.32

0.15

19.63

32.67

n

5

5

5

5

5

5

5

5

5

G3, F & 0.01

Mean

91.80

35.14

0.51

45.00

34.00

7.28

3.60

57.00

108.60

±SD

8.14

1.95

0.03

6.28

8.54

0.16

0.11

11.11

8.11

n

5

5

5

5

5

5

5

5

5

G4, F & 0.03

Mean

101.20*

49.52*

0.52

40.60

34.20

7.38

3.50

62.40

113.60

±SD

17.84

7.88

0.03

5.77

15.11

0.37

0.18

6.69

9.02

n

5

5

5

5

5

5

5

5

5

G1R, F & 0

Mean

98.20

28.50

0.52

73.80

39.40

7.52

3.97

45.00

94.00

±SD

6.91

1.52

0.02

11.28

6.62

0.36

0.22

3.54

8.60

n

5

5

5

5

5

5

5

5

5

G4R, F & 0.03

Mean

110.60

40.98

0.63

67.00

40.40

7.56

3.92

57.80

97.80

±SD

20.18

11.85

0.23

11.25

8.50

0.13

0.06

13.74

13.59

n

5

5

5

5

5

5

5

5

5

 F: Female; R: Recovery; SD: Standard Deviation; n: number of animals; *: Statistically significant (p<0.05)

 

 

 

TABLE 12 (Contd…). SUMMARY OF CLINICAL CHEMISTRY

Refer Appendix 12

Group, Sex & Dose (mg/L)

Alkaline phosphatase

Total Bilirubin

Calcium

Phosphorous

Globulin

Blood Urea Nitrogen

Sodium

Potassium

Chloride

(ALP)

(BIT)

(CAL)

(PHO)

(GLO)

(BUN)

(Na)

(K)

(CLO)

(U/L)

(mg/dL)

(mg/dL)

(mg/dL)

(g/dL)

(mg/dL)

(mmol/L)

(mmol/L)

(mmol/L)

G1, F & 0

Mean

200.40

0.02

10.00

7.58

3.59

18.46

143.34

4.02

110.70

±SD

35.13

0.02

0.31

0.62

0.25

1.94

0.85

0.26

1.70

n

5

5

5

5

5

5

5

5

5

G2, F & 0.003

Mean

179.00

0.03

9.88

7.22

3.79

17.29

142.58

4.12

109.02

±SD

24.21

0.01

0.37

0.38

0.22

1.89

2.66

0.37

1.99

n

5

5

5

5

5

5

5

5

5

G3, F & 0.01

Mean

201.00

0.02

10.20

7.10

3.68

16.40

142.94

3.79

108.82

±SD

27.65

0.02

0.07

0.32

0.17

0.91

1.16

0.05

1.53

n

5

5

5

5

5

5

5

5

5

G4, F & 0.03

Mean

186.80

0.03

9.92

7.06

3.88

23.11*

144.54

3.99

112.36

±SD

55.68

0.01

0.34

0.98

0.20

3.68

1.30

0.14

1.50

n

5

5

5

5

5

5

5

5

5

G1R, F & 0

Mean

67.00

0.03

10.00

5.94

3.55

13.30

141.60

3.42

106.46

±SD

31.44

0.02

0.30

0.38

0.29

0.71

0.95

0.19

0.94

n

5

5

5

5

5

5

5

5

5

G4R, F & 0.03

Mean

95.00

0.03

9.80

5.72

3.64

19.12

141.66

3.82*

106.10

±SD

29.97

0.02

0.21

1.10

0.17

5.53

1.15

0.22

1.75

n

5

5

5

5

5

5

5

5

5

F: Female; R: Recovery; SD: Standard Deviation; n: Number of animals; *: Statistically significant (p<0.05)

 

 

 

 


TABLE 13.SUMMARY OF URINALYSIS

         Refer Appendix 13

Examination

Group, Sex & Dose (mg/L)

G1, M & 0

G2, M & 0.003

G3, M & 0.01

G4, M & 0.03

G1R, M & 0

G4R, M & 0.03

Number of Animals

5

5

5

5

5

5

Physical

Colour

Pale Yellow

5

5

5

5

5

4

Yellow

-

-

-

-

-

1

Appearance

Clear

5

5

5

5

4

3

Turbid

-

-

-

-

1

2

Volume (mL)

Mean

4.8

5.9

6.8

5.9

5.3

4.2

±SD

1.2

1.7

1.3

1.0

0.9

0.6

Chemical

pH

Mean

7.8

7.8

7.7

7.6

7.2

7.4

±SD

0.3

0.3

0.3

0.4

0.7

0.8

Specific Gravity

Mean

1.009

1.010

1.011

1.012

1.013

1.010

±SD

0.002

0.000

0.002

0.003

0.004

0.004

Urobilinogen (mg/dL)

Mean

0.2

0.2

0.2

0.2

0.2

0.2

±SD

0.0

0.0

0.0

0.0

0.0

0.0

Bilirubin (mg/dL)

Neg

1

4

4

3

5

4

1

4

1

1

2

-

1

Ketones (mg/dL)

Neg

2

1

3

-

-

2

5

3

4

2

5

5

3

Blood (Ery/µL)

Neg

5

3

5

4

4

5

Ca10

-

1

-

1

1

-

Ca25

-

1

-

-

-

-

Proteins (mg/dL)

Neg

2

4

2

3

3

3

Trace

1

-

2

2

1

-

30

2

1

1

-

1

1

>=300

-

-

-

-

-

1

Nitrite

Neg

1

3

1

2

4

3

Pos

4

2

4

3

1

2

Leucocytes (Leu/µL)

Neg

-

-

-

1

-

1

Ca15

-

2

1

2

2

2

Ca70

5

3

4

2

3

2

Glucose (mg/dL)

Neg

5

5

5

5

5

5

Micro albumin (MALB) (mg/dL)

Neg

-

1

-

1

-

1

>15

3

1

3

2

2

2

15

2

3

2

2

3

2

Microscopic

Epithelial cells

0

4

5

5

5

2

4

0-1

1

-

-

-

2

-

1-2

-

-

-

-

1

1

Casts

Absent

5

5

5

5

5

5

Crystals

Present

5

5

5

5

5

5

       M: Male; Neg: Negative; Ca: Calculated Approximately; Pos: Positive; SD: Standard Deviation

        

 

 

 

 

 

 

 

 

 

TABLE 13 (Contd…). SUMMARY OF URINALYSIS

Refer Appendix 13

Examination

Group, Sex & Dose (mg/L)

G1, F &

0

G2, F & 0.003

G3, F & 0.01

G4, F &

0.03

G1R, F &

0

G4R, F

&

0.03

Number of Animals

5

5

5

5

5

5

Physical

Colour

Pale Yellow

5

5

5

5

4

5

Yellow

-

-

-

-

1

-

Appearance

Clear

5

5

5

5

4

4

Turbid

-

-

-

-

1

1

Volume (mL)

Mean

5.8

5.1

5.2

5.0

4.0

4.6

±SD

1.5

1.3

0.8

0.9

0.4

1.1

Chemical

pH

Mean

7.3

7.1

7.7

7.8

7.3

7.0

±SD

0.8

1.0

0.3

0.3

0.8

0.8

Specific Gravity

Mean

1.012

1.016

1.010

1.009

1.009

1.012

±SD

0.008

0.007

0.000

0.002

0.004

0.003

Urobilinogen (mg/dL)

Mean

0.2

0.2

0.2

0.6

0.2

0.2

±SD

0.0

0.0

0.0

0.8

0.0

0.0

Bilirubin (mg/dL)

Neg

4

3

5

4

4

5

1

1

2

-

1

1

-

Ketones (mg/dL)

Neg

5

5

5

5

5

5

Blood (Ery/µL)

Neg

5

3

4

4

5

4

Ca10

-

1

1

1

-

-

Ca25

-

1

-

-

-

1

Proteins (mg/dL)

Neg

5

5

4

3

2

4

Trace

-

-

1

1

2

-

30

-

-

-

-

1

1

>=300

-

-

-

1

-

-

Nitrite

Neg

3

2

4

4

3

4

Pos

2

3

1

1

2

1

Leucocytes (Leu/µL)

Neg

-

2

-

-

3

1

Ca15

5

3

2

2

1

4

Ca70

-

-

3

2

1

-

Ca125

-

-

-

1

-

-

Glucose (mg/dL)

Neg

5

5

5

5

5

5

Micro albumin (MALB) (mg/dL)

Neg

1

2

1

1

1

1

>15

-

-

1

2

3

1

15

4

3

3

2

1

3

Microscopic

Epithelial cells

0

4

4

4

3

1

3

0-1

1

-

1

1

3

1

1-2

-

1

-

1

1

1

Casts

Absent

5

5

5

5

5

5

Crystals

Present

5

5

5

5

5

5

       F: Female; Neg: Negative; Ca: Calculated Approximately; Pos: Positive; SD: Standard Deviation

      


TABLE 14.    SUMMARY OF ABSOLUTE ORGAN WEIGHTS (g)

                                                                                                                                                                                                                                                                    Refer Appendix 14

Group, Sex & Dose (mg/L)

Adrenals

Thymus

Spleen

Testes

Heart

Kidneys

Brain

Liver

Lungs

G1, M & 0

Mean

0.0591

0.3086

0.4909

2.8529

0.8080

1.6090

1.8356

6.7114

0.6179

±SD

0.0205

0.0655

0.0487

0.0423

0.1208

0.1735

0.0435

0.5112

0.1409

n

5

5

5

5

5

5

5

5

5

G2, M & 0.003

Mean

0.0491

0.3546

0.4750

3.0797

0.9093

1.8123

1.8330

7.3951

0.5748

±SD

0.0017

0.0335

0.0973

0.1235

0.0870

0.1910

0.0669

0.9794

0.0890

n

5

5

5

5

5

5

5

5

5

G3, M & 0.01

Mean

0.0585

0.2789

0.4677

3.0948

0.8801

1.7441

1.7921

6.8768

0.5045

±SD

0.0101

0.0359

0.0834

0.2170

0.1741

0.1330

0.1249

0.8572

0.1224

n

5

5

5

5

5

5

5

5

5

G4, M & 0.03

Mean

0.0499

0.2645

0.4638

3.0833

0.8060

1.5553

1.7554

6.7946

0.5538

±SD

0.0059

0.0298

0.1002

0.1584

0.1314

0.1997

0.1222

0.7357

0.0879

n

5

5

5

5

5

5

5

5

5

G1R, M & 0

Mean

0.0670

0.3694

0.7428

3.4995

1.3398

2.5617

1.9615

10.6978

0.6410

±SD

0.0091

0.0663

0.1451

0.3605

0.2577

0.5982

0.1373

1.3430

0.0477

n

5

5

5

5

5

5

5

5

5

G4R, M & 0.03

Mean

0.0705

0.4383

0.7226

3.1986

1.4393

2.8197

2.0371

11.1423

0.6347

±SD

0.0132

0.0332

0.0480

0.1484

0.0975

0.3347

0.0799

1.4656

0.0587

n

5

5

5

5

5

5

5

5

5

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

.

 

 

TABLE 14 (Contd…). SUMMARY OF ABSOLUTE ORGAN WEIGHTS (g)

Refer Appendix 14

Group, Sex & Dose (mg/L)

Adrenals

Thymus

Spleen

Ovaries

Uterus

Heart

Kidneys

Brain

Liver

Lungs

G1, F & 0

Mean

0.0688

0.4001

0.5071

0.1062

0.5056

0.7483

1.3784

1.7000

6.4285

0.4590

±SD

0.0084

0.0640

0.0527

0.0343

0.0727

0.1094

0.1386

0.0522

0.5623

0.0574

n

5

5

5

5

5

5

5

5

5

5

G2, F & 0.003

Mean

0.0709

0.3071*

0.4388

0.1335

0.6509

0.7224

1.3771

1.7426

5.7190

0.4886

±SD

0.0109

0.0146

0.0602

0.0338

0.1297

0.0724

0.1948

0.0479

0.6038

0.0660

n

5

5

5

5

5

5

5

5

5

5

G3, F & 0.01

Mean

0.0884

0.3436

0.4720

0.1027

0.6082

0.7089

1.3448

1.7532

6.4444

0.5426

±SD

0.0645

0.0508

0.0590

0.0437

0.1803

0.0492

0.1877

0.0476

0.9024

0.0554

n

5

5

5

5

5

5

5

5

5

5

G4, F & 0.03

Mean

0.0581

0.3067*

0.4379

0.0971

0.4466

0.6583

1.3295

1.7579

5.1555*

0.5497

±SD

0.0085

0.0338

0.1303

0.0283

0.1686

0.0274

0.0703

0.0731

0.8599

0.1192

n

5

5

5

5

5

5

5

5

5

5

G1R, F & 0

Mean

0.0774

0.3357

0.8291

0.1343

1.1745

0.9412

1.7869

1.9118

8.5142

0.5585

±SD

0.0140

0.0703

0.1620

0.0056

0.2419

0.0437

0.1660

0.0276

1.1044

0.1485

n

5

5

5

5

5

5

5

5

5

5

G4R, F & 0.03

Mean

0.0777

0.2541

0.5076*

0.1404

0.7023*

0.9485

1.6055

1.8672

7.3391

0.5311

±SD

0.0149

0.0375

0.0487

0.0314

0.1485

0.0838

0.1722

0.0575

0.6635

0.0730

n

5

5

5

5

5

5

5

5

5

5

 F: Female, R: Recovery; SD: Standard Deviation, n: Number of animals; *: Statistically significant (p<0.05)

 

 

 


TABLE 15.  SUMMARY OF ORGAN WEIGHT RELATIVE TO BODY WEIGHT(%)RECORD

Refer Appendix 15

Group, Sex & Dose (mg/L)

Fasting Body Weight (g)

Adrenals

Thymus

Spleen

Testes

Heart

Kidneys

Brain

Liver

Lungs

G1, M & 0

Mean

219.28

0.0267

0.1414

0.2241

1.3049

0.3676

0.7334

0.8392

3.0624

0.2808

±SD

11.91

0.0082

0.0326

0.0213

0.0893

0.0413

0.0633

0.0519

0.2050

0.0541

n

5

5

5

5

5

5

5

5

5

5

G2, M & 0.003

Mean

221.69

0.0222

0.1606

0.2131

1.3908

0.4104

0.8163

0.8278

3.3281

0.2583

±SD

10.68

0.0011

0.0210

0.0340

0.0630

0.0387

0.0554

0.0382

0.3134

0.0295

n

5

5

5

5

5

5

5

5

5

5

G3, M & 0.01

Mean

217.94

0.0269

0.1279

0.2147

1.4231

0.4042

0.8012

0.8234

3.1585

0.2322

±SD

8.20

0.0050

0.0151

0.0387

0.1334

0.0816

0.0686

0.0685

0.4111

0.0607

n

5

5

5

5

5

5

5

5

5

5

G4, M & 0.03

Mean

218.45

0.0229

0.1213

0.2127

1.4128

0.3682

0.7106

0.8039

3.1065

0.2540

±SD

8.44

0.0029

0.0153

0.0480

0.0842

0.0532

0.0727

0.0529

0.2609

0.0436

n

5

5

5

5

5

5

5

5

5

5

G1R, M & 0

Mean

406.82

0.0165

0.0908

0.1824

0.8601

0.3293

0.6293

0.4820

2.6288

0.1577

±SD

5.43

0.0024

0.0158

0.0342

0.0859

0.0626

0.1442

0.0298

0.3176

0.0131

n

5

5

5

5

5

5

5

5

5

5

G4R, M & 0.03

Mean

408.41

0.0173

0.1073

0.1770

0.7833

0.3525

0.6908

0.4988

2.7301

0.1554

±SD

2.91

0.0033

0.0085

0.0123

0.0383

0.0254

0.0858

0.0190

0.3761

0.0144

n

5

5

5

5

5

5

5

5

5

5

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

 


TABLE 15 (Contd…). SUMMARY OF ORGAN WEIGHT RELATIVE TO BODY WEIGHT (%) RECORD

Refer Appendix 15

Group, Sex & Dose (mg/L)

Fasting Body Weight (g)

Adrenals

Thymus

Spleen

Ovaries

Uterus

Heart

Kidneys

Brain

Liver

Lungs

G1, F & 0

Mean

186.50

0.0369

0.2152

0.2733

0.0562

0.2709

0.4001

0.7379

0.9151

3.4526

0.2470

±SD

11.64

0.0036

0.0370

0.0400

0.0154

0.0321

0.0433

0.0331

0.0779

0.3026

0.0345

n

5

5

5

5

5

5

5

5

5

5

5

G2, F & 0.003

Mean

190.05

0.0374

0.1620*

0.2316

0.0702

0.3417

0.3795

0.7244

0.9178

3.0099

0.2568

±SD

8.48

0.0058

0.0136

0.0362

0.0169

0.0597

0.0268

0.0942

0.0305

0.2928

0.0299

n

5

5

5

5

5

5

5

5

5

5

5

G3, F & 0.01

Mean

186.46

0.0465

0.1837

0.2538

0.0548

0.3264

0.3811

0.7206

0.9417

3.4488

0.2909

±SD

9.23

0.0311

0.0193

0.0359

0.0222

0.0989

0.0353

0.0845

0.0436

0.3509

0.0235

n

5

5

5

5

5

5

5

5

5

5

5

G4, F & 0.03

Mean

186.01

0.0312

0.1648*

0.2341

0.0522

0.2401

0.3541

0.7154

0.9454

2.7706*

0.2964

±SD

6.42

0.0038

0.0158

0.0647

0.0154

0.0926

0.0156

0.0443

0.0350

0.4482

0.0677

n

5

5

5

5

5

5

5

5

5

5

5

G1R, F & 0

Mean

311.50

0.0249

0.1078

0.2670

0.0431

0.3782

0.3023

0.5743

0.6141

2.7364

0.1797

±SD

8.78

0.0046

0.0227

0.0568

0.0022

0.0840

0.0165

0.0595

0.0180

0.3825

0.0500

n

5

5

5

5

5

5

5

5

5

5

5

G4R, F & 0.03

Mean

309.87

0.0250

0.0819

0.1637*

0.0453

0.2268*

0.3061

0.5177

0.6027

2.3667

0.1715

±SD

5.44

0.0045

0.0111

0.0142

0.0098

0.0490

0.0260

0.0500

0.0205

0.1834

0.0247

n

5

5

5

5

5

5

5

5

5

5

5

 F: Female, R: Recovery; SD: Standard Deviation, n: Number of animals; *: Statistically significant (p<0.05)

 


TABLE 16.  SUMMARY OF ORGAN WEIGHT RELATIVE TO BRAIN WEIGHT (%) RECORD

                                                                                                                                                                                                                                                        Refer Appendix 16

Group, Sex & Dose (mg/L)

Adrenals

Thymus

Spleen

Testes

Heart

Kidneys

Liver

Lungs

G1, M & 0

Mean

3.2084

16.8417

26.7271

155.4808

44.0710

87.6913

365.9170

33.6076

±SD

1.0667

3.7434

2.3717

4.1007

7.0256

9.6107

30.7326

7.1803

n

5

5

5

5

5

5

5

5

G2, M & 0.003

Mean

2.6843

19.3593

25.9283

168.0469

49.5460

98.9292

403.3509

31.3037

±SD

0.1704

1.9147

5.3241

4.2249

3.4296

10.3827

50.1715

4.3567

n

5

5

5

5

5

5

5

5

G3, M & 0.01

Mean

3.2578

15.6978

26.2370

172.9034

49.2188

97.6369

384.4100

28.5755

±SD

0.4378

2.9017

4.9883

9.4807

9.2781

9.1659

45.1221

8.7837

n

5

5

5

5

5

5

5

5

G4, M & 0.03

Mean

2.8690

15.0658

26.7382

176.6741*

46.0977

88.6284

389.0618

31.6333

±SD

0.5374

1.2650

7.5037

19.5080

8.4852

9.9253

55.4416

5.2430

n

5

5

5

5

5

5

5

5

G1R, M & 0

Mean

3.4347

18.7629

37.7116

179.2759

68.2623

130.2046

544.9017

32.7719

±SD

0.5718

2.3064

5.4990

23.6753

11.8016

25.8373

48.5062

2.9682

n

5

5

5

5

5

5

5

5

G4R, M & 0.03

Mean

3.4637

21.5828

35.4693

157.2705

70.6704

138.4677

547.5343

31.2402

±SD

0.6484

2.2843

1.8665

10.6981

4.3381

16.2928

75.0901

3.6027

n

5

5

5

5

5

5

5

5

M: Male; R: Recovery; SD: Standard Deviation, n: Number of animals; *: Statistically significant (p<0.05)

 

 

 

TABLE 16 (Contd…). SUMMARY OF ORGAN WEIGHT RELATIVE TO BRAIN WEIGHT (%) RECORD

Refer Appendix 16

Group, Sex & Dose (mg/L)

Adrenals

Thymus

Spleen

Ovaries

Uterus

Heart

Kidneys

Liver

Lungs

G1, F & 0

Mean

4.0588

23.5205

29.8022

6.2720

29.7271

44.1110

81.1947

378.1715

27.0106

±SD

0.5596

3.6232

2.5080

2.0577

4.0718

6.9072

8.8951

31.5449

3.3719

n

5

5

5

5

5

5

5

5

5

G2, F & 0.003

Mean

4.0677

17.6305*

25.2137

7.6464

37.3403

41.4107

78.8936

327.7424

28.0605

±SD

0.5998

0.9366

3.7009

1.8651

7.3467

3.4933

9.7042

27.2146

3.9499

n

5

5

5

5

5

5

5

5

5

G3, F & 0.01

Mean

5.0209

19.6054

26.9066

5.8463

34.9025

40.4713

76.6939

367.4592

30.9520

±SD

3.6063

2.8966

3.1648

2.4545

11.1287

3.1944

10.3619

48.8942

3.0575

n

5

5

5

5

5

5

5

5

5

G4, F & 0.03

Mean

3.3066

17.4195*

24.7852

5.5106

25.3271

37.4696

75.7367

293.0729*

31.3939

±SD

0.4756

1.4201

7.0121

1.5875

9.5655

1.4874

5.0797

46.3627

7.3171

n

5

5

5

5

5

5

5

5

5

G1R, F & 0

Mean

4.0565

17.5985

43.3976

7.0279

61.4346

49.2572

93.5633

445.8532

29.2652

±SD

0.7920

3.9173

8.6843

0.3349

12.5922

2.8374

9.8313

62.6420

8.0534

n

5

5

5

5

5

5

5

5

5

G4R, F & 0.03

Mean

4.1760

13.6221

27.2025*

7.4981

37.7341*

50.7802

85.9015

393.3327

28.4678

±SD

0.8846

2.1080

2.6776

1.5337

8.6957

3.9404

7.7389

37.4725

4.0105

n

5

5

5

5

5

5

5

5

5

 F: Female, R: Recovery; SD: Standard Deviation, n: Number of animals; *: Statistically significant (p<0.05)

 

 

 

 

 

 

 

 

TABLE 17.  SUMMARY OF CHAMBER (EXPOSURE) CONDITIONS

 Refer Appendix17


Group & Conc.

(mg/L)

 

Temp.

 (°C)

Rh

(%)

O2

(%)

CO2

 (mg/L)

Air inlet flow rate

(L/min)*

BZC

(mg/L)

G1/G1R & 0

Mean

22.51

55.70

20.45

615.25

20.00

-

±SD

0.22

0.94

0.22

5.03

0.00

-

G2 & 0.003

Mean

22.53

55.51

20.48

615.34

20.00

0.0031

±SD

0.21

1.08

0.21

4.72

0.00

0.0001

G3 & 0.01

Mean

22.52

55.48

20.46

615.85

20.00

0.011

±SD

0.24

1.01

0.22

5.22

0.00

0.001

G4/G4R & 0.03

Mean

22.31

55.21

20.45

614.76

20.00

0.032

±SD

2.07

1.27

0.21

5.21

0.00

0.0007

*: Values were constant throughout the exposure; hence standard deviation is zero, Temp.: Temperature; Rh: Relative Humidity; O2: Oxygen Concentration;

     CO2: Carbon Dioxide Concentration;BZC: Breathing Zone Concentration:SD: Standard Deviation;R: Recovery.

 

 

 

 

 

 


TABLE 18.SUMMARY OF NOMINAL CONCENTRATION

                                                                                                                              Refer Appendix 18

Day

Group

Test item Used (mg) (a)

Air flow rate (L/minute)(b)

Minute

(min)

(c)

Nominal Concentration

(mg/L)

(a)    / (b) × (c)

1

G1/G1R 

(Air Only)

-

20

    360

-

G2, G3 and G4/G4R 

(Test Item)

1690

20

360

0.23

2650

0.37

3440

0.48

2

G1/G1R

(Air Only)

-

20

360

-

G2, G3 and G4/G4R 

(Test Item)

1750

20

360

0.24

2600

0.36

3650

0.51

 3

G1/G1R 

(Air Only)

-

20

360

-

G2, G3 and G4/G4R 

(Test Item)

1530

20

360

0.21

2570

0.36

3590

0.50

4

G1/G1R 

(Air Only)

-

20

360

-

G2, G3 and G4/G4R 

(Test Item)

1690

20

360

0.23

2700

0.38

3440

0.48

5

G1/G1R 

(Air Only)

-

20

360

-

G2, G3 and G4/G4R 

(Test Item)

1490

20

360

0.21

2670

0.37

3500

0.49

6

G1/G1R 

(Air Only)

-

20

360

-

G2, G3 and G4/G4R 

(Test Item)

1600

20

360

0.22

2780

0.39

3480

0.48

7

G1/G1R 

(Air Only)

-

20

360

-

G2, G3 and G4/G4R 

(Test Item)

1540

20

360

0.21

2680

0.37

3440

0.48


TABLE 18 (Contd…). SUMMARY OF NOMINAL CONCENTRATION

                                                                                                                                                               Refer Appendix 18

Day

Group

Test item Used (mg) (a)

Air flow rate (L/minute)(b)

Minute

(min)

(c)

Nominal Concentration

(mg/L)

(b)    / (b) × (c)

8

G1/G1R 

(Air Only)

-

20

    360

-

G2, G3 and G4/G4R 

(Test Item)

1530

20

360

0.21

2700

0.37

3510

0.49

9

G1/G1R 

(Air Only)

-

20

360

-

G2, G3 and G4/G4R 

(Test Item)

1500

20

360

0.21

3040

0.42

3570

0.50

 10

G1/G1R 

(Air Only)

-

20

360

-

G2, G3 and G4/G4R 

(Test Item)

1730

20

360

0.24

2750

0.38

3650

0.51

11

G1/G1R 

(Air Only)

-

20

360

-

G2, G3 and G4/G4R 

(Test Item)

1780

20

360

0.25

2490

0.35

3440

0.48

12

G1/G1R 

(Air Only)

-

20

360

-

G2, G3 and G4/G4R 

(Test Item)

1820

20

360

0.25

2530

0.35

3410

0.47

13

G1/G1R 

(Air Only)

-

20

360

-

G2, G3 and G4/G4R 

(Test Item)

1720

20

360

0.24

2650

0.37

3680

0.51

14

G1/G1R 

(Air Only)

-

20

360

-

G2, G3 and G4/G4R 

(Test Item)

1780

20

360

0.25

2520

0.35

4040

0.56

 

 

 

 

 

 

 

 

 

 


TABLE 18 (Contd…). SUMMARY OF NOMINAL CONCENTRATION

Refer Appendix 18

Day

Group

Test item Used (mg) (a)

Air flow rate (L/minute)(b)

Minute

(min)

(c)

Nominal Concentration

(mg/L)

(c)    / (b) × (c)

15

G1/G1R 

(Air Only)

-

20

    360

-

G2, G3 and G4/G4R 

(Test Item)

1910

20

360

0.27

2510

0.35

3560

0.49

16

G1/G1R 

(Air Only)

-

20

360

-

G2, G3 and G4/G4R 

(Test Item)

1630

20

360

0.23

2880

0.40

3610

0.50

 17

G1/G1R 

(Air Only)

-

20

360

-

G2, G3 and G4/G4R 

(Test Item)

1660

20

360

0.23

2700

0.37

3750

0.52

18

G1/G1R 

(Air Only)

-

20

360

-

G2, G3 and G4/G4R 

(Test Item)

1510

20

360

0.21

2790

0.39

3690

0.51

19

G1/G1R 

(Air Only)

-

20

360

-

G2, G3 and G4/G4R 

(Test Item)

1530

20

360

0.21

2660

0.37

3590

0.50

20

G1/G1R 

(Air Only)

-

20

360

-

G2, G3 and G4/G4R 

(Test Item)

1720

20

360

0.24

2730

0.38

3630

0.50

 

TABLE 1.      SUMMARY OF GROSS PATHOLOGY FINDINGS

Refer Appendices 1 & 2

Sex

Male

Route of administration

Inhalation

Treatment

Air only

Low Dose

Mid Dose

High Dose

Air only

High Dose Recovery

Group

G1

G2

G3

G4

G1R

G4R

Nominal Target Dose (mg/L)

0

0.003

0.01

0.03

0

0.03

Number of animals

5

5

5

5

5

5

No. of dead rats during treatment

-

-

-

-

-

-

No. of moribund sacrificed rats

-

-

-

-

-

-

No. of terminally sacrificed rats

5

5

5

5

5

5

No. of rats showing gross pathology

-

-

-

-

-

-

 

Sex

Female

Route of administration

Inhalation

Treatment

Air only

Low Dose

Mid Dose

High Dose

Air only

High Dose Recovery

Group

G1

G2

G3

G4

G1R

G4R

Nominal Target Dose (mg/L)

0

0.003

0.01

0.03

0

0.03

Number of animals

5

5

5

5

5

5

No. of dead rats during treatment

-

-

-

-

-

-

No. of moribund sacrificed rats

-

-

-

-

-

-

No. of terminally sacrificed rats

5

5

5

5

5

5

No. of rats showing gross pathology

-

-

-

-

-

-

R: Recovery; - : No incidence.


TABLE 2.      SUMMARY OF HISTOPATHOLOGY FINDINGS– MAIN GROUP

Refer Appendices 3 & 4

Route of administration

Inhalation

Treatment

Air only

High Dose

Nominal Target Dose (mg/L)

0

0.03

Group

G1

G4

Sex

M

F

M

F

Number of Animals

5

5

5

5

Adrenals

Number examined

5

5

5

5

Within normal limits

5

5

5

5

Brain

Number examined

5

5

5

5

Within normal limits

5

5

5

5

Cervical lymph nodes

Number examined

5

5

5

5

Within normal limits

5

5

5

5

Esophagus

Number examined

5

5

5

5

Within normal limits

5

5

5

5

Eyes with optic nerve and eye lids

Number examined

5

5

5

5

Within normal limits

5

5

5

5

Heart

Number examined

5

5

5

5

Within normal limits

5

5

5

5

Hilar lymph nodes

Number examined

5

5

5

5

Within normal limits

5

5

5

5

Kidneys

Number examined

5

5

5

5

Within normal limits

4

5

3

5

Cysts

Present

1

-

2

-

Basophilia, tubules

Minimal

-

-

1

-

Larynx

Number examined

5

5

5

5

Within normal limits

5

5

5

5

Liver

Number examined

5

5

5

5

Within normal limits

5

5

5

5

Lungs

Number examined

5

5

5

5

Within normal limits

5

4

-

-

Pigment, golden brown

Moderate

-

-

5

5

Infiltrate, mononuclear cells

Minimal

-

1

-

-

Mandibular lymph nodes

Number examined

5

5

5

5

Within normal limits

5

5

5

5

Mesenteric lymph nodes

Number examined

5

5

5

5

Within normal limits

5

5

5

5

Nasopharyngeal tissue

Number examined

5

5

5

5

Within normal limits

5

5

5

5

Olfactory bulb

Number examined

5

5

5

5

Within normal limits

5

5

5

5

Ovaries

Number examined

X

5

X

5

Within normal limits

X

5

X

5

Parathyroid

Number examined

5

5

5

5

Within normal limits

5

5

5

5

Seminal vesicles

Number examined

5

X

5

X

Within normal limits

5

X

5

X

M: Male; F: Female; -: no incidence; X: not applicable.


TABLE 2 (Contd…). SUMMARY OF HISTOPATHOLOGY FINDINGS – MAIN GROUP

Refer Appendices 3 & 4

Route of administration

Inhalation

Treatment

Air only

High Dose

Nominal Target Dose (mg/L)

0

0.03

Group

G1

G4

Sex

M

F

M

F

Number of Animals

5

5

5

5

Spinal cord

Number examined

5

5

5

5

Within normal limits

5

5

5

5

Spleen

Number examined

5

5

5

5

Within normal limits

5

5

5

5

Stomach

Number examined

5

5

5

5

Within normal limits

5

5

5

5

Testes

Number examined

5

X

5

X

Within normal limits

4

X

5

X

Degeneration/atrophy, tubule

Moderate

1

X

-

X

Thymus

Number examined

5

5

5

5

Within normal limits

5

5

5

5

Thyroid

Number examined

5

5

5

5

Within normal limits

5

5

5

5

Trachea

Number examined

5

5

5

5

Within normal limits

5

5

5

5

Urinary bladder

Number examined

5

5

5

5

Within normal limits

5

5

5

5

Uterus

Number examined

X

5

X

5

Within normal limits

X

4

X

5

Dilatation, glandular

Minimal

X

1

X

-

M: Male; F: Female; -: no incidence; X: not applicable.


TABLE 3.      SUMMARY OF HISTOPATHOLOGY FINDINGS – RECOVERY GROUP

Refer Appendices 5 & 6

Route of administration

Inhalation

Treatment

Air only

High Dose Recovery

Nominal Target Dose (mg/L)

0

0.03

Group

G1R

G4R

Sex

M

F

M

F

Number of Animals

5

5

5

5

Adrenals

Number examined

5

5

5

5

Within normal limits

5

5

5

5

Brain

Number examined

5

5

5

5

Within normal limits

5

5

5

5

Cervical lymph nodes

Number examined

5

5

5

5

Within normal limits

5

5

5

5

Esophagus

Number examined

5

5

5

5

Within normal limits

5

5

5

5

Eyes with optic nerve and eye lids

Number examined

5

5

5

5

Within normal limits

5

5

5

5

Heart

Number examined

5

5

5

5

Within normal limits

5

5

5

5

Hilar lymph nodes

Number examined

5

5

5

5

Within normal limits

5

5

5

5

Kidneys

Number examined

5

5

5

5

Within normal limits

5

5

3

5

Cysts

Present

-

-

2

-

Basophilia, tubules

Minimal

-

-

1

-

Larynx

Number examined

5

5

5

5

Within normal limits

5

5

5

5

Liver

Number examined

5

5

5

5

Within normal limits

5

5

5

5

Lungs

Number examined

5

5

5

5

Within normal limits

5

5

-

-

Pigment, golden brown

Mild

-

-

-

1

Moderate

-

-

5

4

Mandibular lymph nodes

Number examined

5

5

5

5

Within normal limits

5

5

5

5

Mesenteric lymph nodes

Number examined

5

5

5

5

Within normal limits

5

5

5

5

Nasopharyngeal tissue

Number examined

5

5

5

5

Within normal limits

5

5

5

5

Olfactory bulb

Number examined

5

5

5

5

Within normal limits

5

5

5

5

Ovaries

Number examined

X

5

X

5

Within normal limits

X

5

X

5

Parathyroid

Number examined

5

5

5

5

Within normal limits

4

-

-

-

Hyperplasia

Mild

1

-

-

-

M: Male; F: Female; R: Recovery; -: no incidence; X: not applicable.


TABLE 3 (Contd…). SUMMARY OF HISTOPATHOLOGY FINDINGS – RECOVERY GROUP

Refer Appendices 5 & 6

Route of administration

Inhalation

Treatment

Air only

High Dose Recovery

Nominal Target Dose (mg/L)

0

0.03

Group

G1R

G4R

Sex

M

F

M

F

Number of Animals

5

5

5

5

Seminal vesicles

Number examined

5

X

5

X

Within normal limits

5

X

5

X

Spinal cord

Number examined

5

5

5

5

Within normal limits

5

5

5

5

Spleen

Number examined

5

5

5

5

Within normal limits

5

5

5

5

Stomach

Number examined

5

5

5

5

Within normal limits

5

5

5

5

Testes

Number examined

5

X

5

X

Within normal limits

5

X

5

X

Thymus

Number examined

5

5

5

5

Within normal limits

5

5

5

5

Thyroid

Number examined

5

5

5

5

Within normal limits

5

5

5

5

Trachea

Number examined

5

5

5

5

Within normal limits

5

5

5

5

Urinary bladder

Number examined

5

5

5

5

Within normal limits

5

5

5

5

Uterus

Number examined

X

5

X

5

Within normal limits

X

4

X

4

Dilatation, lumen

Minimal

X

1

X

1

M: Male; F: Female; R: Recovery; -: no incidence; X: not applicable.


TABLE 4.      SUMMARY OF HISTOPATHOLOGY FINDINGS – LOW DOSE AND MID DOSE GROUP

Refer Appendices 7 & 8

Route of administration

Inhalation

Treatment

Low Dose

Mid Dose

Nominal Target Dose (mg/L)

0.003

0.01

Group

G2

G3

Sex

M

F

M

F

Number of Animals

5

5

5

5

Lungs

Number examined

5

5

5

5

Within normal limits

-

-

-

-

Pigment, golden brown

Minimal

3

4

2

3

Mild

2

1

3

2

M: Male; F: Female; -: No incidence.


 

 

 

 

 

 

 

 

 

 

Conclusions:
 Based on the observed results, it is concluded that the No Observed Adverse Effect Concentration (NOAEC) of test item,Novoperm Orange HLwas 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..
Executive summary:

The objective of this study was to determine the toxic potential of the test item when administered for 6 hours/day, 5 days per week, for                4 consecutive weeks by flow-past nose-only inhalation route to Sprague Dawley rats. This study provides information on toxic effects, target organs, the possibility of cumulative effects, the reversibility of effects (after 56 days recovery period), and an estimate of the No Observed Adverse Effects Concentration (NOAEC).A total of 60 (30 males + 30 females) healthy young Sprague Dawley rats were distributed to four main groups viz., control (G1), low dose (G2), mid dose (G3), high dose (G4) and two recovery groups [(G1R (Air only) and G4R (high dose)]. Each main and recovery group comprised of 10 animals (5 males and 5 females). Animals allocated to groups G2, G3 and G4/G4R were exposed to test item for 6 hours per day, 5 days per week, for 4 consecutive weeks., at a nominal target concentration of 0.003, 0.01 and 0.03 mg/L. Animals of the control group (G1/G1R) received air only inhalation for 6 hours/day for 4 consecutive weeks. The inhalation exposure of test item/air was achieved by a flow-past, nose-only dynamic inhalation exposure system supplied by CH Technologies, USA.All animals were observedonce daily for clinical signs and twice daily for mortality. Body weightwas recorded before exposure (day 1and twice weekly for initial two weeks during treatment period and thereafter once weekly.Feed consumption was recorded weekly.Ophthalmoscopic examinationswereperformed duringtheacclimatizationperiodfor all groups, andduringWeek4for main groups (G1and G4),and duringWeek12for recovery groups (G1Rand G4R).Neurological/Functionaltestswereperformed duringWeek4forG1 and G4groupsand duringWeek12for recovery groupanimals.At the end of treatment and recovery periods, all animals were fasted overnight (water was availablead libitum), and the next day, blood, urine, and Broncho alveolar lavage fluid (BALF) samples were collected and analysed. Subsequently, the animals were sacrificed and subjected to gross pathological examination, and the organs were collected, weighed, and preserved. The tissues/organs in vehicle control and high dose group animals including recovery animals were subjected to histopathological examinations.The data recorded for all exposure days relating to the chamber conditions like particle size, temperature, relative humidity, oxygen, and carbon dioxide concentrations determined during the exposure period were found within the specified range.No treatment-related changes in body weight, percent change in body weight with respect to day 1, feed consumption,or ophthalmology were noted.No adverse effects were observed in the neurological/functional examination tests.No adverse effects were observed in haematology, clinical chemistry, coagulation, BALF analysis,or urinalysis parameters. No toxicologically significant changes were observed infasting body weight, absoluteorgan weight, or relative organ weight.There were no test item-related, microscopic findings in animals evaluated after terminal and recovery period in the study.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 of0.03mg/L. (Nikula KJet.al., 2014).


Conclusion: Based on the observed results, it is concluded that the No Observed Adverse Effect Concentration (NOAEC) of test item,Novoperm Orange HLwas 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.


 


 


 

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEC
30 mg/m³
Study duration:
subacute
Experimental exposure time per week (hours/week):
30
Species:
rat
Quality of whole database:
reliable

Repeated dose toxicity: inhalation - local effects

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

DETAILS OF FOOD AND WATER QUALITY:

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

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

TEST ATMOSPHERE
- Samples taken from breathing zone: yes

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

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly once

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

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

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

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

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

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

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

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

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

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

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

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

TABLE 1.   SUMMARY OF CLINICAL SIGNS AND MORTALITY RECORD