2-[[1-[[(2,3-dihydro-2-oxo-1H-benzimidazol-5-yl)amino]carbonyl]-2-oxopropyl]azo]benzoic acid

Endpoint summary

Description of key information

The results of a subchronic oral repeated dose study study indicated that the oral administration of test item for 90 consecutive days in Wistar rats at dose levels of 111, 333 and 1000 mg/kg/day doses did not cause any toxicological effect on general health, body weights, food consumption, haematology, clinical chemistry, coagulation parameters, terminal fasting body weight, organ weights and histopathology in both sexes. Yellow colour faecal matter was observed at all the tested doses in both sexes during the treatment period. Grossly, yellow colored intestinal contents noted in 333 mg/kg/day and 1000 mg/kg/day rats (both sex). Yellow colour of faecal matter and intestinal contents was attributed to the physical appearance of the test item.

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 is considered to be 1000 mg/kg/day under the test conditions and doses employed.

Based on the results observed in a subacute inhalation study, it is concluded that the No Observed Adverse Effect Concentration (NOAEC) of test item was found to be 0.03 mg/L (30 mg/m³) when exposed for 6 hours/day, 5 days/week, for 4 weeks by flow-past nose-only inhalation route to Sprague Dawley rats.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

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: 189.42 to 236.69 g & Females : 149.11 to 186.01g
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 (SC-37).
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 69) 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.: G19479. 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 presence 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 89) and at the end of recovery period (Day 114)for recovery groups. Before examination, mydriasis 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 clean 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 following 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 nonhomogeneous 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 (non-normal 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):

Yellow colour faecal matter were observed at all the tested doses in both sexes. This could be due to physical nature of the test item. No mortalities observed at any of the tested doses in both sexes

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Treatment did not affect the mean body weights and net body weight gains at all the doses tested in either sex. Incidence of significantly lower body weight gain was observed during Days 8-15 in females at 1000 mg/kg/day. This significant difference was considered toxicologically not significant as the mean body weight were comparable to vehicle control

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

In males, the food consumption was significantly higher during Days 1-8 in all the toxicity main groups and during Days 29-36, 50-57, 57-64 and 85-90 at 333 mg/kg/day dose. At 1000 mg/kg/day, significantly lower food consumption was observed during Days 15-22 and 78-85. At 1000 mg/kg/day recovery group, significantly higher food consumption was observed during Days 8-15, 43-50 and 85-90 during the treatment period and significantly higher food consumption was observed during Days 111-118 during recovery period.
In females, the food consumption was significantly lower during Days 50-57 at 333 mg/kg/day dose. At 1000 mg/kg/day recovery group, significantly higher food consumption was observed during Days 8-15, 36-43 and 50-57 during the treatment period and during Days 90-97 during recovery period.
These significant differences were not considered toxicologically relevant as the body weights were not altered by the treatment.

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

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:

The results of the study indicated that the oral administration of test item C.I. Pigment Yellow 194 for 90 consecutive days in Wistar rats at dose levels of 111, 333 and 1000 mg/kg/day doses did not cause any toxicological effect on general health, body weights, food consumption, haematology, clinical chemistry, coagulation parameters, terminal fasting body weight, organ weights and histopathology in both sexes. Yellow colour faecal matter was observed at all the tested doses in both sexes during the treatment period. Grossly, yellow colored intestinal contents noted in 333 mg/kg/day and 1000 mg/kg/day rats (both sex). Yellow colour of faecal matter and intestinal contents was attributed to the physical appearance of the test item.
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 Yellow 194 is considered to be 1000 mg/kg/day under the test conditions and doses employed.

Executive summary:

C.I Pigment Yellow 194  administered orally by gavage to Wistar rats when for a 90 day and the reversibilityof any effects was assessed during a  28days recovery period. 

The test item was suspended in 0.5% Carboxymethylcellulose Sodium salt (medium viscosity) in Milli-Q^®Water and administered to rats at the graduated dose levels of 0, 111, 333 and 1000 mg/kg/day. 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.

All rat were 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 for main groups (Day 84) and towards the end of recovery period (Day 114) for recovery groups.The clinical laboratory investigations such as haematology, coagulation, clinical chemistry, hormone analysis and urine analysis were performed at termination. Vaginal smears were examined in the female rats and the stage of oestrous cycle was recorded prior to necropsy.

All rats 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 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. 

The following results were obtained:

·        Clinical Signs and Mortality:Yellowish colour faecal matter was 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 ineithersex.

·        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:Yellow colored intestinal contents (ileum/cecum /colon/rectum) were observed in males and/or females at 333 mg/kg/day and 1000 mg/kg/day. The gross finding was attributed to yellow color (physical appearance) of the test item. On the contrary, the intestinal contents were normal (comparable to vehicle control) in 111 mg/kg/day males/females. This difference was likely attributed to reduction in total quantity of the administered test item as compared to the 333 mg/kg/day or 1000 mg/kg/day groups. The yellowish intestinal content was not a feature in recovery rats.

·        Histopathology:There were no test item-related microscopic changes at any of the doses tested in either sex. The qualitative assessment of stages of spermatogenesis and evaluation of interstitial testicular structures did not show any remarkable alterations at the highest dose tested.

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 Yellow 194) is considered to be 1000 mg/kg/day.

Reference 2

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

See Read across document in chapter 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: 189.42 to 236.69 g & Females : 149.11 to 186.01g
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 (SC-37).
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 69) 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.: G19479. 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 presence 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 89) and at the end of recovery period (Day 114)for recovery groups. Before examination, mydriasis 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 clean 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 following 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 nonhomogeneous 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 (non-normal 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):

Yellow colour faecal matter were observed at all the tested doses in both sexes. This could be due to physical nature of the test item. No mortalities observed at any of the tested doses in both sexes

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Treatment did not affect the mean body weights and net body weight gains at all the doses tested in either sex. Incidence of significantly lower body weight gain was observed during Days 8-15 in females at 1000 mg/kg/day. This significant difference was considered toxicologically not significant as the mean body weight were comparable to vehicle control

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

In males, the food consumption was significantly higher during Days 1-8 in all the toxicity main groups and during Days 29-36, 50-57, 57-64 and 85-90 at 333 mg/kg/day dose. At 1000 mg/kg/day, significantly lower food consumption was observed during Days 15-22 and 78-85. At 1000 mg/kg/day recovery group, significantly higher food consumption was observed during Days 8-15, 43-50 and 85-90 during the treatment period and significantly higher food consumption was observed during Days 111-118 during recovery period.
In females, the food consumption was significantly lower during Days 50-57 at 333 mg/kg/day dose. At 1000 mg/kg/day recovery group, significantly higher food consumption was observed during Days 8-15, 36-43 and 50-57 during the treatment period and during Days 90-97 during recovery period.
These significant differences were not considered toxicologically relevant as the body weights were not altered by the treatment.

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

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:

The results of the study indicated that the oral administration of test item C.I. Pigment Yellow 194 for 90 consecutive days in Wistar rats at dose levels of 111, 333 and 1000 mg/kg/day doses did not cause any toxicological effect on general health, body weights, food consumption, haematology, clinical chemistry, coagulation parameters, terminal fasting body weight, organ weights and histopathology in both sexes. Yellow colour faecal matter was observed at all the tested doses in both sexes during the treatment period. Grossly, yellow colored intestinal contents noted in 333 mg/kg/day and 1000 mg/kg/day rats (both sex). Yellow colour of faecal matter and intestinal contents was attributed to the physical appearance of the test item.
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 Yellow 194 is considered to be 1000 mg/kg/day under the test conditions and doses employed.

Executive summary:

C.I Pigment Yellow 194  administered orally by gavage to Wistar rats when for a 90 day and the reversibilityof any effects was assessed during a  28days recovery period. 

The test item was suspended in 0.5% Carboxymethylcellulose Sodium salt (medium viscosity) in Milli-Q^®Water and administered to rats at the graduated dose levels of 0, 111, 333 and 1000 mg/kg/day. 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.

All rat were 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 for main groups (Day 84) and towards the end of recovery period (Day 114) for recovery groups.The clinical laboratory investigations such as haematology, coagulation, clinical chemistry, hormone analysis and urine analysis were performed at termination. Vaginal smears were examined in the female rats and the stage of oestrous cycle was recorded prior to necropsy.

All rats 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 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. 

The following results were obtained:

·        Clinical Signs and Mortality:Yellowish colour faecal matter was 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 ineithersex.

·        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:Yellow colored intestinal contents (ileum/cecum /colon/rectum) were observed in males and/or females at 333 mg/kg/day and 1000 mg/kg/day. The gross finding was attributed to yellow color (physical appearance) of the test item. On the contrary, the intestinal contents were normal (comparable to vehicle control) in 111 mg/kg/day males/females. This difference was likely attributed to reduction in total quantity of the administered test item as compared to the 333 mg/kg/day or 1000 mg/kg/day groups. The yellowish intestinal content was not a feature in recovery rats.

·        Histopathology:There were no test item-related microscopic changes at any of the doses tested in either sex. The qualitative assessment of stages of spermatogenesis and evaluation of interstitial testicular structures did not show any remarkable alterations at the highest dose tested.

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 Yellow 194) is considered to be 1000 mg/kg/day.

Reference 3

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

See Read across document in chapter 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: 189.42 to 236.69 g & Females : 149.11 to 186.01g
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 (SC-37).
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 69) 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.: G19479. 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 presence 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 89) and at the end of recovery period (Day 114)for recovery groups. Before examination, mydriasis 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 clean 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 following 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 nonhomogeneous 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 (non-normal 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):

Yellow colour faecal matter were observed at all the tested doses in both sexes. This could be due to physical nature of the test item. No mortalities observed at any of the tested doses in both sexes

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Treatment did not affect the mean body weights and net body weight gains at all the doses tested in either sex. Incidence of significantly lower body weight gain was observed during Days 8-15 in females at 1000 mg/kg/day. This significant difference was considered toxicologically not significant as the mean body weight were comparable to vehicle control

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

In males, the food consumption was significantly higher during Days 1-8 in all the toxicity main groups and during Days 29-36, 50-57, 57-64 and 85-90 at 333 mg/kg/day dose. At 1000 mg/kg/day, significantly lower food consumption was observed during Days 15-22 and 78-85. At 1000 mg/kg/day recovery group, significantly higher food consumption was observed during Days 8-15, 43-50 and 85-90 during the treatment period and significantly higher food consumption was observed during Days 111-118 during recovery period.
In females, the food consumption was significantly lower during Days 50-57 at 333 mg/kg/day dose. At 1000 mg/kg/day recovery group, significantly higher food consumption was observed during Days 8-15, 36-43 and 50-57 during the treatment period and during Days 90-97 during recovery period.
These significant differences were not considered toxicologically relevant as the body weights were not altered by the treatment.

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

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:

The results of the study indicated that the oral administration of test item C.I. Pigment Yellow 194 for 90 consecutive days in Wistar rats at dose levels of 111, 333 and 1000 mg/kg/day doses did not cause any toxicological effect on general health, body weights, food consumption, haematology, clinical chemistry, coagulation parameters, terminal fasting body weight, organ weights and histopathology in both sexes. Yellow colour faecal matter was observed at all the tested doses in both sexes during the treatment period. Grossly, yellow colored intestinal contents noted in 333 mg/kg/day and 1000 mg/kg/day rats (both sex). Yellow colour of faecal matter and intestinal contents was attributed to the physical appearance of the test item.
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 Yellow 194 is considered to be 1000 mg/kg/day under the test conditions and doses employed.

Executive summary:

C.I Pigment Yellow 194  administered orally by gavage to Wistar rats when for a 90 day and the reversibilityof any effects was assessed during a  28days recovery period. 

The test item was suspended in 0.5% Carboxymethylcellulose Sodium salt (medium viscosity) in Milli-Q^®Water and administered to rats at the graduated dose levels of 0, 111, 333 and 1000 mg/kg/day. 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.

All rat were 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 for main groups (Day 84) and towards the end of recovery period (Day 114) for recovery groups.The clinical laboratory investigations such as haematology, coagulation, clinical chemistry, hormone analysis and urine analysis were performed at termination. Vaginal smears were examined in the female rats and the stage of oestrous cycle was recorded prior to necropsy.

All rats 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 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. 

The following results were obtained:

·        Clinical Signs and Mortality:Yellowish colour faecal matter was 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 ineithersex.

·        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:Yellow colored intestinal contents (ileum/cecum /colon/rectum) were observed in males and/or females at 333 mg/kg/day and 1000 mg/kg/day. The gross finding was attributed to yellow color (physical appearance) of the test item. On the contrary, the intestinal contents were normal (comparable to vehicle control) in 111 mg/kg/day males/females. This difference was likely attributed to reduction in total quantity of the administered test item as compared to the 333 mg/kg/day or 1000 mg/kg/day groups. The yellowish intestinal content was not a feature in recovery rats.

·        Histopathology:There were no test item-related microscopic changes at any of the doses tested in either sex. The qualitative assessment of stages of spermatogenesis and evaluation of interstitial testicular structures did not show any remarkable alterations at the highest dose tested.

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 Yellow 194) is considered to be 1000 mg/kg/day.

Reference 4

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 2^ndmonth (Day 34) and 3^rdmonth (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.

Reference 5

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 chapter 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 2^ndmonth (Day 34) and 3^rdmonth (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 conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

reliable

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

repeated dose toxicity: inhalation, other

Remarks:

28-Day Inhalation

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

15 March 2021 to 12 October 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

See read across document in chapter 13

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)

Version / remarks:

adopted on 25 June 2018

GLP compliance:

yes (incl. QA statement)

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 bred animals
- Females (if applicable) nulliparous and non-pregnant: [yes]
- Age at study initiation: 7 weeks
- Weight at study initiation: Males: 140.06 g to 159.97 g; Females: 120.63 g to 139.41 g
- Fasting period before study: No
- Housing: Maximum of three animals per cage were housed in a standard polypropylene cage (size: L 430 x B 285 x H 150 mm) with stainless steel mesh top grill having facilities for holding pelleted feed and drinking water in water bottle fitted with stainless steel sipper tube. Clean sterilized corn cob was provided as bedding material.
- Diet (e.g. ad libitum): Altromin Maintenance diet for rats and mice (manufactured by Altromin Spezialfutter GmbH & Co. KG).
- Water (e.g. ad libitum): Deep bore-well water passed through a reverse osmosis unit was provided in plastic water bottles with stainless steel sipper tubes.
- Acclimation period: 7 Days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.6°C to 22.9°C
- Humidity (%): 46% to 65%
- Air changes (per hr): 12 to 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours fluorescent light and 12 hours dark cycle

IN-LIFE DATES: From: 15 March 2021 To: 10 August 2021

Route of administration:

inhalation: dust

Type of inhalation exposure:

nose only

Vehicle:

air

Mass median aerodynamic diameter (MMAD):

>= 2 - ca. 2 µm

Geometric standard deviation (GSD):

3

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: flow-past, nose-only dynamic inhalation exposure system supplied by CH Technologies, USA.
- Method of holding animals in test chamber: The animals were confined separately in restraint tubes which were positioned radially around the flow-past, nose-only dynamic inhalation exposure chamber.
- Source and rate of air: Air flow from the generation system into the chamber was controlled and monitored through control panel (supplied by CH Technologies, USA) using calibrated flow-meters (rotameters) and outlet of the chamber air flow was controlled by rotameter. The actual flow rate of chamber inlet was 20 L/min with 60 psi pressure and outlet air from chamber was 15 L/min maintained throughout the exposure period. The air inlet flow rate was recorded hourly once during exposure (i.e. ± 10 minutes) after equilibration period (T95) 0.11min.
- Method of conditioning air: Compressed 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: 22 ± 3°C, 30 to 70%, 60psi
- Air flow rate: 20L/min
- Air change rate: 12 to 15 air changes per cycle
- Method of particle size determination: Cascade impactor
- Treatment of exhaust air: NaOH

TEST ATMOSPHERE
- Brief description of analytical method used: No
- Samples taken from breathing zone: yes

Duration of treatment / exposure:

Animals were exposed to aerosolized test item continuously for 6 hours per day, 5 days per week, for a total of 20 actual exposures, after equilibration period of the chamber concentration.
Following the 28 days exposure period, the animals in the recovery groups (G1R and G4R) were not given any treatment and was maintained for 56 days post treatment period and observed for reversibility or persistence of toxic effects.

Frequency of treatment:

6 hours per day, 5 days per week

Dose / conc.:

0 mg/L air (nominal)

Remarks:

Air Only

Dose / conc.:

0.001 mg/L air (nominal)

Remarks:

Low Dose

Dose / conc.:

0.003 mg/L air (nominal)

Remarks:

Mid Dose

Dose / conc.:

0.03 mg/L air (nominal)

Remarks:

High Dose

No. of animals per sex per dose:

5 Males + 5 Females

Control animals:

yes, concurrent no treatment

Details on study design:

- Dose selection rationale:
- Rationale for selecting satellite groups: Following the 28 days exposure period, the animals in the recovery groups (G1R and G4R) were not given any treatment and was maintained for 56 days post treatment period and observed for reversibility or persistence of toxic effects.
- Post-exposure recovery period in satellite groups: 56 Days

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All the animals were observed for clinical signs and pre-terminal deaths at 1.5 hrs (±10 mins), 3 hrs (±10 mins), 4.5 hrs (±10 mins) and 6 hrs (±10 mins) during exposure period. Post-exposure clinical signs and 30 to 40 minutes and 1 hour (±10 mins) following exposure to the test chemical, thereafter once a day for clinical signs and twice daily for mortality.
- Cage side observations checked in table No.1

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once in a week

BODY WEIGHT: Yes
- Time schedule for examinations: Individual animal body weights were recorded at receipt, prior to exposure (Day 1) and twice weekly thereafter for first two weeks. Since there was no significant body weight effects in the first two weeks, body weights were recorded weekly thereafter (post exposure period) for remaining period of the study. Fasting body weight of all the animals of all the groups were recorded at their scheduled terminal sacrifice.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Ophthalmological examination was carried out once during acclimatization (Pre-treatment) and during week 4 in control and high dose group animals. No, treatment related ocular changes are noted in high dose during week 4, the examination was not extended to lower dose groups (G2, G3) of main study animals and during week 12 for recovery group animals (G1R and G4R).
- Dose groups that were examined: G1/G1R and G4/G4R

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood samples were collected from all the surviving rats of each group on day 27 (within 24 hours after the last given dose) and day 85 of recovery groups respectively.
- Anaesthetic used for blood collection: Yes
- Animals fasted: Yes
- How many animals: 60
- Parameters checked in table [No.10] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood samples were collected from all the surviving rats of each group on day 27 (within 24 hours after the last given dose) and day 85 of recovery groups respectively.
- Animals fasted: Yes
- How many animals: 60
- Parameters checked in table [No.12] were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: Urine was collected and analyzed from all rats on day 27 (within 24 hours after the last given dose). The animals were kept in urine collection cages overnight for urine collection. Animal was not given access to feed but water was provided ad libitum during their stay in urine collection cages.
The recovery group animal’s urine was collected on day 85.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked in table [No.13] were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Neurological/Functional examination was carried out during Week 4 and 12 for vehicle control and high dose animals. Since no apparent treatment related effects persisted in high dose animals, the examination was not carried out for low and mid dose groups (G2 and G3) during Week 4 of treatment period.
- Dose groups that were examined: G1/G1R and G4/G4R
- Battery of functions tested: sensory activity / grip strength / motor activity.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table No. 19)
HISTOPATHOLOGY: Yes (see table No. 20)

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:

no effects observed

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

Description (incidence):

All animals survived to scheduled sacrifice, and there were no mortality attributable to the test item. Post-exposure, all animals were normal throughout the experimental period. No mortalities were noted during the recovery period.

Body weight and weight changes:

no effects observed

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:

no effects observed

Description (incidence and severity):

No statistically significant treatment-related variations were observed in all the tested dose groups when compared with control group.

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, decrease in HCT (G2) was noted. In females decrease in APTT (G2, G3 and G4), MCV and MCH (G2 and G4) was noted.
In recovery males decrease in Percent monocytes (G4R) was noted. However, in female no significance observed.
All the noted variations in main group are considered incidental in the absence of dose responsiveness. Variations noted at the end of recovery period are considered incidental as no such variations were noted in recovery females.

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 vehicle control group. However, the following statistically significant variations were noted.
In main group males, increase in chloride (G4) was noted; In females no significance was noted.
In recovery group no significance was observed.
The observed 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 adverse treatment-related effects were observed in urinalysis parameters when compared to the control group. However, a single incidence of a statistically significant decrease in specific gravity was observed in G4R females. This effect, which was minimal in nature, lacked dose-dependency and was therefore not attributed to the test item.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

There were no adverse effects during neurological/functional examinations were observed in main group (G1 and G4) and recovery group (G1R and G4R), however in females (G4R) excessive grooming was noted, this is considered as incidental due to lack of dose response.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

In main group males, decrease in absolute weight of testis in G2; increase in absolute and relative weight of lungs in G2 and G4 was noted. In females decrease in absolute and relative weight of spleen in G3 was noted.
In recovery group, increase in absolute and relative weight of thymus (G4R); decrease in absolute and relative weight in spleen (G4R) was noted. In females, increase in absolute and relative weight of ovaries (G4R) were noted.
In main group males, statistically significant increase in relative weight of lungs in G2 and G4 was noted. In females, decrease in relative weight of spleen (G3) and liver (G2); increase in relative weight of lungs (G4) were noted.
In recovery group males, decrease in relative weight of spleen (G4R); increase in relative weight of thymus (G4R) was noted. In females increase in relative weight of ovaries (G4R) was noted.
All the observed changes are considered incidental in the absence of gross and histopathological changes in the high dose group animals and also lack dose dependency.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No treatment related gross pathological lesions were observed in main group and recovery group animals.

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related, microscopic findings in animals evaluated after terminal and recovery period in the study.
Few microscopic findings observed in this study such as ultimobranchial cyst in thyroid gland, epithelial cysts in thymus and all other findings were considered incidental as they occurred randomly across the dose groups including concurrent controls and/or were expected in laboratory rats.

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

BALF Fluid: The bronchio-alveolar lavage fluid measured at termination revealed the following statistically significant changes: In main group males, increase in percent eosinophils and basophils (G3) was noted; In females, increase in percent monocytes (G4) was noted.
In recovery group, females increase in LDH (G4R) was noted; however, in males no significance was noted.
There were no test item-related microscopic findings in animals evaluated after terminal and recovery period in the study and the noted variations are considered non adverse effect.

Key result

Dose descriptor:

NOAEC

Effect level:

>= 0.03 - ca. 0.03 mg/L air (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 efficiency

gross pathology

haematology

histopathology: non-neoplastic

mortality

ophthalmological examination

organ weights and organ / body weight ratios

urinalysis

other: BALF findings

Key result

Critical effects observed:

no

TABLE 1.   SUMMARY OF CLINICAL SIGNS AND MORTALITY RECORD

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 & 0.001	5	N	0/5
G3, M & 0.003	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.

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

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 & 0.001	5	N	0/5
G3, F & 0.003	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.

TABLE 2.  SUMMARY OF BODY WEIGHTS (g) RECORD

Group, Sex & Dose (mg/L)		Body Weight (g) on Days
		1	5	8	12	15	22	26
G1, M & 0	Mean	187.88	191.93	205.03	209.22	220.95	234.65	241.23
	±SD	14.19	14.38	13.69	13.43	13.45	13.00	12.55
	n	5	5	5	5	5	5	5
G2, M & 0.001	Mean	183.58	187.41	199.18	203.48	214.50	228.10	235.87
	±SD	14.65	14.59	13.65	13.86	13.30	14.17	14.78
	n	5	5	5	5	5	5	5
G3, M & 0.003	Mean	182.16	185.60	197.45	201.89	214.35	228.81	235.88
	±SD	15.60	15.80	14.39	13.60	13.57	14.20	13.89
	n	5	5	5	5	5	5	5
G4, M & 0.03	Mean	180.15	183.99	196.54	201.16	213.32	227.63	235.65
	±SD	18.66	18.08	17.15	16.54	15.36	14.94	14.81
	n	5	5	5	5	5	5	5
G1, F & 0	Mean	152.09	155.06	161.34	163.66	172.01	182.69	189.18
	±SD	12.49	12.10	10.71	10.09	10.81	11.06	10.92
	n	5	5	5	5	5	5	5
G2, F & 0.001	Mean	153.99	157.68	163.82	166.47	175.30	184.89	190.90
	±SD	11.36	11.61	11.46	11.06	11.32	11.43	11.75
	n	5	5	5	5	5	5	5
G3, F & 0.003	Mean	149.86	153.07	160.44	162.55	170.62	180.12	186.94
	±SD	11.55	12.04	11.07	10.79	10.78	10.64	11.15
	n	5	5	5	5	5	5	5
G4, F & 0.03	Mean	151.90	154.59	160.20	162.81	170.66	180.51	186.39
	±SD	8.04	7.91	8.06	7.73	7.95	9.00	9.00
	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

Group, Sex & Dose (mg/L)		Body Weight (g) on Days
		1	5	8	12	15	22	26	29	36	43	50	57	64	71	78	84
G1R, M & 0	Mean	184.58	188.92	201.70	205.86	217.92	232.09	242.91	250.80	272.02	294.25	317.10	339.64	362.85	386.35	411.61	432.52
	±SD	10.63	11.00	10.49	9.66	9.90	9.61	9.14	9.32	9.71	10.66	10.84	10.19	10.54	10.56	10.87	10.69
	n	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5
G4R, M & 0.03	Mean	182.85	186.62	199.13	203.34	215.44	230.12	243.82	251.75	272.84	295.09	317.85	340.56	363.70	386.94	412.13	433.06
	±SD	12.36	12.69	11.96	13.79	13.22	13.17	12.42	12.49	11.83	12.80	13.47	13.68	13.72	13.78	14.37	14.48
	n	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5
G1R, F & 0	Mean	151.42	154.17	159.60	162.05	170.53	180.30	187.50	192.72	208.02	223.72	239.22	254.79	271.47	288.34	305.89	321.44
	±SD	12.00	11.75	10.93	10.98	10.61	8.85	8.38	8.72	9.15	8.87	9.39	9.33	9.70	9.90	9.79	9.86
	n	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5
G4R, F & 0.03	Mean	151.49	154.31	160.21	162.55	173.23	182.00	189.45	194.62	209.88	225.43	241.03	256.53	273.35	290.30	307.74	323.36
	±SD	11.10	11.31	10.58	9.78	11.74	11.05	10.57	10.69	10.98	10.90	10.94	10.91	11.02	10.83	10.45	10.36
	n	5	5	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 animals.

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

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	2.16	9.19	11.44	17.71	25.05	28.59
	±SD	0.52	1.38	1.95	2.09	2.93	3.31
	n	5	5	5	5	5	5
G2, M & 0.001	Mean	2.10	8.58	10.93	16.97	24.38	28.62
	±SD	0.42	1.46	1.86	2.19	2.28	2.25
	n	5	5	5	5	5	5
G3, M & 0.003	Mean	1.89	8.50	10.98	17.87	25.84	29.76
	±SD	0.53	1.67	2.17	3.16	3.61	4.10
	n	5	5	5	5	5	5
G4, M & 0.03	Mean	2.18	9.25	11.86	18.72	26.75	31.25
	±SD	0.62	1.77	2.42	3.76	4.74	5.28
	n	5	5	5	5	5	5
G1, F & 0	Mean	1.99	6.19	7.75	13.25	20.30	24.59
	±SD	0.53	1.70	2.28	2.33	2.66	3.28
	n	5	5	5	5	5	5
G2, F & 0.001	Mean	2.40	6.41	8.15	13.90	20.15	24.06
	±SD	0.28	0.53	1.01	1.18	2.05	1.85
	n	5	5	5	5	5	5
G3, F & 0.003	Mean	2.13	7.12	8.54	13.96	20.34	24.89
	±SD	0.72	1.16	1.75	2.21	3.07	2.90
	n	5	5	5	5	5	5
G4, F & 0.03	Mean	1.78	5.48	7.22	12.39	18.86	22.74
	±SD	0.29	0.59	1.35	1.44	0.90	1.30
	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

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	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	2.35	9.31	11.59	18.14	25.84	31.73	36.01	47.54	59.58	71.99	84.25	96.85	109.61	123.32	134.68
	±SD	0.30	0.61	1.51	1.92	2.46	3.09	3.31	3.90	3.97	4.48	5.40	5.85	6.55	7.11	7.80
	n	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5
G4R, M & 0.03	Mean	2.06	8.94	11.20	17.86	25.92	33.48	37.82	49.42	61.60	74.08	86.54	99.24	111.99	125.80	137.28
	±SD	0.45	0.81	0.61	0.99	1.38	3.24	3.40	4.36	4.67	5.39	5.93	6.81	7.54	8.13	8.78
	n	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5
G1R, F & 0	Mean	1.84	5.48	7.11	12.75	19.33	24.13	27.57	37.71	48.14	58.41	68.75	79.81	90.99	102.65	112.97
	±SD	0.41	1.23	1.71	2.42	4.43	4.95	4.92	5.32	6.27	6.72	7.66	8.36	9.11	10.22	11.06
	n	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5
G4R, F & 0.03	Mean	1.87	5.81	7.39	14.41	20.25	25.20	28.63	38.75	49.05	59.39	69.67	80.81	92.06	103.64	113.99
	±SD	0.23	1.11	1.55	2.31	2.97	3.11	3.53	4.27	4.67	5.22	6.01	6.67	7.64	8.66	9.19
	n	5	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

Group, Sex & Dose (mg/L)		Week 1	Week 2	Week 3	Week 4
G1, M & 0	Mean	18.74	18.84	18.68	32.63
	±SD	2.77	2.75	2.72	4.74
	n	2	2	2	2
G2, M & 0.001	Mean	18.60	18.66	18.49	32.58
	±SD	2.42	2.74	2.71	4.67
	n	2	2	2	2
G3, M & 0.003	Mean	18.63	18.74	18.55	32.63
	±SD	2.60	2.99	2.92	4.79
	n	2	2	2	2
G4, M & 0.03	Mean	18.67	18.48	18.56	32.65
	±SD	2.62	2.67	2.59	4.99
	n	2	2	2	2
G1, F & 0	Mean	14.20	14.55	14.39	32.70
	±SD	1.66	1.72	1.61	4.72
	n	2	2	2	2
G2, F & 0.001	Mean	14.46	14.37	14.33	32.70
	±SD	1.72	1.44	1.54	4.86
	n	2	2	2	2
G3, F & 0.003	Mean	14.22	14.39	14.35	32.66
	±SD	1.37	1.57	1.41	4.71
	n	2	2	2	2
G4, F & 0.03	Mean	14.12	14.22	14.36	32.57
	±SD	1.47	1.64	1.60	4.74
	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

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	18.58	18.64	18.42	18.94	18.81	19.28	19.29	19.31	19.34	19.25	19.39	22.32
	±SD	2.54	2.78	2.63	3.14	3.13	3.09	3.30	3.52	3.64	3.55	3.65	4.15
	n	2	2	2	2	2	2	2	2	2	2	2	2
G4R, M & 0.03	Mean	18.63	18.68	18.50	18.63	18.66	19.20	19.21	19.32	19.45	19.33	19.46	22.34
	±SD	2.65	2.89	2.76	2.78	2.98	3.14	3.30	3.21	3.30	3.24	3.29	3.79
	n	2	2	2	2	2	2	2	2	2	2	2	2
G1R, F & 0	Mean	14.24	14.26	14.25	14.15	14.30	15.14	15.26	15.30	15.57	15.58	15.67	18.02
	±SD	1.57	1.28	1.39	1.84	1.88	1.82	1.84	2.09	2.21	2.01	1.97	2.33
	n	2	2	2	2	2	2	2	2	2	2	2	2
G4R, F & 0.03	Mean	14.07	14.19	14.19	14.15	14.36	14.74	14.93	15.16	15.33	15.27	15.43	17.69
	±SD	1.48	1.88	1.67	1.78	1.94	2.25	2.19	2.27	2.43	2.11	2.13	2.41
	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)

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

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	7.4	6.6	7.8	7.6
	±SD	1.5	0.9	1.9	1.1
Numbers of Urination	Mean	1.4	1.4	1.4	1.6
	±SD	0.5	0.5	0.5	0.5
Number of Defecation	Mean	1.0	1.0	1.0	0.8
	±SD	0.7	0.7	0.7	0.4
Clonic involuntary movement		1	1	1	1
Tonic involuntary movement		1	1	1	1
Stereotype Behaviour		1	1	1	1
Excessive Grooming	Mean	3.8	4.0	4.2	4.4
	±SD	0.4	1.0	0.8	0.5
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	98.2	98.2	98.5	98.5
	±SD	0.2	0.2	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

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

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

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	7.8	7.6	8.8	9.0
	±SD	0.8	1.7	0.8	1.0
Numbers of Urination	Mean	1.2	1.2	1.0	1.4
	±SD	0.8	0.4	0.7	0.5
Number of Defecation	Mean	1.0	1.0	1.2	1.0
	±SD	0.7	0.7	0.4	0.7
Clonic involuntary movement		1	1	1	1
Tonic involuntary movement		1	1	1	1
Stereotype Behaviour		1	1	1	1
Excessive Grooming	Mean	4.2	3.4	4.0	5.4*
	±SD	0.8	0.9	0.7	0.5
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	97.9	97.9	98.7	98.7
	±SD	0.2	0.2	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; *: Statistically significant (p<0.05).

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

# Week 4 & 12
Group, Sex & Dose (mg/L)		Hind Limb Foot Splay (cm)
G1, M & 0	Mean	6.0
	±SD	0.4
	n	5
G4, M & 0.03	Mean	5.7
	±SD	0.2
	n	5
G1, F & 0	Mean	6.4
	±SD	0.5
	n	5
G4, F & 0.03	Mean	5.9
	±SD	0.6
	n	5
G1R, M & 0	Mean	8.3
	±SD	0.4
	n	5
G4R, M & 0.03	Mean	7.8
	±SD	0.5
	n	5
G1R, F & 0	Mean	6.5
	±SD	0.4
	n	5
G4R, F & 0.03	Mean	5.8
	±SD	0.8
	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

# Week 4 & 12
Group, Sex & Dose (mg/L)		Movement Counts (no.)
G1, M & 0	Mean	2114.4
	±SD	61.2
	n	5
G4, M & 0.03	Mean	2067.8
	±SD	75.0
	n	5
G1, F & 0	Mean	2179.2
	±SD	71.7
	n	5
G4, F & 0.03	Mean	2164.4
	±SD	26.9
	n	5
G1R, M & 0	Mean	2074.0
	±SD	54.0
	n	5
G4R, M & 0.03	Mean	2117.0
	±SD	26.3
	n	5
G1R, F & 0	Mean	2213.2
	±SD	27.6
	n	5
G4R, F & 0.03	Mean	2221.8
	±SD	9.4
	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 8. SUMMARY OF GRIP STRENGTH (kgf) MEASUREMENT RECORD

# Week 4 & 12
Group, Sex & Dose (mg/L)		Fore limb	Hind limb
G1, M & 0	Mean	1.352	0.330
	±SD	0.032	0.021
	n	5	5
G4, M & 0.03	Mean	1.373	0.354
	±SD	0.034	0.017
	n	5	5
G1, F & 0	Mean	1.358	0.316
	±SD	0.020	0.018
	n	5	5
G4, F & 0.03	Mean	1.364	0.337
	±SD	0.009	0.019
	n	5	5
G1R, M & 0	Mean	1.563	0.536
	±SD	0.036	0.020
	n	5	5
G4R, M & 0.03	Mean	1.550	0.531
	±SD	0.029	0.025
	n	5	5
G1R, F & 0	Mean	1.418	0.371
	±SD	0.053	0.023
	n	5	5
G4R, F & 0.03	Mean	1.399	0.351
	±SD	0.056	0.017
	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					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					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	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; F: Female.

TABLE 10.     SUMMARY OF HAEMATOLOGY RECORD

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)
		(10^3cells/µL)	(10^6cells/µL)	(g/dL)	(%)	(fL)	(pg)	(g/dL)	(10^3cells/µL)	(fL)
G1, M & 0	Mean	10.44	8.93	17.14	53.50	60.02	19.22	32.06	972.00	5.90
	±SD	2.75	0.41	0.82	2.05	3.62	1.14	0.84	131.79	0.25
	n	5	5	5	5	5	5	5	5	5
G2, M & 0.001	Mean	9.54	8.57	15.86	50.28*	58.84	18.54	31.54	865.20	6.08
	±SD	3.23	0.51	0.51	1.88	4.30	1.15	0.33	28.49	0.41
	n	5	5	5	5	5	5	5	5	5
G3, M & 0.003	Mean	10.98	8.30	16.30	52.12	63.28	19.72	31.24	760.80	7.22
	±SD	3.97	0.81	0.87	1.58	5.95	1.10	1.25	348.58	1.47
	n	5	5	5	5	5	5	5	5	5
G4, M & 0.03	Mean	12.13	8.88	16.24	52.10	58.76	18.30	31.18	877.80	6.66
	±SD	3.91	0.56	0.92	1.78	2.46	0.60	0.76	118.40	1.02
	n	5	5	5	5	5	5	5	5	5
G1R, M & 0	Mean	9.06	8.85	16.46	50.18	56.66	18.60	32.86	835.00	6.78
	±SD	2.95	0.22	0.74	2.10	2.28	0.89	0.48	171.39	0.22
	n	5	5	5	5	5	5	5	5	5
G4R, M & 0.03	Mean	8.17	9.08	16.00	48.82	53.80	17.62	32.78	757.20	6.70
	±SD	1.98	0.33	0.85	2.43	2.94	1.04	0.55	350.57	0.22
	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

Group, Sex & Dose (mg/L)		Reticulocyte Count	Neutrophils	Lymphocytes	Monocytes	Eosinophils	Basophils	Absolute Reticulocyte Count
		(Retic)	(Neut)	(Lymph)	(Mono)	(Eos)	(Baso)	(Retic)
		(%)	(%)	(%)	(%)	(%)	(%)	(10^9cells/L)
G1, M & 0	Mean	4.03	20.72	72.38	4.02	0.66	1.02	356.90
	±SD	1.89	4.02	3.94	1.89	0.42	0.38	160.41
	n	5	5	5	5	5	5	5
G2, M & 0.001	Mean	4.09	27.06	65.18	5.10	0.68	0.80	342.64
	±SD	1.83	2.48	3.20	0.62	0.13	0.10	124.07
	n	5	5	5	5	5	5	5
G3, M & 0.003	Mean	7.29	21.04	71.66	4.18	0.94	1.06	571.88
	±SD	5.52	6.75	7.23	1.17	0.27	0.55	374.97
	n	5	5	5	5	5	5	5
G4, M & 0.03	Mean	4.66	21.72	71.72	4.16	0.88	0.62	406.30
	±SD	2.03	6.20	6.74	0.88	0.58	0.22	156.82
	n	5	5	5	5	5	5	5
G1R, M & 0	Mean	2.75	24.76	68.60	3.74	1.36	0.48	243.48
	±SD	0.51	6.03	5.00	0.65	0.41	0.16	49.00
	n	5	5	5	5	5	5	5
G4R, M & 0.03	Mean	2.24	26.74	68.40	2.26*	1.68	0.28	203.34
	±SD	0.23	5.60	4.67	0.61	0.71	0.13	17.59
	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

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)
		(10^3cells/µL)	(10^3cells/µL)	(10^3cells/µL)	(10^3cells/µL)	(10^3cells/µL)	(Seconds)	(Seconds)
G1, M & 0	Mean	2.14	7.57	0.42	0.06	0.10	15.72	20.68
	±SD	0.63	2.10	0.20	0.03	0.03	1.40	0.77
	n	5	5	5	5	5	5	5
G2, M & 0.001	Mean	2.55	6.25	0.48	0.06	0.08	16.12	20.48
	±SD	0.84	2.21	0.13	0.03	0.02	0.56	2.41
	n	5	5	5	5	5	5	5
G3, M & 0.003	Mean	2.21	8.01	0.43	0.10	0.11	16.94	26.86
	±SD	0.78	3.43	0.12	0.03	0.07	2.33	8.44
	n	5	5	5	5	5	5	5
G4, M & 0.03	Mean	2.75	8.57	0.52	0.10	0.07	16.32	23.94
	±SD	1.41	2.46	0.23	0.07	0.03	1.11	4.54
	n	5	5	5	5	5	5	5
G1R, M & 0	Mean	2.12	6.32	0.36	0.13	0.04	15.32	21.20
	±SD	0.29	2.46	0.17	0.06	0.02	1.49	1.80
	n	5	5	5	5	5	5	5
G4R, M & 0.03	Mean	2.14	5.63	0.19	0.13	0.03	17.96	26.26
	±SD	0.43	1.63	0.09	0.05	0.02	5.13	6.31
	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

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)
		(10^3cells/µL)	(10^6cells/µL)	(g/dL)	(%)	(fL)	(pg)	(g/dL)	(10^3cells/µL)	(fL)
G1, F & 0	Mean	9.06	8.85	16.60	49.98	56.50	18.84	33.30	1023.80	6.02
	±SD	2.15	0.71	0.75	3.61	1.04	1.19	2.14	93.66	0.13
	n	5	5	5	5	5	5	5	5	5
G2, F & 0.001	Mean	9.75	9.77	17.18	52.46	53.74*	17.58*	32.74	1092.80	6.16
	±SD	4.30	1.21	2.04	6.09	1.24	0.39	0.21	213.31	0.30
	n	5	5	5	5	5	5	5	5	5
G3, F & 0.003	Mean	9.40	9.63	17.08	52.46	54.48	17.76	32.58	980.00	6.38
	±SD	3.58	0.96	1.70	5.61	1.70	0.51	0.57	262.16	0.49
	n	5	5	5	5	5	5	5	5	5
G4, F & 0.03	Mean	9.93	9.26	16.10	49.62	53.60*	17.38*	32.44	992.60	6.60
	±SD	2.52	0.32	0.60	1.67	1.50	0.72	0.62	290.58	0.52
	n	5	5	5	5	5	5	5	5	5
G1R, F & 0	Mean	8.38	8.72	16.10	47.82	54.82	18.50	33.70	711.40	6.58
	±SD	1.44	0.65	1.11	3.32	0.93	0.51	0.73	276.36	0.29
	n	5	5	5	5	5	5	5	5	5
G4R, F & 0.03	Mean	8.96	8.55	15.82	47.44	55.50	18.52	33.36	854.20	6.66
	±SD	1.56	0.41	0.91	3.04	1.58	0.56	0.54	202.83	0.23
	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 10 (Contd…). SUMMARY OF HAEMATOLOGY RECORD

Group, Sex & Dose (mg/L)		Reticulocyte Count	Neutrophils	Lymphocytes	Monocytes	Eosinophils	Basophils	Absolute Reticulocyte Count
		(Retic)	(Neut)	(Lymph)	(Mono)	(Eos)	(Baso)	(Retic)
		(%)	(%)	(%)	(%)	(%)	(%)	(10^9cells/L)
G1, F & 0	Mean	2.43	21.12	72.62	3.46	1.26	0.64	214.50
	±SD	0.43	4.43	4.70	0.96	0.68	0.24	40.96
	n	5	5	5	5	5	5	5
G2, F & 0.001	Mean	1.72	27.40	66.90	2.64	1.32	0.84	166.78
	±SD	0.56	6.67	7.47	0.66	0.34	0.61	47.63
	n	5	5	5	5	5	5	5
G3, F & 0.003	Mean	2.15	24.56	70.40	2.38	1.06	0.76	204.00
	±SD	0.54	5.33	5.11	1.22	0.43	0.62	37.56
	n	5	5	5	5	5	5	5
G4, F & 0.03	Mean	1.91	26.70	67.40	2.78	1.58	0.44	176.24
	±SD	0.61	3.96	5.05	0.88	0.64	0.11	51.51
	n	5	5	5	5	5	5	5
G1R, F & 0	Mean	1.84	22.58	71.16	2.90	2.10	0.38	161.28
	±SD	0.29	5.49	6.51	1.01	1.42	0.04	33.92
	n	5	5	5	5	5	5	5
G4R, F & 0.03	Mean	1.95	23.14	71.68	2.38	1.64	0.34	165.02
	±SD	0.54	5.94	6.51	0.31	0.32	0.09	40.29
	n	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

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)
		(10^3cells/µL)	(10^3cells/µL)	(10^3cells/µL)	(10^3cells/µL)	(10^3cells/µL)	(Seconds)	(Seconds)
G1, F & 0	Mean	1.94	6.55	0.31	0.12	0.06	16.82	25.28
	±SD	0.68	1.48	0.09	0.07	0.04	1.78	1.76
	n	5	5	5	5	5	5	5
G2, F & 0.001	Mean	2.51	6.70	0.25	0.13	0.07	17.50	20.36*
	±SD	0.74	3.44	0.09	0.06	0.03	3.15	1.60
	n	5	5	5	5	5	5	5
G3, F & 0.003	Mean	2.24	6.66	0.25	0.09	0.08	16.44	19.46*
	±SD	0.82	2.75	0.21	0.01	0.06	2.49	2.35
	n	5	5	5	5	5	5	5
G4, F & 0.03	Mean	2.66	6.69	0.28	0.15	0.04	17.50	17.86*
	±SD	0.80	1.70	0.13	0.06	0.01	4.63	2.02
	n	5	5	5	5	5	5	5
G1R, F & 0	Mean	1.89	5.97	0.24	0.18	0.03	16.12	22.70
	±SD	0.54	1.25	0.08	0.12	0.01	0.82	4.20
	n	5	5	5	5	5	5	5
G4R, F & 0.03	Mean	2.10	6.39	0.21	0.15	0.03	16.94	24.66
	±SD	0.81	1.07	0.05	0.05	0.01	1.48	5.92
	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

Group, Sex & Dose (mg/L)		Total Leucocyte Count	Neutrophils	Lymphocytes	Monocytes	Eosinophils	Basophils	Absolute Neutrophils
		(WBC)	(Neut)	(Lymph)	(Mono)	(Eos)	(Baso)	(Neut)
		(10^3cells/µL)	(%)	(%)	(%)	(%)	(%)	(10^3cells/µL)
G1, M & 0	Mean	0.36	17.16	73.14	2.00	0.24	1.60	0.06
	±SD	0.16	5.63	7.02	1.27	0.29	1.21	0.03
	n	5	5	5	5	5	5	5
G2, M & 0.001	Mean	0.54	22.56	67.02	2.66	0.70	3.46	0.14
	±SD	0.42	4.76	7.90	0.82	0.93	2.57	0.12
	n	5	5	5	5	5	5	5
G3, M & 0.003	Mean	0.15	23.48	65.82	3.22	4.22*	8.06*	0.02
	±SD	0.13	20.92	26.49	4.01	3.80	3.11	0.02
	n	5	5	5	5	5	5	5
G4, M & 0.03	Mean	0.15	25.36	66.18	2.70	1.96	2.16	0.04
	±SD	0.06	9.32	12.11	2.65	2.09	1.27	0.02
	n	5	5	5	5	5	5	5
G1R, M & 0	Mean	0.34	17.08	57.66	1.10	0.90	5.56	0.06
	±SD	0.12	3.07	20.72	0.49	1.33	3.29	0.03
	n	5	5	5	5	5	5	5
G4R, M & 0.03	Mean	0.40	17.76	56.22	1.78	0.88	3.66	0.07
	±SD	0.22	9.61	12.23	1.80	0.75	2.14	0.06
	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

Group, Sex & Dose (mg/L)		Absolute Lymphocytes	Absolute Monocytes	Absolute Eosinophils	Absolute Basophils	Lactate Dehydrogenase	Total Protein
		(Lymph)	(Mono)	(Eos)	(Baso)	(LDH)	(TPR)
		(10^3cells/µL)	(10^3cells/µL)	(10^3cells/µL)	(10^3cells/µL)	(u/L)	(g/dL)
G1, M & 0	Mean	0.26	0.01	0.00	0.01	63.76	322.20
	±SD	0.11	0.00	0.00	0.01	27.81	108.59
	n	5	5	5	5	5	5
G2, M & 0.001	Mean	0.34	0.01	0.00	0.02	83.96	291.48
	±SD	0.24	0.01	0.00	0.01	53.17	142.06
	n	5	5	5	5	5	5
G3, M & 0.003	Mean	0.12	0.00	0.00	0.01	52.34	306.36
	±SD	0.10	0.00	0.00	0.01	45.59	105.45
	n	5	5	5	5	5	5
G4, M & 0.03	Mean	0.10	0.00	0.00	0.00	34.26	281.34
	±SD	0.05	0.01	0.00	0.00	9.63	105.25
	n	5	5	5	5	5	5
G1R, M & 0	Mean	0.21	0.00	0.00	0.02	72.74	253.26
	±SD	0.12	0.00	0.00	0.02	29.08	178.66
	n	5	5	5	5	5	5
G4R, M & 0.03	Mean	0.22	0.01	0.00	0.01	149.78	200.61
	±SD	0.11	0.01	0.00	0.01	122.02	89.26
	n	5	5	5	5	5	5

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

TABLE 11 (Contd…). SUMMARY OF BALF RECORD

Group, Sex & Dose (mg/L)		Total Leucocyte Count	Neutrophils	Lymphocytes	Monocytes	Eosinophils	Basophils	Absolute Neutrophils
		(WBC)	(Neut)	(Lymph)	(Mono)	(Eos)	(Baso)	(Neut)
		(10^3cells/µL)	(%)	(%)	(%)	(%)	(%)	(10^3cells/µL)
G1, F & 0	Mean	0.25	22.28	64.02	1.18	3.18	2.42	0.05
	±SD	0.13	1.84	8.09	0.62	2.18	1.52	0.03
	n	5	5	5	5	5	5	5
G2, F & 0.001	Mean	0.21	24.72	58.52	1.88	3.64	7.48	0.05
	±SD	0.09	11.62	21.00	0.67	4.05	6.51	0.03
	n	5	5	5	5	5	5	5
G3, F & 0.003	Mean	0.32	23.14	65.12	2.02	1.72	1.94	0.08
	±SD	0.17	6.81	9.88	0.69	1.53	0.98	0.06
	n	5	5	5	5	5	5	5
G4, F & 0.03	Mean	0.23	16.04	71.72	3.52*	3.48	4.42	0.04
	±SD	0.17	8.52	11.69	1.93	2.34	4.54	0.03
	n	5	5	5	5	5	5	5
G1R, F & 0	Mean	0.22	21.06	53.16	0.98	1.72	6.22	0.04
	±SD	0.09	6.39	7.11	1.31	1.64	2.01	0.02
	n	5	5	5	5	5	5	5
G4R, F & 0.03	Mean	0.21	17.64	61.56	1.86	0.90	4.18	0.03
	±SD	0.07	6.61	16.43	1.10	0.53	1.54	0.02
	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

Group, Sex & Dose (mg/L)		Absolute Lymphocytes	Absolute Monocytes	Absolute Eosinophils	Absolute Basophils	Lactate Dehydrogenase	Total Protein
		(Lymph)	(Mono)	(Eos)	(Baso)	(LDH)	(TPR)
		(10^3cells/µL)	(10^3cells/µL)	(10^3cells/µL)	(10^3cells/µL)	(u/L)	(g/dL)
G1, F & 0	Mean	0.16	0.00	0.01	0.00	40.70	272.13
	±SD	0.10	0.00	0.00	0.01	23.61	89.97
	n	5	5	5	5	5	5
G2, F & 0.001	Mean	0.13	0.00	0.01	0.02	63.46	302.96
	±SD	0.08	0.00	0.01	0.02	32.58	89.90
	n	5	5	5	5	5	5
G3, F & 0.003	Mean	0.21	0.01	0.00	0.00	51.52	355.10
	±SD	0.10	0.01	0.01	0.01	21.60	108.66
	n	5	5	5	5	5	5
G4, F & 0.03	Mean	0.16	0.01	0.00	0.01	46.32	346.50
	±SD	0.11	0.01	0.01	0.01	23.88	251.82
	n	5	5	5	5	5	5
G1R, F & 0	Mean	0.12	0.00	0.00	0.01	46.52	235.94
	±SD	0.06	0.00	0.00	0.01	12.39	96.20
	n	5	5	5	5	5	5
G4R, F & 0.03	Mean	0.13	0.00	0.00	0.01	91.78*	151.51
	±SD	0.05	0.00	0.00	0.00	26.14	37.86
	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 CHEMISTRY RECORD

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	91.60	31.90	0.41	42.00	44.60	6.82	3.09	53.20	94.20
	±SD	8.88	2.88	0.03	8.40	8.85	0.48	0.07	13.94	10.66
	n	5	5	5	5	5	5	5	5	5
G2, M & 0.001	Mean	85.00	31.50	0.40	49.60	41.20	7.00	3.15	57.40	103.20
	±SD	8.51	1.64	0.01	6.43	12.76	0.20	0.15	7.57	12.77
	n	5	5	5	5	5	5	5	5	5
G3, M & 0.003	Mean	97.00	32.28	0.39	47.60	49.20	6.90	3.10	53.40	108.20
	±SD	15.70	4.40	0.02	11.01	8.23	0.30	0.11	6.19	7.76
	n	5	5	5	5	5	5	5	5	5
G4, M & 0.03	Mean	86.60	35.32	0.38	50.40	48.00	6.64	2.99	71.00	109.00
	±SD	9.71	11.73	0.03	12.30	13.67	0.43	0.17	15.89	21.51
	n	5	5	5	5	5	5	5	5	5
G1R, M & 0	Mean	77.20	34.62	0.52	53.60	29.80	7.52	3.37	62.20	106.60
	±SD	10.08	6.83	0.05	7.47	4.71	0.29	0.18	7.26	17.56
	n	5	5	5	5	5	5	5	5	5
G4R, M & 0.03	Mean	89.00	36.06	0.53	52.60	33.60	7.66	3.50	62.00	104.40
	±SD	6.71	1.99	0.05	8.68	9.02	0.15	0.33	13.62	14.06
	n	5	5	5	5	5	5	5	5	5

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

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

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	202.00	0.01	9.78	7.30	3.73	14.89	142.10	3.87	109.74
	±SD	79.50	0.02	0.33	0.63	0.45	1.34	0.83	0.18	0.91
	n	5	5	5	5	5	5	5	5	5
G2, M & 0.001	Mean	178.80	0.04	9.92	7.24	3.85	14.70	142.12	3.97	110.44
	±SD	21.75	0.02	0.29	0.43	0.15	0.76	0.89	0.16	0.73
	n	5	5	5	5	5	5	5	5	5
G3, M & 0.003	Mean	184.40	0.04	9.94	7.14	3.80	15.06	141.14	3.96	109.42
	±SD	47.26	0.05	0.21	0.55	0.33	2.05	0.91	0.29	1.27
	n	5	5	5	5	5	5	5	5	5
G4, M & 0.03	Mean	188.80	0.03	9.80	7.44	3.65	16.48	142.92	3.94	111.54*
	±SD	57.73	0.02	0.12	0.36	0.27	5.47	0.91	0.09	0.52
	n	5	5	5	5	5	5	5	5	5
G1R, M & 0	Mean	142.60	0.05	9.60	6.08	4.15	16.16	144.46	3.54	105.24
	±SD	31.91	0.01	0.28	0.47	0.26	3.19	0.27	0.34	1.42
	n	5	5	5	5	5	5	5	5	5
G4R, M & 0.03	Mean	163.80	0.05	9.52	5.36	4.16	16.83	144.52	3.55	106.68
	±SD	44.64	0.01	0.36	0.92	0.30	0.93	0.64	0.12	0.63
	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

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	95.80	29.54	0.39	53.60	48.60	7.08	3.35	42.60	91.20
	±SD	9.86	4.95	0.04	11.74	5.50	0.36	0.25	7.44	8.76
	n	5	5	5	5	5	5	5	5	5
G2, F & 0.001	Mean	100.20	28.98	0.37	54.40	50.80	7.32	3.33	44.20	96.00
	±SD	2.68	2.77	0.01	6.62	14.79	0.22	0.13	6.61	6.63
	n	5	5	5	5	5	5	5	5	5
G3, F & 0.003	Mean	102.20	30.38	0.35	59.60	37.40	7.28	3.34	46.80	105.40
	±SD	5.45	5.89	0.03	14.59	5.94	0.28	0.11	12.19	10.31
	n	5	5	5	5	5	5	5	5	5
G4, F & 0.03	Mean	108.00	32.98	0.37	58.20	45.20	7.20	3.16	38.20	92.00
	±SD	11.98	7.09	0.07	14.75	14.13	0.61	0.18	9.98	27.06
	n	5	5	5	5	5	5	5	5	5
G1R, F & 0	Mean	90.80	40.48	0.59	73.60	37.40	8.14	3.92	41.60	88.60
	±SD	8.64	2.53	0.04	27.56	10.43	0.58	0.32	5.03	14.03
	n	5	5	5	5	5	5	5	5	5
G4R, F & 0.03	Mean	98.00	36.66	0.57	72.40	38.20	8.14	3.95	40.80	85.60
	±SD	13.69	4.61	0.06	19.49	5.36	0.42	0.26	5.89	15.04
	n	5	5	5	5	5	5	5	5	5

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

TABLE 12 (Contd…). SUMMARY OF CLINICAL CHEMISTRY

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	105.20	0.02	10.40	7.02	3.73	13.79	140.90	4.12	111.32
	±SD	23.99	0.02	0.31	0.54	0.24	2.31	0.99	0.29	2.77
	n	5	5	5	5	5	5	5	5	5
G2, F & 0.001	Mean	96.00	0.03	10.14	6.98	3.99	13.53	141.66	4.05	111.20
	±SD	12.41	0.01	0.32	0.87	0.34	1.29	1.60	0.70	1.14
	n	5	5	5	5	5	5	5	5	5
G3, F & 0.003	Mean	140.60	0.04	10.28	6.80	3.94	14.18	141.68	3.82	111.46
	±SD	71.25	0.01	0.36	0.94	0.33	2.75	1.44	0.23	1.94
	n	5	5	5	5	5	5	5	5	5
G4, F & 0.03	Mean	129.80	0.03	10.34	7.20	4.04	15.39	142.58	4.07	112.06
	±SD	26.81	0.02	0.30	0.35	0.46	3.31	1.00	0.20	0.86
	n	5	5	5	5	5	5	5	5	5
G1R, F & 0	Mean	80.00	0.06	10.02	5.12	4.22	18.89	143.94	3.56	106.40
	±SD	19.27	0.01	0.49	0.79	0.32	1.18	0.88	0.41	1.02
	n	5	5	5	5	5	5	5	5	5
G4R, F & 0.03	Mean	62.20	0.07	10.06	4.72	4.19	17.11	143.88	3.17	106.60
	±SD	10.69	0.02	0.26	0.85	0.47	2.15	1.39	0.29	1.04
	n	5	5	5	5	5	5	5	5	5

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

TABLE 13.  SUMMARY OF URINALYSIS

Examination	Group, Sex & Dose (mg/L)		G1, M & 0	G2, M & 0.001	G3, M & 0.003	G4, M & 0.03	G1R, M & 0	G4R, M & 0.03
	Number of Animals		5	5	5	5	5	5
Physical	Colour	Pale Yellow	4	3	3	4	5	5
		Yellow	1	2	2	1	-	-
	Appearance	Clear	4	3	2	3	5	5
		Turbid	1	2	3	2	-	-
	Volume (mL)	Mean	5.5	5.9	5.7	5.4	6.5	6.5
		±SD	0.9	0.7	1.3	0.4	0.6	0.8
Chemical	pH	Mean	6.4	6.0	6.8	6.1	7.5	7.4
		±SD	1.6	0.9	1.3	1.0	0.6	0.5
	Specific Gravity	Mean	1.009	1.007	1.007	1.006	1.018	1.016
		±SD	0.002	0.004	0.003	0.002	0.007	0.007
	Urobilinogen (mg/dL)	Mean	0.2	0.2	0.2	0.2	0.2	0.4
		±SD	0.0	0.0	0.0	0.0	0.0	0.4
	Bilirubin (mg/dL)	Neg	5	5	5	5	5	5
	Ketones (mg/dL)	Neg	2	4	2	2	-	2
		5	2	1	3	3	5	2
		15	1	-	-	-	-	1
	Blood (Ery/µL)	Neg	-	-	-	-	1	1
		Ca10	1	-	-	-	-	1
		Ca25	3	2	1	-	2	2
		Ca80	1	3	4	5	1	1
		>=Ca200	-	-	-	-	1	-
	Proteins (mg/dL)	Neg	-	-	-	2	-	1
		Trace	2	4	3	1	-	1
		30	3	1	1	2	2	3
		100	-	-	1	-	2	-
		>=300	-	-	-	-	1	-
	Nitrite	Neg	3	5	4	5	4	5
		Pos	2	-	1	-	1	-
	Leucocytes (Leu/µL)	Neg	2	3	-	1	-	1
		Ca15	3	2	5	4	2	1
		Ca70	-	-	-	-	3	2
		Ca125	-	-	-	-	-	1
	Glucose (mg/dL)	Neg	5	5	5	5	5	5
	Micro albumin (MALB) (mg/dL)	Neg	-	-	-	2	-	-
		>15	5	5	5	3	5	4
		15	-	-	-	-	-	1
Microscopic	Epithelial cells	0	1	-	1	1	4	3
		0-1	2	4	3	3	1	2
		0-2	1	-	-	-	-	-
		1-2	1	1	1	1	-	-
	Casts	Absent	5	5	5	5	5	5
	Crystals	Absent	2	1	1	2	-	-
		Present	3	4	4	3	5	5

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

TABLE 13 (Contd…). SUMMARY OF URINALYSIS

Examination	Group, Sex & Dose (mg/L)		G1, F & 0	G2, F & 0.001	G3, F & 0.003	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	4	4	4	5	5
		Yellow	-	1	1	1	-	-
	Appearance	Clear	5	3	4	4	5	5
		Turbid	-	2	1	1	-	-
	Volume (mL)	Mean	6.1	6.6	7.6	6.6	6.6	6.9
		±SD	2.3	0.8	2.3	2.9	1.0	1.3
Chemical	pH	Mean	6.6	7.1	6.8	6.4	7.0	7.6
		±SD	1.7	1.3	1.2	1.0	0.4	0.7
	Specific Gravity	Mean	1.006	1.005	1.005	1.005	1.013	1.009*
		±SD	0.002	0.000	0.000	0.000	0.003	0.002
	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	5	5	5	5	5	5
	Ketones (mg/dL)	Neg	5	5	5	5	5	5
	Blood (Ery/µL)	Neg	4	1	1	-	4	4
		Ca10	-	-	-	3	1	-
		Ca25	-	3	4	2	-	1
		Ca80	1	1	-	-	-	-
	Proteins (mg/dL)	Neg	1	3	2	4	5	3
		Trace	3	2	3	1	-	1
		30	1	-	-	-	-	-
		100	-	-	-	-	-	1
	Nitrite	Neg	4	4	4	5	3	5
		Pos	1	1	1	-	2	-
	Leucocytes (Leu/µL)	Neg	1	-	-	-	4	4
		Ca15	4	5	5	5	1	-
		Ca70	-	-	-	-	-	1
	Glucose (mg/dL)	Neg	5	5	5	5	5	5
	Micro albumin (MALB) (mg/dL)	Neg	1	3	2	4	3	3
		>15	4	2	3	1	-	2
		15	-	-	-		2	-
Microscopic	Epithelial cells	0	1	1	2	2	5	4
		0-1	3	1	1	3	-	1
		1-2	1	3	2	-	-	-
	Casts	Absent	4	5	4	5	5	5
		Present	1	-	1	-	-	-
	Crystals	Absent	2	1	-	3	-	-
		Present	3	4	5	2	5	5

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

*: Statistically significant (p<0.05).

TABLE 14.      SUMMARY OF ABSOLUTE ORGAN WEIGHTS (g)

Group, Sex & Dose (mg/L)		Adrenals	Thymus	Spleen	Testes	Heart	Kidneys	Brain	Liver	Lungs
G1, M & 0	Mean	0.0546	0.3164	1.0157	3.2814	0.9708	1.9636	1.9756	8.1699	0.5472
	±SD	0.0079	0.0341	0.0992	0.2444	0.0559	0.1238	0.1009	0.6837	0.0516
	n	5	5	5	5	5	5	5	5	5
G2, M & 0.001	Mean	0.0507	0.2643	1.1574	2.8052*	1.0410	1.9459	1.9383	8.4021	0.7314*
	±SD	0.0057	0.0492	0.2344	0.2205	0.0990	0.1978	0.1199	0.5872	0.0658
	n	5	5	5	5	5	5	5	5	5
G3, M & 0.003	Mean	0.0525	0.2908	1.1117	2.9190	0.9237	1.7474	1.8780	7.0657	0.5565
	±SD	0.0037	0.0359	0.1191	0.1732	0.0443	0.0918	0.0720	0.4982	0.0609
	n	5	5	5	5	5	5	5	5	5
G4, M & 0.03	Mean	0.0542	0.2789	0.9743	3.4094	1.0249	1.9576	1.9070	7.4683	0.7014*
	±SD	0.0049	0.0236	0.0965	0.4453	0.0507	0.1801	0.0340	0.9456	0.0711
	n	5	5	5	5	5	5	5	5	5
G1R, M & 0	Mean	0.0609	0.2117	1.3692	3.6598	1.4328	2.8445	2.0602	11.2475	0.7140
	±SD	0.0042	0.0517	0.0633	0.3034	0.1040	0.5484	0.0512	1.7149	0.0767
	n	5	5	5	5	5	5	5	5	5
G4R, M & 0.03	Mean	0.0631	0.2829*	1.1172*	3.3671	1.3194	2.3350	2.0685	10.1517	0.6762
	±SD	0.0048	0.0365	0.1003	0.2875	0.0756	0.1815	0.0608	0.0759	0.0505
	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 14 (Contd…). SUMMARY OF ABSOLUTE ORGAN WEIGHTS (g)

Group, Sex & Dose (mg/L)		Adrenals	Thymus	Spleen	Ovaries	Uterus	Heart	Kidneys	Brain	Liver	Lungs
G1, F & 0	Mean	0.0623	0.3702	0.6049	0.1205	0.4089	0.7567	1.4280	1.7656	6.2227	0.5146
	±SD	0.0037	0.0701	0.1064	0.0129	0.0518	0.0741	0.1903	0.0929	0.4228	0.0453
	n	5	5	5	5	5	5	5	5	5	5
G2, F & 0.001	Mean	0.0594	0.3682	0.4791	0.1181	0.5253	0.7450	1.3006	1.8402	5.5576	0.5163
	±SD	0.0080	0.0627	0.1005	0.0094	0.1533	0.0599	0.1535	0.0842	0.3985	0.0639
	n	5	5	5	5	5	5	5	5	5	5
G3, F & 0.003	Mean	0.0561	0.2862	0.3462*	0.0987	0.3390	0.7369	1.2861	1.7616	5.6863	0.5077
	±SD	0.0070	0.0741	0.0372	0.0138	0.0602	0.1018	0.1160	0.0763	0.3377	0.0388
	n	5	5	5	5	5	5	5	5	5	5
G4, F & 0.03	Mean	0.0560	0.2707	0.4679	0.1156	0.3450	0.7066	1.2147	1.7537	6.0287	0.6040
	±SD	0.0056	0.0276	0.0857	0.0180	0.0603	0.0985	0.1357	0.0714	0.6349	0.0824
	n	5	5	5	5	5	5	5	5	5	5
G1R, F & 0	Mean	0.0809	0.2637	0.6039	0.1321	0.7511	0.9010	1.5550	1.8811	7.9632	0.4801
	±SD	0.0111	0.0441	0.0799	0.0168	0.2097	0.0950	0.0643	0.0875	0.4811	0.0316
	n	5	5	5	5	5	5	5	5	5	5
G4R, F & 0.03	Mean	0.0778	0.2352	0.5206	0.1647*	0.7788	0.9655	1.5697	1.8020	7.2701	0.4812
	±SD	0.0035	0.0293	0.0647	0.0077	0.2014	0.0968	0.1387	0.1353	0.9500	0.0610
	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

Group, Sex & Dose (mg/L)		Fasting Body Weight (g)	Adrenals	Thymus	Spleen	Testes	Heart	Kidneys	Brain	Liver	Lungs
G1, M & 0	Mean	229.55	0.0237	0.1385	0.4480	1.4392	0.4259	0.8641	0.8683	3.5879	0.2396
	±SD	20.95	0.0018	0.0167	0.0808	0.1679	0.0480	0.1288	0.1105	0.4917	0.0270
	n	5	5	5	5	5	5	5	5	5	5
G2, M & 0.001	Mean	225.52	0.0225	0.1174	0.5200	1.2504	0.4629	0.8621	0.8604	3.7368	0.3261*
	±SD	15.25	0.0022	0.0207	0.1333	0.1495	0.0523	0.0501	0.0388	0.3325	0.0437
	n	5	5	5	5	5	5	5	5	5	5
G3, M & 0.003	Mean	228.80	0.0230	0.1278	0.4869	1.2795	0.4045	0.7658	0.8230	3.1008	0.2440
	±SD	14.36	0.0012	0.0204	0.0550	0.1057	0.0231	0.0593	0.0548	0.3321	0.0305
	n	5	5	5	5	5	5	5	5	5	5
G4, M & 0.03	Mean	229.21	0.0236	0.1220	0.4252	1.4888	0.4480	0.8539	0.8344	3.2547	0.3068*
	±SD	15.64	0.0009	0.0117	0.0314	0.1825	0.0220	0.0491	0.0454	0.3036	0.0339
	n	5	5	5	5	5	5	5	5	5	5
G1R, M & 0	Mean	423.01	0.0144	0.0499	0.3236	0.8657	0.3385	0.6744	0.4873	2.6686	0.1692
	±SD	11.01	0.0008	0.0116	0.0088	0.0748	0.0194	0.1410	0.0183	0.4805	0.0227
	n	5	5	5	5	5	5	5	5	5	5
G4R, M & 0.03	Mean	418.89	0.0151	0.0676*	0.2670*	0.8047	0.3150	0.5574	0.4939	2.4239	0.1613
	±SD	6.76	0.0013	0.0089	0.0268	0.0797	0.0187	0.0407	0.0156	0.0366	0.0098
	n	5	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 15 (Contd…). SUMMARY OF ORGAN WEIGHT RELATIVE TO BODY WEIGHT (%) RECORD

Group, Sex & Dose (mg/L)		Fasting Body Weight (g)	Adrenals	Thymus	Spleen	Ovaries	Uterus	Heart	Kidneys	Brain	Liver	Lungs
G1, F & 0	Mean	183.73	0.0340	0.2021	0.3302	0.0659	0.2228	0.4117	0.7780	0.9621	3.3918	0.2801
	±SD	10.58	0.0031	0.0399	0.0616	0.0093	0.0267	0.0288	0.0967	0.0446	0.2396	0.0176
	n	5	5	5	5	5	5	5	5	5	5	5
G2, F & 0.001	Mean	184.83	0.0323	0.1989	0.2584	0.0639	0.2833	0.4037	0.7078	0.9991	3.0179	0.2812
	±SD	11.27	0.0057	0.0301	0.0468	0.0023	0.0773	0.0323	0.1163	0.0846	0.3169	0.0489
	n	5	5	5	5	5	5	5	5	5	5	5
G3, F & 0.003	Mean	181.71	0.0308	0.1592	0.1918*	0.0546	0.1868	0.4045	0.7093	0.9707	3.1331	0.2800
	±SD	11.37	0.0027	0.0487	0.0310	0.0094	0.0339	0.0379	0.0690	0.0360	0.1629	0.0240
	n	5	5	5	5	5	5	5	5	5	5	5
G4, F & 0.03	Mean	180.35	0.0312	0.1505	0.2599	0.0643	0.1925	0.3923	0.6760	0.9740	3.3548	0.3360
	±SD	8.36	0.0040	0.0179	0.0485	0.0111	0.0407	0.0568	0.0940	0.0590	0.4502	0.0522
	n	5	5	5	5	5	5	5	5	5	5	5
G1R, F & 0	Mean	313.71	0.0258	0.0840	0.1930	0.0423	0.2400	0.2871	0.4958	0.5996	2.5426	0.1531
	±SD	9.95	0.0035	0.0141	0.0286	0.0065	0.0704	0.0272	0.0176	0.0176	0.2084	0.0095
	n	5	5	5	5	5	5	5	5	5	5	5
G4R, F & 0.03	Mean	315.75	0.0247	0.0746	0.1648	0.0522*	0.2452	0.3060	0.4971	0.5706	2.2994	0.1527
	±SD	10.67	0.0011	0.0098	0.0187	0.0037	0.0568	0.0317	0.0403	0.0362	0.2605	0.0212
	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

Group, Sex & Dose (mg/L)		Adrenals	Thymus	Spleen	Testes	Heart	Kidneys	Liver	Lungs
G1, M & 0	Mean	2.7788	16.0226	51.3579	166.1722	49.2449	99.5469	413.9069	27.8307
	±SD	0.4854	1.5547	3.4144	10.1365	3.9319	7.2237	33.9594	3.9036
	n	5	5	5	5	5	5	5	5
G2, M & 0.001	Mean	2.6222	13.6994	60.4701	145.6095	53.8465	100.3085	435.2792	37.9309*
	±SD	0.3220	2.6851	15.6939	19.4023	5.9632	6.4199	47.2753	5.0597
	n	5	5	5	5	5	5	5	5
G3, M & 0.003	Mean	2.7980	15.4923	59.2262	155.4821	49.2030	93.0155	376.2989	29.7121
	±SD	0.2108	1.8488	6.3794	8.1774	2.0680	1.8959	23.2915	3.8733
	n	5	5	5	5	5	5	5	5
G4, M & 0.03	Mean	2.8385	14.6201	51.0374	178.7947	53.7343	102.6157	391.1467	36.7839*
	±SD	0.2285	1.1804	4.1897	23.3721	2.2678	8.8050	44.0085	3.7514
	n	5	5	5	5	5	5	5	5
G1R, M & 0	Mean	2.9629	10.3105	66.5051	177.9219	69.6012	137.7537	545.6673	34.6491
	±SD	0.2716	2.6969	3.8161	17.8584	5.7242	24.4075	79.4291	3.5038
	n	5	5	5	5	5	5	5	5
G4R, M & 0.03	Mean	3.0525	13.6603*	53.9801*	162.9183	63.8029	112.8276	491.1223	32.6866
	±SD	0.2426	1.5808	4.1095	15.1124	3.4811	7.0959	14.6572	2.1792
	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

Group, Sex & Dose (mg/L)		Adrenals	Thymus	Spleen	Ovaries	Uterus	Heart	Kidneys	Liver	Lungs
G1, F & 0	Mean	3.5292	20.9151	34.2411	6.8497	23.1571	42.7947	80.6817	352.3827	29.1361
	±SD	0.1907	3.3835	5.4867	0.8875	2.5721	2.2916	7.4942	13.8956	1.8218
	n	5	5	5	5	5	5	5	5	5
G2, F & 0.001	Mean	3.2258	20.0988	26.2348	6.4392	28.7329	40.5900	70.6182	302.3913*	28.0970
	±SD	0.4068	3.9272	6.4878	0.7038	9.0904	4.1657	6.8405	23.1828	3.5362
	n	5	5	5	5	5	5	5	5	5
G3, F & 0.003	Mean	3.1860	16.3556	19.7471*	5.6262	19.2871	41.7060	73.1094	323.1543	28.8421
	±SD	0.3606	4.8290	2.9899	0.9687	3.6096	4.1403	7.2204	21.4424	2.1647
	n	5	5	5	5	5	5	5	5	5
G4, F & 0.03	Mean	3.1976	15.4181	26.5831	6.6137	19.7461	40.2033	69.2865	343.4061	34.3678*
	±SD	0.3393	1.1681	3.8553	1.1811	3.8810	4.5301	7.6436	28.5407	3.6477
	n	5	5	5	5	5	5	5	5	5
G1R, F & 0	Mean	4.2971	14.0269	32.2042	7.0595	40.0258	47.8431	82.7173	424.6109	25.5294
	±SD	0.4989	2.4052	4.8172	1.1360	11.8751	3.5805	2.8224	39.7625	1.4101
	n	5	5	5	5	5	5	5	5	5
G4R, F & 0.03	Mean	4.3383	13.1595	29.1223	9.1710*	43.1295	53.6468	87.3998	403.2624	26.7929
	±SD	0.3998	2.2296	5.0961	0.7043	10.4359	4.6557	8.9537	40.2997	3.4916
	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

Group & Conc. (mg/L)		Temp.  (°C)	Rh (%)	O2 (%)	CO2  (mg/L)	Air inlet flow rate (L/min)*	BZC (mg/L)
G1/G1R & 0	Mean	22.55	55.52	20.39	617.48	20.00	-
	±SD	0.31	1.90	0.19	5.43	0.00	-
G2 & 0.001	Mean	22.66	55.95	20.47	619.67	20.00	0.0011
	±SD	0.31	1.35	0.21	4.84	0.00	0.0001
G3 & 0.003	Mean	22.64	55.88	20.50	619.73	20.00	0.0032
	±SD	0.31	1.32	0.23	5.31	0.00	0.0001
G4/G4R & 0.03	Mean	22.59	55.10	20.38	617.74	20.00	0.032
	±SD	0.31	1.59	0.20	5.12	0.00	0.001

    *: 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.

TABLE 18.    SUMMARY OF NOMINAL CONCENTRATION

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)	1150	20	360	0.16
		4370			0.61
		5230			0.73
2	G1/G1R (Air Only)	-	20	360	-
	G2, G3 and G4/G4R  (Test Item)	1120	20	360	0.16
		4440			0.62
		5110			0.71
3	G1/G1R  (Air Only)	-	20	360	-
	G2, G3 and G4/G4R  (Test Item)	1200	20	360	0.17
		4380			0.61
		5140			0.71
4	G1/G1R  (Air Only)	-	20	360	-
	G2, G3 and G4/G4R  (Test Item)	1160	20	360	0.16
		4350			0.60
		5160			0.72
5	G1/G1R  (Air Only)	-	20	360	-
	G2, G3 and G4/G4R  (Test Item)	1180	20	360	0.16
		4430			0.62
		5200			0.72
6	G1/G1R  (Air Only)	-	20	360	-
	G2, G3 and G4/G4R  (Test Item)	1090	20	360	0.15
		4340			0.60
		5180			0.72
7	G1/G1R  (Air Only)	-	20	360	-
	G2, G3 and G4/G4R  (Test Item)	1170	20	360	0.16
		4430			0.62
		5190			0.72

TABLE 18 (Contd…). SUMMARY OF NOMINAL CONCENTRATION

Day	Group	Test item Used (mg) (a)	Air flow rate (L/minute)(b)	Minute (min) (c)	Nominal Concentration (mg/L) (a)    / (b) × (c)
8	G1/G1R  (Air Only)	-	20	360	-
	G2, G3 and G4/G4R  (Test Item)	1140	20	360	0.16
		4570			0.63
		5200			0.72
9	G1/G1R  (Air Only)	-	20	360	-
	G2, G3 and G4/G4R  (Test Item)	1150	20	360	0.16
		4330			0.60
		5280			0.73
10	G1/G1R  (Air Only)	-	20	360	-
	G2, G3 and G4/G4R  (Test Item)	1190	20	360	0.17
		4410			0.61
		5110			0.71
11	G1/G1R  (Air Only)	-	20	360	-
	G2, G3 and G4/G4R  (Test Item)	1130	20	360	0.16
		4440			0.62
		5210			0.72
12	G1/G1R  (Air Only)	-	20	360	-
	G2, G3 and G4/G4R  (Test Item)	1170	20	360	0.16
		4580			0.64
		5290			0.73
13	G1/G1R  (Air Only)	-	20	360	-
	G2, G3 and G4/G4R  (Test Item)	1190	20	360	0.17
		4320			0.60
		5150			0.72
14	G1/G1R  (Air Only)	-	20	360	-
	G2, G3 and G4/G4R  (Test Item)	1160	20	360	0.16
		4440			0.62
		5120			0.71

TABLE 18 (Contd…). SUMMARY OF NOMINAL CONCENTRATION

Day	Group	Test item Used (mg) (a)	Air flow rate (L/minute)(b)	Minute (min) (c)	Nominal Concentration (mg/L) (a)    / (b) × (c)
15	G1/G1R  (Air Only)	-	20	360	-
	G2, G3 and G4/G4R  (Test Item)	1170	20	360	0.16
		4400			0.61
		5170			0.72
16	G1/G1R  (Air Only)	-	20	360	-
	G2, G3 and G4/G4R  (Test Item)	1170	20	360	0.16
		4460			0.62
		5210			0.72
17	G1/G1R  (Air Only)	-	20	360	-
	G2, G3 and G4/G4R  (Test Item)	1190	20	360	0.17
		4400			0.61
		5270			0.73
18	G1/G1R  (Air Only)	-	20	360	-
	G2, G3 and G4/G4R  (Test Item)	1140	20	360	0.16
		4360			0.61
		5200			0.72
19	G1/G1R  (Air Only)	-	20	360	-
	G2, G3 and G4/G4R  (Test Item)	1190	20	360	0.17
		4590			0.64
		5230			0.73
20	G1/G1R  (Air Only)	-	20	360	-
	G2, G3 and G4/G4R  (Test Item)	1160	20	360	0.16
		4590			0.64
		5270			0.73

TABLE 19.     SUMMARY OF GROSS PATHOLOGY FINDINGS

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 Concentration (mg/L)	0	0.001*	0.003*	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 Concentration (mg/L)	0	0.001*	0.003*	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	-	-	1	-	-	-
Uterus– small in size	-	-	1	-	-	-

R: Recovery; *: Above doses were provided by the sponsor; - : No incidence.

TABLE 20.      SUMMARY OF HISTOPATHOLOGY FINDINGS – MAIN GROUP

Route of administration		Inhalation
Treatment		Air only		High Dose
Nominal Target Concentration (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	4	3	4
Cyst	Present	1	1	2	-
Cast	Minimal	1	-	-	-
Basophilia, tubules	Minimal	-	-	1	-
Dilatation, 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(left lobe)	Number examined	5	5	5	5
	Within normal limits	5	5	5	5
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

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

TABLE 20 (Contd..). SUMMARY OF HISTOPATHOLOGY FINDINGS – MAIN GROUP

Route of administration		Inhalation
Treatment		Air only		High Dose
Nominal Target Concentration (mg/L)		0		0.03
Group		G1		G4
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	4		X
Inflammation	Mild	-	X	1		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	4	X	5	X
Atrophy, tubules	Minimal	1	X	-	X
Thymus	Number examined	5	5	5	5
	Within normal limits	5	3	5	4
Cyst, epithelial	Present	-	2	-	1
Thyroid	Number examined	5	5	5	5
	Within normal limits	5	3	3	5
Cyst, ultimobranchial	Present	-	1	2	-
Ectopic tissue, thymus	Present	-	1	-	-
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	5	X	4
Dilatation, lumen	Minimal	X	-	X	1

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

TABLE 21. SUMMARY OF HISTOPATHOLOGY FINDINGS – RECOVERY GROUP

Route of administration		Inhalation
Treatment		Air only		High Dose Recovery
Nominal Target Concentration (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	4	5	4	5
Ectopic tissue, adrenocortical	Present	1	-	1	-
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	4	5	5	5
Cellularity, increased, lymphocytes	Mild	1	-	-	-
Esophagus	Number examined	5	5	5	5
	Within normal limits	5	5	5