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Description of key information

Based on the results of an OECD 407 subacute study, the no-observed-adverse-effect-level (NOAEL) was considered to be 1000 mg/kg body weight/day

The results of the subchronic oral 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.

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.

Based on the observed results of a 28 day inhalation study in rats, it is concluded that the No Observed Adverse Effect Concentration (NOAEC) of test item was found to be 0.03 mg/L when exposed for 6 hours/day, 5 days/week, for 4 weeks by flow-past nose-only inhalation route to Sprague Dawley rats.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 15 Sep 2011 to 29 May 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study (OECD 407, GLP-compliant)
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Japanese Guidelines for Screening Toxicity Testing of Chemicals: Testing Methods for New Substances, enacted July 13, 1974, amended December 5, 1986.
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Animals: Rat, RccHanTM: WIST(SPF)
- Rationale: Recognized by international guidelines as a recommended test system.
- Source: Harlan Laboratories B.V., Kreuzelweg 53, 5961 NM Horst / Netherlands
- Age (at Delivery): 7 weeks
- Body Weight Range (at Acclimatization): 192 to 207 g (mean 198 g) in males and 122 to 150 g (mean 137 g) in females
- Housing: In groups of five in Makrolon type-4 cages with wire mesh tops and standard softwood bedding (J. Rettenmaier & Söhne GmbH & Co. KG, 73494 Rosenberg / Germany, imported by Provimi Kliba AG, 4303 Kaiseraugst / Switzerland) including paper enrichment (Enviro-dri from Lillico, Biotechnology, Surrey, UK)
- Diet: Pelleted standard Harlan Teklad 2914C (batch nos. 44/11 and 46/11) rat / mouse maintenance diet (Provimi Kliba AG, 4303 Kaiseraugst / Switzerland) was available ad libitum. The feed batches were analyzed for contaminants.
- Water: Community tap-water from Itingen was available ad libitum in water bottles.
- Acclimation period: yes, 6 days
- Randomization: Randomly allocated to groups by body weight.

ENVIRONMENTAL CONDITIONS
Standard laboratory conditions, continuously monitored.
- Temperature (°C): 22 +/- 3
- Humidity (%): 30 to 70
- Air changes (per hr): . Air-conditioned with 10 - 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12/12 (with at least eight hours music during the light period)

IN-LIFE DATES: From: 22 SEP 2011 To: 20 OCT 2011 (recovery groups of control and high dose group were necropsied at 3rd NOV 2011)
Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
Method:
- Oral, by gavage
- Rationale for Method: Administration by gavage is a common and accepted route of exposure for studies of this type.
- Frequency of Administration: Daily.
- Dose Levels:
Group 1: 0 mg/kg/day
Group 2: 100 mg/kg/day
Group 3: 300 mg/kg/day
Group 4: 1000 mg/kg/day
- Rationale for Dose Level Selection:The dose levels were selected based on a previous dose range finding toxicity study in Wistar rats, Harlan Laboratories study.
- Dose Volume: 10 mL/kg body weight
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The application formulations investigated during the study were found to comprise test item in the range of 87.1% to 105.3%, and met the required content limit of ±20% with reference to the nominal content. Because single results found did not deviate more than 10% (<15%) from the corresponding mean, dose formulations with test item were considered to be homogeneous.
The test item was found to be stable in application formulations of group 4 (concentration of 100 mg/mL) when kept four hours or eight days under room temperature: recoveries met the variation limit of 10% from the time-zero (homogeneity) mean.
However, the result of stability in groups 2 and 3 exceeded the acceptance criteria. In application formulations of group 2 (10 mg/mL), the maximum deviation of time-zero mean was found to be 24.0% for 4 hours stability and 23.4% for eight day stability, as in application formulations of group 3 (30 mg/mL), the maximum deviation of time-zero mean was found to be 14.8% for 4 hours stability and 16.1% for eight day stability.
Although it is not possible to determine the exact reason for exceeding the acceptance criteria, smaller dose levels commonly yield greater deviation from time zero after 4 hours and changes in the recovery do not change between sampling intervals (4 hours and 8 days). A possible explanation is an undetermined degree of reactivity with a constant amount of another substance such as oxygen. It is also possible that adsorption to the surface of the sample vessel may have occurred. In addition, a lack of stability does not stop after 4 hours.
In conclusion, the results indicate the accurate use of the test item and bidistilled water as vehicle during this study. Application formulations were found to be homogeneously prepared.
Duration of treatment / exposure:
28 days
Frequency of treatment:
Once daily
Remarks:
Doses / Concentrations:
0 , 100, 300, 1000 mg/kg/day
Basis:
actual ingested
No. of animals per sex per dose:
Group 1 (0 mg/kg/day): 10 males / 10 females
Group 2 (100 mg/kg/day): 5 males / 5 females
Group 3 (300 mg/kg/day): 5 males / 5 females
Group 4 (1000 mg/kg/day): 10 males / 10 females
Control animals:
yes
Details on study design:
The purpose of this oral toxicity study was to assess the cumulative toxicity of the test item when administered daily to rats by gavage for a period of 28 days. The reversibility of treatment-related changes was assessed after a treatment-free 14-day recovery period.

Positive control:
No
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily (for viability/mortality)

CLINICAL OBSERVATIONS: Yes
- Time schedule: daily (during acclimatisation period, before test item administration during treatment period and during recovery period; twice daily during days 1 to 3 of treatment period)

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION: yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes ( for details see "any othe information on materials and methods")
- Time schedule for collection of blood: At the end of the dosing and the treatment-free recovery period

CLINICAL CHEMISTRY: Yes ( for details see "any othe information on materials and methods)
- Time schedule for collection of blood: At the end of the dosing and the treatment-free recovery period

URINALYSIS: Yes ( for details see "any othe information on materials and methods")

NEUROBEHAVIOURAL EXAMINATION: Yes ( for details see "any othe information on materials and methods")
- Time schedule for examinations:during week 4
- Battery of functions tested: grip strength / locomotor activity

OTHER:Histological examinations were performed on organs and tissues from all control and high dose animals, and all gross lesions from all animals. The stage of estrus was also determined by the pathologist.
Sacrifice and pathology:
GROSS PATHOLOGY:Yes ( for details see "any othe information on materials and methods")
HISTOPATHOLOGY: Yes (performed on organs and tissues from all control and high dose animals, and all gross lesions from all animals)
A description of all abnormalities is included. Attempts were made to correlate gross observations with microscopic findings.
Statistics:
The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.
Fisher's exact-test.
Clinical signs:
no effects observed
Description (incidence and severity):
one test item-unrelated mortality
Mortality:
no mortality observed
Description (incidence):
one test item-unrelated mortality
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Not test item related, considered to be secondary findings after aspiration
Histopathological findings: neoplastic:
not examined
Details on results:
Observations
Viability / Mortality
All males and females survived the treatment period. On day 1 of the recovery period, female no. 58 (previously treated with 1000 mg/kg/day) died during anesthesia for blood sampling.

Daily Observations
No test item-related adverse findings were noted during the treatment period and no late effects were noted during the recovery period. At all dose levels, yellow staining of the feces was noted with dose-dependent onset; this was considered to be a passive effect and is commonly noted following oral administration of a dyestuff. In males and females at 100 mg/kg/day, this was noted on day 25; at 300 mg/kg/day on day 7 and from day 25-28 and at 1000 mg/kg/day from day 7 onwards.
Minor scabbing was noted on the shoulders of one male treated with 1000 mg/kg/day on days 26-28 of treatment and in one female treated with 300 mg/kg/day on day 28 of treatment. These findings were considered to be unrelated to the test item.
During the recovery period, fecal staining was reversible from day 3 onwards.

Weekly Behavioral Observations
No findings were evident during the weekly behavioral observations performed at weeks 1, 2 or 3 of treatment.

Functional Observational Battery (Screen)
No findings were evident during the functional observational battery performed at week 4 of treatment.

Grip Strength
The mean fore- and hind limb grip strength values of the test item-treated males and females compared favorably with those of the respective control values.

Locomotor Activity
The mean locomotor activity values of the test item-treated males and females were considered to be unaffected. During the first measurement interval (0-10 minutes), significantly higher values were noted in females (p<0.05) treated with 1000 mg/kg/day when compared with the controls. All other values recorded during subsequent measurement intervals were similar to those of the control females.

Food Consumption
There were no test item-related changes in the mean daily food consumption at any dose level.
The mean daily food consumption of the test item-treated females decreased slightly during the recovery period, but this was considered to be an artifact caused by the coincidental removal (and necropsy) of the cage with the higher mean food consumption.

Body Weights
The mean absolute and relative body weights of the test item-treated rats compared favorably with their respective control values during the treatment period. During the recovery period, significantly lower (p<0.01) mean body weight gain values were noted in the males (days 8 and 14) and females (day 8) previously treated with 1000 mg/kg/day.

Clinical Laboratory Investigations
Hematology
There were no test item-related effects upon the hematology parameters of males or females at any dose level.
At 1000 mg/kg/day, significantly elevated mean corpuscular volume (p<0.05) was noted at the end of the treatment period in males when compared with the controls. This difference remained within the range of the historical control data and was considered to be incidental.
The mean absolute and relative reticulocyte counts of the males treated with 1000 mg/kg/day were significantly elevated when compared with the controls. However, when compared with the historical control data, the control values were lower and therefore the increased values were considered to be an artifact.
All other changes of statistical significance were noted in the low- or middle-dose groups and were without dose dependence. Therefore these differences were considered to be unrelated to the test item.
After the recovery period, the hematology parameters of the males compared favorably. In females previously treated with 1000 mg/kg/day, significantly lower mean corpuscular volume (p<0.05), the mean absolute eosinophil count (p<0.01) and mean absolute basophil count (p<0.05) were noted when compared with the respective control values, but all values remained within the ranges of the historical control values.

Clinical Biochemistry
There were no test item-related effects upon the clinical biochemistry parameters of males or females at any dose level.
After the treatment period, the mean sodium level was significantly elevated in males treated with 1000 mg/kg/day (p<0.05) when compared with the controls and marginally exceeded the upper range of the historical control data. No toxicological relevance was associated with this isolated difference.
All clinical biochemistry values recorded in the females treated with 1000 mg/kg/day were similar to those of the control females. All other changes of statistical significance were noted in the low- or middle-dose groups and were without dose dependence. Therefore these differences were considered to be unrelated to the test item.
After the recovery period, males which were previously treated with the test item at 1000 mg/kg/day showed significantly elevated glucose levels (p<0.05) and elevated triglycerides (p<0.01) which remained within the ranges of the respective historical control values. The mean phospholipid level was significantly elevated (p<0.01) and exceeded the upper limit of the historical control data, but in the absence of similar difference after the end of the treatment period, was considered to be of no toxicological relevance. In females, the mean phospholipid level was significantly reduced (p<0.05) when compared with control females. Significant reductions of creatine kinase (p<0.05), calcium (p<0.01), phosphorus (p<0.01) protein (p<0.05) and albumin (p<0.05) were noted, all of which remained within the range of the historical control data.

Urinalysis
There were no test item-related effects upon the urinalysis parameters of males or females at any dose level.

Pathology
Organ Weights
At the end of the treatment period, there were no statistically significant differences in the mean absolute and relative organ weights at any dose level. During the recovery period, the mean absolute adrenal weights and mean absolute uterus weights (including oviducts) were significantly elevated (p<0.05) in females previously treated with 1000 mg/kg/day. The mean heart-to-body weight ratio of these females was marginally, but significantly, reduced (p<0.05), whereas significantly higher organ-to-brain weights were noted for pituitary (p<0.05), thyroids (p<0.05), adrenals (p<0.05) and uterus/oviducts (p<0.01) when compared with the controls. Insofar as these findings were not evident at the end of the treatment period and were not accompanied by microscopical changes of morphology, all were considered to be unrelated to the treatment with the test item.

Macroscopic Findings
After the treatment period, a small number of macroscopical changes were noted in rats at all dose levels, with no clear dose dependent incidence.
In males, discoloration and/or inflated lungs were noted in single males at 100 and 1000 mg/kg/day. Inflated lungs were also recorded in two females at 100 mg/kg/day. Lung foci were recorded for one female at 1000 mg/kg/day.
Thymic foci and/or discoloration were noted in on control female, one female at 100 mg/kg/day, two females at 300 mg/kg/day and one male at 1000 mg/kg/day.
Enlargement and/or discoloration of the lymph nodes were noted in one male treated with 100 mg/kg/day and in one male treated with 1000 mg/kg/day.
Uterine dilation was recorded in one control female and single females at 300 mg/kg/day and 1000 mg/kg/day. Skin sores were recorded in one female at 300 mg/kg/day and in one male at 1000 mg/kg/day,
After the recovery period, one male previously treated with 1000 mg/kg/day had smaller testes and epididymides and a female had discoloration of the lungs and ovaries. These findings were not considered to be related to late effects of the test item.

Microscopic Findings
Lung, Trachea
In male number 14 (100 mg/kg/day) and in female number 53 (1000 mg/kg/day), fine granular yellowish foreign material was found to be present in alveolar macrophages. Similar foreign material was found in inflammatory (granulomatous) lesions of the lung of male number 25 (1000 mg/kg/day) and in the submucosal areas of the trachea of this animal.

Lymph Nodes
The above animals showed macroscopically enlarged and yellowish discolored bronchial and mediastinal lymph nodes. During microscopic examination lymphoid hyperplasia along with accumulated fine granular, yellowish foreign material was recorded.

Other Findings
The remaining findings recorded were within the range of normal background lesions which may be recorded in animals of this strain and age.
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No toxicologically relevant findings were noted up to the dose level of 1000 mg/kg bw/day, the highest dose level tested.
Critical effects observed:
not specified
Conclusions:
Based on the results of this OECD 407 subacute study, a no-observed-effect-level (NOEL) could not be established but the no-observed-adverse-effect-level (NOAEL) was considered to be 1000 mg/kg body weight/day.
Executive summary:

In this subacute toxicity study, the test material was administered daily by oral gavage to Wistar rats of both sexes at dose levels of 100, 300 and 1000 mg/kg body weight/day for a period of 28 days. A control group was treated similarly with the vehicle, bidistilled water, only. The groups comprised 5 animals per sex which were sacrificed after 28 days of treatment. Additional 5 rats per sex and group were used at 0 and 1000 mg/kg. These animals were treated for 28 days and then allowed a 14-day treatment-free recovery period after which they were sacrificed.

Clinical signs, outside cage observation, food consumption and body weights were recorded periodically during acclimatization, the treatment and recovery periods. Functional observational battery, locomotor activity and grip strength were performed during week 4. At the end of the dosing and the treatment-free recovery period, blood samples were withdrawn for hematology and plasma chemistry analyses. Urine samples were collected for urinalyses. All animals were killed, necropsied and examined post mortem. Histological examinations were performed on organs and tissues from all control and high dose animals, and all gross lesions from all animals.

There were no toxicologically relevant deaths. One high dose female died during anesthesia for blood sampling.

No test item-related adverse findings were noted in the daily clinical observations performed during the treatment period and no late effects were noted during the recovery period. At all dose levels, yellow staining of the feces was noted with dose-dependent onset; this was considered to

be a passive effect and is commonly noted following oral administration of a dyestuff. No findings were evident during the weekly behavioral observations performed at weeks 1, 2 or 3 of treatment.

No findings were evident during the functional observational battery performed at week 4 of treatment.

The mean fore- and hind limb grip strength values of the test item-treated males and females compared favorably with those of the respective control values.

The mean locomotor activity values of the test item-treated males and females were considered to be unaffected.

There were no test item-related changes in the mean daily food consumption at any dose level.

The mean absolute and relative body weights of the test item-treated rats compared favorably with their respective control values during the treatment period. Minor differences seen during the recovery period were considered to be of no toxicological relevance.

There were no test item-related effects upon the hematology paramters of males or females at any dose level.

There were no test item-related effects upon the clinical biochemistry parameters of males or females at any dose level.

There were no test item-related effects upon the urinalysis parameters of males or females at any dose level.

At the end of the treatment period, there were no statistically significant differences in the mean absolute and relative organ weights at any dose level. During the recovery period, the mean absolute adrenal weights and mean absolute uterus weights (including oviducts) were elevated in females previously treated with 1000 mg/kg/day. The mean heart-to-body weight ratio of these females was marginally reduced, whereas higher organ-tobrain weights were noted for pituitary, thyroids, adrenals and uterus/oviducts when compared with the controls. Insofar as these findings were not evident at the end of the treatment period and were not accompanied by microscopical changes of morphology, all were considered to be

unrelated to the treatment with the test item.

After the treatment period, a small number of macroscopical changes were noted in rats at all dose levels, with no clear dose dependent incidence.

Microscopical changes were noted in the in lungs, trachea, bronchial and mediastinal lymph nodes of two single animals, and were considered to be largely secondary changes that were not indicative of systemic toxicity.

The only test item-related, yet non-adverse findings were restricted to staining of the feces in rats at all dose levels, which was considered to be a typical passive effect following large oral doses of a dyestuff.

Based on the results of this study, a no-observed-effect-level (NOEL) could no be established but the no-observed-adverse-effect-level (NOAEL) was considered to be 1000 mg/kg body weight/day.

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.

Endpoint:
sub-chronic toxicity: oral
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
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: 189.42 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.

Endpoint:
sub-chronic toxicity: oral
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Justification for type of information:
See Read Across Justification document in Chapter 13
Reason / purpose for cross-reference:
read-across source
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.

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
From 15 Sep 2011 to 29 May 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study (OECD 407, GLP-compliant)
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 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Japanese Guidelines for Screening Toxicity Testing of Chemicals: Testing Methods for New Substances, enacted July 13, 1974, amended December 5, 1986.
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Animals: Rat, RccHanTM: WIST(SPF)
- Rationale: Recognized by international guidelines as a recommended test system.
- Source: Harlan Laboratories B.V., Kreuzelweg 53, 5961 NM Horst / Netherlands
- Age (at Delivery): 7 weeks
- Body Weight Range (at Acclimatization): 192 to 207 g (mean 198 g) in males and 122 to 150 g (mean 137 g) in females
- Housing: In groups of five in Makrolon type-4 cages with wire mesh tops and standard softwood bedding (J. Rettenmaier & Söhne GmbH & Co. KG, 73494 Rosenberg / Germany, imported by Provimi Kliba AG, 4303 Kaiseraugst / Switzerland) including paper enrichment (Enviro-dri from Lillico, Biotechnology, Surrey, UK)
- Diet: Pelleted standard Harlan Teklad 2914C (batch nos. 44/11 and 46/11) rat / mouse maintenance diet (Provimi Kliba AG, 4303 Kaiseraugst / Switzerland) was available ad libitum. The feed batches were analyzed for contaminants.
- Water: Community tap-water from Itingen was available ad libitum in water bottles.
- Acclimation period: yes, 6 days
- Randomization: Randomly allocated to groups by body weight.

ENVIRONMENTAL CONDITIONS
Standard laboratory conditions, continuously monitored.
- Temperature (°C): 22 +/- 3
- Humidity (%): 30 to 70
- Air changes (per hr): . Air-conditioned with 10 - 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12/12 (with at least eight hours music during the light period)

IN-LIFE DATES: From: 22 SEP 2011 To: 20 OCT 2011 (recovery groups of control and high dose group were necropsied at 3rd NOV 2011)
Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
Method:
- Oral, by gavage
- Rationale for Method: Administration by gavage is a common and accepted route of exposure for studies of this type.
- Frequency of Administration: Daily.
- Dose Levels:
Group 1: 0 mg/kg/day
Group 2: 100 mg/kg/day
Group 3: 300 mg/kg/day
Group 4: 1000 mg/kg/day
- Rationale for Dose Level Selection:The dose levels were selected based on a previous dose range finding toxicity study in Wistar rats, Harlan Laboratories study.
- Dose Volume: 10 mL/kg body weight
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The application formulations investigated during the study were found to comprise test item in the range of 87.1% to 105.3%, and met the required content limit of ±20% with reference to the nominal content. Because single results found did not deviate more than 10% (<15%) from the corresponding mean, dose formulations with test item were considered to be homogeneous.
The test item was found to be stable in application formulations of group 4 (concentration of 100 mg/mL) when kept four hours or eight days under room temperature: recoveries met the variation limit of 10% from the time-zero (homogeneity) mean.
However, the result of stability in groups 2 and 3 exceeded the acceptance criteria. In application formulations of group 2 (10 mg/mL), the maximum deviation of time-zero mean was found to be 24.0% for 4 hours stability and 23.4% for eight day stability, as in application formulations of group 3 (30 mg/mL), the maximum deviation of time-zero mean was found to be 14.8% for 4 hours stability and 16.1% for eight day stability.
Although it is not possible to determine the exact reason for exceeding the acceptance criteria, smaller dose levels commonly yield greater deviation from time zero after 4 hours and changes in the recovery do not change between sampling intervals (4 hours and 8 days). A possible explanation is an undetermined degree of reactivity with a constant amount of another substance such as oxygen. It is also possible that adsorption to the surface of the sample vessel may have occurred. In addition, a lack of stability does not stop after 4 hours.
In conclusion, the results indicate the accurate use of the test item and bidistilled water as vehicle during this study. Application formulations were found to be homogeneously prepared.
Duration of treatment / exposure:
28 days
Frequency of treatment:
Once daily
Remarks:
Doses / Concentrations:
0 , 100, 300, 1000 mg/kg/day
Basis:
actual ingested
No. of animals per sex per dose:
Group 1 (0 mg/kg/day): 10 males / 10 females
Group 2 (100 mg/kg/day): 5 males / 5 females
Group 3 (300 mg/kg/day): 5 males / 5 females
Group 4 (1000 mg/kg/day): 10 males / 10 females
Control animals:
yes
Details on study design:
The purpose of this oral toxicity study was to assess the cumulative toxicity of the test item when administered daily to rats by gavage for a period of 28 days. The reversibility of treatment-related changes was assessed after a treatment-free 14-day recovery period.

Positive control:
No
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily (for viability/mortality)

CLINICAL OBSERVATIONS: Yes
- Time schedule: daily (during acclimatisation period, before test item administration during treatment period and during recovery period; twice daily during days 1 to 3 of treatment period)

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION: yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes ( for details see "any othe information on materials and methods")
- Time schedule for collection of blood: At the end of the dosing and the treatment-free recovery period

CLINICAL CHEMISTRY: Yes ( for details see "any othe information on materials and methods)
- Time schedule for collection of blood: At the end of the dosing and the treatment-free recovery period

URINALYSIS: Yes ( for details see "any othe information on materials and methods")

NEUROBEHAVIOURAL EXAMINATION: Yes ( for details see "any othe information on materials and methods")
- Time schedule for examinations:during week 4
- Battery of functions tested: grip strength / locomotor activity

OTHER:Histological examinations were performed on organs and tissues from all control and high dose animals, and all gross lesions from all animals. The stage of estrus was also determined by the pathologist.
Sacrifice and pathology:
GROSS PATHOLOGY:Yes ( for details see "any othe information on materials and methods")
HISTOPATHOLOGY: Yes (performed on organs and tissues from all control and high dose animals, and all gross lesions from all animals)
A description of all abnormalities is included. Attempts were made to correlate gross observations with microscopic findings.
Statistics:
The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.
Fisher's exact-test.
Clinical signs:
no effects observed
Description (incidence and severity):
one test item-unrelated mortality
Mortality:
no mortality observed
Description (incidence):
one test item-unrelated mortality
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Not test item related, considered to be secondary findings after aspiration
Histopathological findings: neoplastic:
not examined
Details on results:
Observations
Viability / Mortality
All males and females survived the treatment period. On day 1 of the recovery period, female no. 58 (previously treated with 1000 mg/kg/day) died during anesthesia for blood sampling.

Daily Observations
No test item-related adverse findings were noted during the treatment period and no late effects were noted during the recovery period. At all dose levels, yellow staining of the feces was noted with dose-dependent onset; this was considered to be a passive effect and is commonly noted following oral administration of a dyestuff. In males and females at 100 mg/kg/day, this was noted on day 25; at 300 mg/kg/day on day 7 and from day 25-28 and at 1000 mg/kg/day from day 7 onwards.
Minor scabbing was noted on the shoulders of one male treated with 1000 mg/kg/day on days 26-28 of treatment and in one female treated with 300 mg/kg/day on day 28 of treatment. These findings were considered to be unrelated to the test item.
During the recovery period, fecal staining was reversible from day 3 onwards.

Weekly Behavioral Observations
No findings were evident during the weekly behavioral observations performed at weeks 1, 2 or 3 of treatment.

Functional Observational Battery (Screen)
No findings were evident during the functional observational battery performed at week 4 of treatment.

Grip Strength
The mean fore- and hind limb grip strength values of the test item-treated males and females compared favorably with those of the respective control values.

Locomotor Activity
The mean locomotor activity values of the test item-treated males and females were considered to be unaffected. During the first measurement interval (0-10 minutes), significantly higher values were noted in females (p<0.05) treated with 1000 mg/kg/day when compared with the controls. All other values recorded during subsequent measurement intervals were similar to those of the control females.

Food Consumption
There were no test item-related changes in the mean daily food consumption at any dose level.
The mean daily food consumption of the test item-treated females decreased slightly during the recovery period, but this was considered to be an artifact caused by the coincidental removal (and necropsy) of the cage with the higher mean food consumption.

Body Weights
The mean absolute and relative body weights of the test item-treated rats compared favorably with their respective control values during the treatment period. During the recovery period, significantly lower (p<0.01) mean body weight gain values were noted in the males (days 8 and 14) and females (day 8) previously treated with 1000 mg/kg/day.

Clinical Laboratory Investigations
Hematology
There were no test item-related effects upon the hematology parameters of males or females at any dose level.
At 1000 mg/kg/day, significantly elevated mean corpuscular volume (p<0.05) was noted at the end of the treatment period in males when compared with the controls. This difference remained within the range of the historical control data and was considered to be incidental.
The mean absolute and relative reticulocyte counts of the males treated with 1000 mg/kg/day were significantly elevated when compared with the controls. However, when compared with the historical control data, the control values were lower and therefore the increased values were considered to be an artifact.
All other changes of statistical significance were noted in the low- or middle-dose groups and were without dose dependence. Therefore these differences were considered to be unrelated to the test item.
After the recovery period, the hematology parameters of the males compared favorably. In females previously treated with 1000 mg/kg/day, significantly lower mean corpuscular volume (p<0.05), the mean absolute eosinophil count (p<0.01) and mean absolute basophil count (p<0.05) were noted when compared with the respective control values, but all values remained within the ranges of the historical control values.

Clinical Biochemistry
There were no test item-related effects upon the clinical biochemistry parameters of males or females at any dose level.
After the treatment period, the mean sodium level was significantly elevated in males treated with 1000 mg/kg/day (p<0.05) when compared with the controls and marginally exceeded the upper range of the historical control data. No toxicological relevance was associated with this isolated difference.
All clinical biochemistry values recorded in the females treated with 1000 mg/kg/day were similar to those of the control females. All other changes of statistical significance were noted in the low- or middle-dose groups and were without dose dependence. Therefore these differences were considered to be unrelated to the test item.
After the recovery period, males which were previously treated with the test item at 1000 mg/kg/day showed significantly elevated glucose levels (p<0.05) and elevated triglycerides (p<0.01) which remained within the ranges of the respective historical control values. The mean phospholipid level was significantly elevated (p<0.01) and exceeded the upper limit of the historical control data, but in the absence of similar difference after the end of the treatment period, was considered to be of no toxicological relevance. In females, the mean phospholipid level was significantly reduced (p<0.05) when compared with control females. Significant reductions of creatine kinase (p<0.05), calcium (p<0.01), phosphorus (p<0.01) protein (p<0.05) and albumin (p<0.05) were noted, all of which remained within the range of the historical control data.

Urinalysis
There were no test item-related effects upon the urinalysis parameters of males or females at any dose level.

Pathology
Organ Weights
At the end of the treatment period, there were no statistically significant differences in the mean absolute and relative organ weights at any dose level. During the recovery period, the mean absolute adrenal weights and mean absolute uterus weights (including oviducts) were significantly elevated (p<0.05) in females previously treated with 1000 mg/kg/day. The mean heart-to-body weight ratio of these females was marginally, but significantly, reduced (p<0.05), whereas significantly higher organ-to-brain weights were noted for pituitary (p<0.05), thyroids (p<0.05), adrenals (p<0.05) and uterus/oviducts (p<0.01) when compared with the controls. Insofar as these findings were not evident at the end of the treatment period and were not accompanied by microscopical changes of morphology, all were considered to be unrelated to the treatment with the test item.

Macroscopic Findings
After the treatment period, a small number of macroscopical changes were noted in rats at all dose levels, with no clear dose dependent incidence.
In males, discoloration and/or inflated lungs were noted in single males at 100 and 1000 mg/kg/day. Inflated lungs were also recorded in two females at 100 mg/kg/day. Lung foci were recorded for one female at 1000 mg/kg/day.
Thymic foci and/or discoloration were noted in on control female, one female at 100 mg/kg/day, two females at 300 mg/kg/day and one male at 1000 mg/kg/day.
Enlargement and/or discoloration of the lymph nodes were noted in one male treated with 100 mg/kg/day and in one male treated with 1000 mg/kg/day.
Uterine dilation was recorded in one control female and single females at 300 mg/kg/day and 1000 mg/kg/day. Skin sores were recorded in one female at 300 mg/kg/day and in one male at 1000 mg/kg/day,
After the recovery period, one male previously treated with 1000 mg/kg/day had smaller testes and epididymides and a female had discoloration of the lungs and ovaries. These findings were not considered to be related to late effects of the test item.

Microscopic Findings
Lung, Trachea
In male number 14 (100 mg/kg/day) and in female number 53 (1000 mg/kg/day), fine granular yellowish foreign material was found to be present in alveolar macrophages. Similar foreign material was found in inflammatory (granulomatous) lesions of the lung of male number 25 (1000 mg/kg/day) and in the submucosal areas of the trachea of this animal.

Lymph Nodes
The above animals showed macroscopically enlarged and yellowish discolored bronchial and mediastinal lymph nodes. During microscopic examination lymphoid hyperplasia along with accumulated fine granular, yellowish foreign material was recorded.

Other Findings
The remaining findings recorded were within the range of normal background lesions which may be recorded in animals of this strain and age.
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No toxicologically relevant findings were noted up to the dose level of 1000 mg/kg bw/day, the highest dose level tested.
Critical effects observed:
not specified
Conclusions:
Based on the results of this OECD 407 subacute study, a no-observed-effect-level (NOEL) could not be established but the no-observed-adverse-effect-level (NOAEL) was considered to be 1000 mg/kg body weight/day.
Executive summary:

In this subacute toxicity study, the test material was administered daily by oral gavage to Wistar rats of both sexes at dose levels of 100, 300 and 1000 mg/kg body weight/day for a period of 28 days. A control group was treated similarly with the vehicle, bidistilled water, only. The groups comprised 5 animals per sex which were sacrificed after 28 days of treatment. Additional 5 rats per sex and group were used at 0 and 1000 mg/kg. These animals were treated for 28 days and then allowed a 14-day treatment-free recovery period after which they were sacrificed.

Clinical signs, outside cage observation, food consumption and body weights were recorded periodically during acclimatization, the treatment and recovery periods. Functional observational battery, locomotor activity and grip strength were performed during week 4. At the end of the dosing and the treatment-free recovery period, blood samples were withdrawn for hematology and plasma chemistry analyses. Urine samples were collected for urinalyses. All animals were killed, necropsied and examined post mortem. Histological examinations were performed on organs and tissues from all control and high dose animals, and all gross lesions from all animals.

There were no toxicologically relevant deaths. One high dose female died during anesthesia for blood sampling.

No test item-related adverse findings were noted in the daily clinical observations performed during the treatment period and no late effects were noted during the recovery period. At all dose levels, yellow staining of the feces was noted with dose-dependent onset; this was considered to

be a passive effect and is commonly noted following oral administration of a dyestuff. No findings were evident during the weekly behavioral observations performed at weeks 1, 2 or 3 of treatment.

No findings were evident during the functional observational battery performed at week 4 of treatment.

The mean fore- and hind limb grip strength values of the test item-treated males and females compared favorably with those of the respective control values.

The mean locomotor activity values of the test item-treated males and females were considered to be unaffected.

There were no test item-related changes in the mean daily food consumption at any dose level.

The mean absolute and relative body weights of the test item-treated rats compared favorably with their respective control values during the treatment period. Minor differences seen during the recovery period were considered to be of no toxicological relevance.

There were no test item-related effects upon the hematology paramters of males or females at any dose level.

There were no test item-related effects upon the clinical biochemistry parameters of males or females at any dose level.

There were no test item-related effects upon the urinalysis parameters of males or females at any dose level.

At the end of the treatment period, there were no statistically significant differences in the mean absolute and relative organ weights at any dose level. During the recovery period, the mean absolute adrenal weights and mean absolute uterus weights (including oviducts) were elevated in females previously treated with 1000 mg/kg/day. The mean heart-to-body weight ratio of these females was marginally reduced, whereas higher organ-tobrain weights were noted for pituitary, thyroids, adrenals and uterus/oviducts when compared with the controls. Insofar as these findings were not evident at the end of the treatment period and were not accompanied by microscopical changes of morphology, all were considered to be

unrelated to the treatment with the test item.

After the treatment period, a small number of macroscopical changes were noted in rats at all dose levels, with no clear dose dependent incidence.

Microscopical changes were noted in the in lungs, trachea, bronchial and mediastinal lymph nodes of two single animals, and were considered to be largely secondary changes that were not indicative of systemic toxicity.

The only test item-related, yet non-adverse findings were restricted to staining of the feces in rats at all dose levels, which was considered to be a typical passive effect following large oral doses of a dyestuff.

Based on the results of this study, a no-observed-effect-level (NOEL) could no be established but the no-observed-adverse-effect-level (NOAEL) was considered to be 1000 mg/kg body weight/day.

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

 

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

No Observed Adverse Effect Level (NOAEL):

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

Endpoint:
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 document in Cahpter 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.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
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 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, 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.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

No Observed Adverse Effect Level (NOAEL):

As there were no treatment-related adverse effects observed up to the highest dose the No Observed Adverse Effect Level (NOAEL)for systemic toxicityof the test item 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 without restriction

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
repeated dose toxicity: inhalation, other
Remarks:
28-Day Inhalation
Type of information:
experimental study
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
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)

(103cells/µL)

(106cells/µL)

(g/dL)

(%)

(fL)

(pg)

(g/dL)

(103cells/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)

(%)

(%)

(%)

(%)

(%)

(%)

(109cells/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)

(103cells/µL)

(103cells/µL)

(103cells/µL)

(103cells/µL)

(103cells/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)

(103cells/µL)

(106cells/µL)

(g/dL)

(%)

(fL)

(pg)

(g/dL)

(103cells/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)

(%)

(%)

(%)

(%)

(%)

(%)

(109cells/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)

(103cells/µL)

(103cells/µL)

(103cells/µL)

(103cells/µL)

(103cells/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)

(103cells/µL)

(%)

(%)

(%)

(%)

(%)

(103cells/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)

(103cells/µL)

(103cells/µL)

(103cells/µL)

(103cells/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)

(103cells/µL)

(%)

(%)

(%)

(%)

(%)

(103cells/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)

(103cells/µL)

(103cells/µL)

(103cells/µL)

(103cells/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

-

-

-