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Toxicological information

Repeated dose toxicity: inhalation

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Administrative data

Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
12/2019 - 06/2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 413 (90-Day (Subchronic) Inhalation Toxicity Study
Version / remarks:
adopted on 25 June 2018 with exception to particle size distribution (as test item is highly unstable and volatile in nature)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: vapour
Details on test material:
- Physical state: vapour
Specific details on test material used for the study:
Name of Test Item: Diethyl Ether [CAS No. 60-29-7]
Chemical Name (IUPAC): Diethyl Ether

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
Rat (Rattus norvegicus), Sprague Dawley as is the preferred laboratory rodent species for inhalation toxicity assessment and also recommended by the various regulatory guidelines. Animals were in-house bred animals at the age of 8 weeks at the time of randomization.
Sex:
male/female
Details on test animals and environmental conditions:
Animal Identification: During acclimatization period by cage cards and Tail marking by black permanent marker pen and during treatment period by cage cards and Body marking by turmeric solution and group marking by turmeric solution.
Husbandry
Environmental Conditions: Animals were housed under standard laboratory conditions in an environmentally monitored, air-conditioned room with adequate fresh air supply (12 to 15 air changes per hour), room temperature 19.7 °C to 23.1 °C and relative humidity 41% to 66%, with 12 hours fluorescent light and 12 hours dark cycle. The temperature and relative humidity were recorded once daily.
Housing: Two animals of same sex 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 water bottle. Clean sterilized corn cob was provided as bedding material.
Feed: Altromin Maintenance diet for rats and mice (manufactured by Altromin Spezialfutter GmbH & Co. KG) was provided ad libitum to the animals throughout the experimental period (except during restraining and exposure period). The contaminant analysis test report for the feed is included as an Annex to the original report.
Water: Water was provided ad libitum throughout the acclimatization and experimental period. Deep bore-well water passed through Reverse osmosis unit was provided in plastic water bottles with stainless steel sipper tubes (except during restraining and exposure period). The contaminant analysis test reports for the water and bedding material nearest to the experimental period are present s Annexes to the original report.
Acclimatization of Animals: Healthy young adult animals were acclimatized for seven days to laboratory conditions prior to treatment and were observed for clinical signs once daily and mortality twice daily. During the acclimatization period all the animals was restrained for one hour (±10 minutes) in the restraining tubes for two different days and observed for one hour (±10 minutes). The rectal temperature of all the animals was measured before and after restraining activity. Veterinary examination of all the animals were performed on the day of receipt and on day of randomization. Ophthalmoscopic examination of all the animals was performed on the day of grouping.
Grouping and Allocation: The animals taken (55 males and 55 females) for the experiment were weighed and arranged in ascending order of their body weights. These body weight stratified rats were distributed to all the experimental groups using Microsoft Excel Spread sheet, such that body weight variation of animals selected for the experiment did not exceed ±20% (+6.77% to -11.28% for males and +8.14% to -7.23% for females) of the mean body weight of each sex. Remaining 5 males and 5 females with extreme body weight range were excluded from the study during grouping. The grouping was done two days prior to the initiation of treatment. Body weight of the animals was analysed statistically for mean body weight to rule out any statistically significant difference between groups within each sex.

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure:
nose only
Vehicle:
air
Details on inhalation exposure:
The test item was administered through inhalation route by flow-past, nose-only dynamic inhalation exposure unit. The vapours generated by the Volatile Organic Compound (VOC) generator was passed through the pipeline into the inhalation chamber. The inhalation chamber was maintaining positive pressure at the inlet and negative pressure at the exposure port. The generated vapour was mixed with dilution air and air was passed through the inlet of the inhalation chamber with high pressure and where in turn the same air was passed inside the chamber of animal’s breathing ports (at a low pressure).
Analytical verification of doses or concentrations:
no
Remarks:
Doses were verified by gravimetric measures
Details on analytical verification of doses or concentrations:
Exposure System Monitoring: The chamber temperature, relative humidity, oxygen and carbon dioxide concentration was measured during exposure at an empty port of the exposure chamber. The air flow meter (rotameter) was used to regulate and measure, inlet and outlet air of the chamber.
Determination of Nominal Concentration: The nominal concentration was calculated weekly according to “mass of test item disseminated into the exposure system during the generation period divided by the total airflow through the inhalation chamber during the same time period”. It was determined as follows: Nominal Concentration (mg/L) = Total amount of test item used (during 6 hour exposure) / Air flow rate (L/minute) × Time (minute)
The nominal concentration for air only (G1 and G1R) and test item exposure groups (G2, G3, G4 and G4R) were as follows:
Group G1 & G1R received air only, G2 received 2.23 – 2.46 mg/L, G3 received 4.46 - 4.92 mg/L and G4 & G4R received 6.67 – 7.39 mg/L (all nominal)
Determination of Breathing Zone Concentration in ppm (Actual Concentration): Actual aerosol concentration was determined in ppm by using Gray Wolf Sensing Solution probe, supplied by CH Technologies, USA. The 0.84 L/min critical orifice was used to draw the test item from the chamber through the Gray Wolf Sensing Solution probe from the inhalation chamber for one minute at animal breathing zone. It was carried out at least four times during exposure, initially before keeping the animals into the chamber and two hours once [i.e. 120 minutes (±15 minutes), 240 minutes (±15 minutes) and 360 minutes (±15 minutes)] after equilibration period for each exposure day.
Actual Concentration (ppm) = Test item in ppm detected by Gray Wolf Sensing Solution probe / Volume of air passed through the Sensing Solution probe * Time (minutes)
Determination of Particle Size Distribution: The particle size distribution was not measured because the test item was liquid and highly volatile in nature.
Relative Humidity, Temperature, Oxygen and Carbon Dioxide Concentration: The Indoor Air Quality probe (IAQ probe) supplied by GrayWolf Sensing Solutions, was used to monitor continuous monitoring of relative humidity, temperature, oxygen and carbon dioxide content in the chamber and recorded four times for each group during exposure (±20 minutes from breathing zone concentration) after equilibration period during each exposure day. Relative humidity, temperature, oxygen and carbon dioxide content were maintained 30% to 70%, 22 ±3 °C, at least 19% and less than 1%, respectively.
Exposure Air Flow Rate: Air flow through the generation system into the chamber was controlled and monitored through control panel (supplied by CH Technologies, USA) and outlet of the chamber air flow was controlled by rotameter. The actual air flow rate of chamber inlet and outlet was 20 L/min and 10 L/min, respectively. The air inlet flow rate was recorded during each exposure day (i.e ±20 minutes from BZC sampling).
Treatment of Exhaust Air: Exhaust air was treated with 1% w/v sodium hydroxide solution (manufactured by: SD Fine Chem Limited, Batch No.: L18A/1018/1711/08, Expiry date: 11/2023) with coolant and passed through absorbent cotton before evacuated into the atmosphere.
Duration of treatment / exposure:
The exposure period was 90 days. Following the exposure period, the animals in the recovery groups (G1R and G4R) were not exposed to the test item, maintained for a 28 days post treatment period and observed for reversibility or persistence of toxic effects.
Frequency of treatment:
Animals were exposed to the test item by vapour inhalation continuously for 6 hours per day, 5 days per week, for a total of 65 actual exposures, after equilibration period of the chamber concentration.
Doses / concentrationsopen allclose all
Dose / conc.:
0 ppm (nominal)
Remarks:
G1 control group (air only); males Re2081 - Re2090; females Re2121 - Re2130 and G1R Recovery control group; males Re2161 - Re2165; females Re2171 - Re2175
Dose / conc.:
500 ppm (nominal)
Remarks:
G2 Low dose group; males Re2091 - Re2100, females Re2131 - Re2140
Dose / conc.:
1 000 ppm (nominal)
Remarks:
G3 Mid dose group; males Re2101 - Re2110; females Re2141 - Re2150
Dose / conc.:
1 500 ppm (nominal)
Remarks:
G4 High dose group; males Re2111 - Re2120; females Re2151 - Re2160 and G4R High dose recovery group; males Re2166 - Re2170; females Re2176 - Re2180
No. of animals per sex per dose:
G1, G2, G3 and G4 groups consisted of 10 males and 10 females each; the recovery groups G1R and G4R consisted of 5 males and 5 females each.
Control animals:
yes
Details on study design:
Technical Pre-Test: A technical pre-test, without animals was conducted to assess and establish the feasibility of maintaining the recommended test atmosphere and stability as well as achieving the target concentration in ppm. During the technical pre-test, target concentration of test item was achieved by using Volatile Organic Compound generator. The individual achieved concentration varied within ±10% of mean concentration. The data generated during technical pre-test was not included in the study report however maintained with the study raw data file.
Dose Selection and Justification: The dose levels of 500 (G2), 1000 (G3) and 1500 (G4/G4R) ppm were considered for low, mid and high dose groups based on available literature and 14-day dose range finding study (Bioneeds Study No.: BIO-ITX 059). The legal air born permissible exposure limit is 400 ppm averaged over an 8 hour work shift as per OSHA (Occupational Safety and Health Administration). In BIO-ITX 059, the dose levels of 2000, 4000 and 6000/7000 ppm for 6 hours respectively were administered for 5 days a week for two weeks. In the high dose (6000/7000 ppm), during exposure, all animals exhibited slow response to tail pinch from Day 3 onwards. Soft stool was noted in few animals intermittently from Day 3 onwards till Day 10. Post exposure, all animals were normal throughout the experimental period. Statistically significant decrease in relative neutropils in G2 and G4 females; increase in relative lymphocytes in G2 and G4 females; decrease in relative monocytes in G4 females; decrease in absolute monocytes in G4 females; decrease in absolute eosinophil in G2 and G3 females was noted. Hence, based on the chamber validation, stability of test item concentration and 14-day dose range finding study, dose levels of 500 (G2), 1000 (G3) and 1500 (G4/G4R) ppm were selected as low, mid and high doses in the study. In addition, air only groups (G1/G1R) were included which were administered air alone.
Inhalation Exposure System: Inhalation exposure was conducted using a flow-past, nose-only dynamic inhalation exposure system supplied by CH Technologies, USA, with a provision of at least 12 air changes per hour. The exposure unit consists of stackable exposure tiers plus top and bottom sections or plates for introduction and exhaust of test item. Each tier has 12 exposure ports which were used for exposing up to 10 animals and sampling of test atmosphere. All parts (except o-ring seals) are constructed of stainless steel. The volume of each inner plenum of inhalation chamber is 0.76 liters (11 cm diameter and 8 cm height) that consisted of total 12 port holes (one tier) and it was increased based on the requirement. Each tier is made of an inner plenum and an outer plenum that are connected to each other through rectangular trumpets (tubes) and connector cones. The chamber is equipped with a bottom inlet section (plate) and a top exhaust section. Both sections are designed to connect easily to other equipment through swivels. The animals were restrained in the transparent polycarbonate restraining tubes. The chamber was made to sit on a rotating table that allowed easy access to observe all animals at each port during exposure. This unit ensured uniform test item distribution, provides a continuous “fresh” test item to each animal and precludes re-breathing of the exhaled air. The chamber provided continuous supply of fresh aerosol to all the restrained animals through the trumpet from inner plenum of chamber. A slight positive pressure was maintained in inner plenum of chamber. The animals were confined separately in restraint tubes which were positioned radially around the flow-past, nose-only dynamic inhalation exposure chamber. The outlet air from chamber exhaust was treated with 1% w/v sodium hydroxide solution and passed through absorbent cotton before being evacuated in the atmosphere.
Vapour Generation was performed by a VOC Generator, Supplied by CH Technologies, USA (model CSSCPC11). It generates the vapours with the help of control panel. The control panel controls the vapours based on the carrier air and dilution air. The carrier air 10 L/min and dilution air 10 L/min was maintained throughout the study. Liquid test item was passed by using syringe pump through the stainless-steel column. The stainless-steel column was filled with borosilicate glass beads, the beads to increase the surface area for vapourization. The generated vapour was mixed with the dilution air which was passed into the bottom of the inhalation chamber through the connected pipeline. VOC liquid reservoir temperature rises; this rise in temperature makes the percolating liquid to evaporate around the surface of the glass beads. An automatically controlled heating tape wrapped around the stainless-steel column provides the necessary heat to evaporate the liquid.

Examinations

Observations and examinations performed and frequency:
The following observations were done during the experimental period.
Clinical Signs and Mortality: All animals were observed for clinical signs before exposure and 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 at 30 to 40 minutes and 1.5 hour (±10 mins) following exposure to the test item and thereafter once a day for clinical signs and twice daily for mortality.
Body Weight: Individual animal body weights were recorded at receipt, prior to exposure (Day 1) and twice weekly thereafter for the first 4 weeks. As no significant body weight effects in the first 4 weeks were observed body weights were recorded weekly thereafter (post exposure period) for the remaining period of the study. Fasting body weight of all animals was recorded at their scheduled terminal sacrifice.
Feed Consumption: Individual animal feed consumption was recorded weekly, coinciding with the body weights of the respective animals. Feed intake per rat (g/rat/day) was calculated using the amount of feed given and left over in each cage and the number of rats in each cage.
Neurological/Functional Examination: Neurological/Functional examination was carried out during week 13 for control and high dose main group animals (G1 and G4) and during week 17 for all recovery group animals (G1R and G4R).
The following Neurological/Functional observations were performed:
- Home Cage Observations: Animals in the cage were observed for convulsions, tremors and palpebral closure and the scores were recorded.
- Handling Observations: The animals were observed for the ease of removal from the cage and ease of handling of animals in hand. During removal from the cage, the animals were observed for the ease to remove out of cage. During handling observation, the animals were observed for the resistance, lacrimation, red and crusty deposits around eyes, nose and mouth.
- Open Field Observations: Animals were placed in an open field arena and observed for their mobility, gait, arousal, rearing, urination, defecation, stereotypes and excessive grooming.
- Sensory Observations: Animals were observed for startle response, touch response, pupil response, response to nociceptive stimuli and righting reflexes.
- Neuromuscular Observations: As a part of neuromuscular observations, the animals were dropped on to the paper from a height approximately 30 cm with painted paws to record the Hind Limb Foot Splay.
- Physiological Observation (Rectal temperature): Physiological temperature of the animals was recorded using a calibrated digital thermometer.
- Grip Strength: Animals were evaluated for their hind and fore limb grip strength.
- Locomotor Activity: Each animal was kept in activity cage for ten minutes and the activity readings were recorded.
- Ophthalmoscopic Examination: Ophthalmological examination was performed during week 13 for vehicle control and high dose main group animals (G1 and G4) and during week 17 for recovery group animals (G1R and G4R). As no treatment related ocular changes were observed in high dose group animals, the examination was not extended to low and mid dose group (G2 and G3) animals.
- Clinical Pathology: Blood samples were collected from all the surviving rats on experimental day 90 (within 24 hours after the last given dose) and 120 for main and recovery groups respectively. The animals were fasted overnight (approximately at least 16 to 18 hours) before blood collection. Water was provided ad libitum during fasting period. Blood samples were collected from the animals separately into the tubes containing K2 EDTA and sodium heparin tubes for hematology and clinical chemistry respectively, Sodium citrate tubes for Prothrombin time (PT) and activated partial thromboplastin time (APTT) parameters. Blood samples were collected from retro-orbital plexus puncture under mild isoflurane anesthesia with the help of a fine capillary tube.
- Haematology: The following Haematology parameters were estimated using Advia 2120 Hematology system (Siemens Limited): Hemoglobin concentration (HGB) [g/dL], Hematocrit (HCT) [%], Erythrocyte count (RBC) [10⁶ cells/µL], Total leukocyte count (WBC) [10³ cells/µL], Mean corpuscular volume (MCV) [fL], Mean corpuscular hemoglobin (MCH) [pg], Mean corpuscular hemoglobin concentration (MCHC) [g/dL], Platelet count (PLT) [10³ cells/µL], Mean platelet volume (MPV) [fL], Absolute reticulocyte count [10⁹ cells/L], Reticulocyte count [%], Differential leukocyte count (DLC) [%], Absolute differential leucocytes count (DLC) [10³ cells/µL], Prothrombin Time (PT) and Activated Partial Thromboplastin Time (APTT) was estimated by coagulation analyzer [Tulip diagnostics (p) Ltd., India].
- Clinical Chemistry: The following clinical chemistry parameters were analysed using the “Rx Daytona+ Clinical Chemistry Analyzer” (Randox Laboratories): Alanine aminotransferase (ALT) [U/L], Aspartate aminotransferase (AST) [U/L], Alkaline phosphatase (ALP) [U/L], Albumin [g/dL], Urea [mg/dL], Blood Urea Nitrogen (Calculated) [mg/dL], Calcium [mg/dL], Creatinine [mg/dL], Total protein [g/dL], Total bilirubin [mg/dL], Glucose [mg/dL], Total cholesterol [mg/dL], Triglycerides [mg/dL], Phosphorous [mg/dL], Globulin (calculated), [g/dL]. Plasma Sodium [mmol/L], Potassium [mmol/L] and Chloride [mmol/L] was estimated using Electrolyte analyzer (Medica Corporation/Diamond Diagnostics). The plasma was separated by centrifuging the blood samples at 5000 rpm for 10 minutes for determining the clinical chemistry and at 1500 rpm for 15 minutes for determining the PT and APTT parameters.
- Urinalysis: Urine was collected from all rats of each sex/group of main groups and recovery groups on experimental day 89 and 120, respectively. The animals were kept in urine collection cages for overnight and fasted but water was provided ad libitum during this period. The overnight urine volume (mL) collected from these animals was measured. Urine volume (mL), appearance and the color was examined by naked eye. The urine was analyzed using “Urine analyzer” (Dirui Industrial Company Ltd.) for the following parameters: Blood [Ery/µL], Bilirubin [mg/dL], Urobilinogen [mg/dL], Ketones [mg/dL], Protein [mg/dL], Glucose [mg/dL], Leucocytes [Leu/µL], Microalbumin [mg/dL]. In addition, nitrite, pH and specific gravity was analyzed. After analysis of the above parameters, the urine was subjected for centrifugation at 1500 rpm for 3 minutes. The urine was subjected for microscopic examination for urine sediments.
Sacrifice and pathology:
- Gross Necropsy: On day 90 and day 120, all the animals of main and recovery groups, respectively, were subjected to necropsy and detailed gross pathological examination was carried out. The animals were fasted overnight (water was provided ad libitum) prior to scheduled necropsy. On the day of terminal sacrifice, the body weight of all the fasted animals were recorded prior to exsanguination. The animals were euthanized by intraperitoneal administration of sodium thiopentone followed by exsanguination (lethal dose). The animals were sacrificed within 24 hours after the end of exposure period. Necropsy included an examination of external surfaces, external orifices, abdominal, thoracic and cranial cavities, organs and tissues.
- Tissue Collection: Organs and tissues were collected, trimmed off any adherent tissue, as appropriate. All the tissues mentioned in the table below, from all animals, preserved in 10% v/v Neutral Buffered Formalin and modified Davidson’s fixative for testes.
- Organ Weights: The following organs from all animals at the scheduled sacrifices were trimmed off any adherent tissue and fat, as appropriate and weighed wet as soon as possible to avoid drying. The following organs were weighed: 1. Kidneys*, 2. Adrenals*, 3. Spleen, 4. Heart, 5. Liver, 6. Brain, 7. Lungs (Left), 8. Testes*/Ovaries*, 9. Thymus, 10.Epididymides/Uterus, 11. Thyroid (*: Paired organs were weighed together). Organ weight ratio was calculated against fasting body weight and absolute brain weight.
- Bone Marrow Smear Examination: Bone marrow smear (from one femur) was prepared for all the animals at the time of necropsy, fixed in methanol and stained with Giemsa stain. As treatment related changes were not noticed in haematology parameters and no microscopic changes in tissues/organs such as thymus, spleen and lymph node; bone marrow smears were not evaluated.
- Histopathological Examination: Histopathological examination was conducted on all the tissues from the concurrent air control (G1) and high dose (G4) group animals. All organs and tissue samples as defined in the study plan were processed, embedded in paraffin, cut at a thickness of 4 to 6 micrometers and stained with hematoxylin and eosin. Special attention was paid to respiratory tract histopathology examination. Unlavaged lung lobe (left lobe) was sectioned at three levels, nasopharyngeal tissues sectioned at four levels which include the nasopharyngeal duct, for examination of the squamous, transitional (non-ciliated respiratory), respiratory (ciliated respiratory) and olfactory epithelium, and the draining lymphatic tissue. Three levels of the larynx were examined and one of these levels included the base of the epiglottis. Two levels of the trachea were examined including one longitudinal section through the carina of the bifurcation of the extra-pulmonary bronchi and one transverse section.
- Bronchoalveolar Lavage Fluid (BALF): Broncho alveolar lavage fluid (BALF) from the right lung of all rats was analyzed for total protein, lactate dehydrogenase (LDH), total cells and differential cells. BALF was collected within 24 hours after the last given dose.
Statistics:
The raw data was subjected to statistical analysis. The computer printout of the data (in the form of appendix) were verified with the original raw data. After verification, the data was 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 were done for different treatment groups comparing with the control group data. All analysis 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).

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
There were no treatment related clinical signs noted during exposure in main and recovery groups. Post exposure, all animals were normal throughout the experimental period. No clinical signs were noted during recovery period. For details refer to Table 1.
Mortality:
no mortality observed
Description (incidence):
There was no mortality/morbidity noted during exposure in main and recovery groups. Post exposure, all animals were normal throughout the experimental period. No clinical signs were noted during recovery period. For details refer to Table 1.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment related variations were noted in body weights at any of the doses tested in either sex. However, statistically significant higher percent change in body weight with respect to day 1 was noted in G3M (during days 1 - 15, 1 - 26, 1 - 29 and 1 - 36), G2F and G3F (during days 1 - 26, 1 - 29 and 1 - 36) was noted. In G4F, statistically significant lower percent change in body weight with respect to day 1 (during days 1 - 5) was noted. The observed variations are considered incidental as the mean body weights were unaffected, and also the magnitude of variations were minimal. For details refer to Tables 2 & 3.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
No treatment related changes in feed consumption was observed; however, statistically significant decrease in feed consumption was noted in G4F (week 1) and G4RM (week 1); increase in feed consumption was noted in G2F (week 3). The noted variations in feed consumption without any concomitant variation in mean body weight is considered incidental. For details refer to Table 4.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
No ocular changes were observed during ophthalmoscopic examination. For details refer to Table 9.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No toxicologically significant treatment related changes were observed in haematology parameters when compared to vehicle control group. However, the following statistically significant (p<0.05) variations were noted.
In main group males, increase in MPV (G2) and decrease in PT (G2, G3 and G4) was noted. In main group females, increase in WBC (G3), MPV (G4), absolute and % monocytes (G3F) was noted. Decrease in PT is not of toxicologically significance and all other variations are considered incidental in the absence of dose relationship.
In recovery group males decrease in RBC and Platelet (G4R); increase in WBC was noted.
There were no comparable significant changes in related parameters like HGB, HCT retic with respect to RBC and MPV with respect to Platelets. Similarly, there were no corresponding significant (p<0.05) increase in absolute differential counts of leucocytes. Moreover, all these values fall within the in-house historical control data [RBC (10⁶ cells/µL): 6.90 - 8.95, PLT (10³ cells/µL): 647 - 974, WBC (10³ cells/µL): 6.60 – 15.31]. For details refer to Table 10.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
No toxicologically significant treatment related changes were observed in clinical chemistry parameters when compared to vehicle control group. However, the following statistically significant (p<0.05) variations were noted.
In main group males, total protein was decreased in G3 and G4. In main group females, percent monocytes and total protein was increased in G3; LDH was increased in G2 and G3 groups.
In the absence of microscopic and macroscopic changes in liver and kidneys, decrease in total protein in G3 and G4 males is considered incidental. The variations noted in main group females is considered incidental in the absence of dose responsiveness. For details refer to Table 11.
No toxicologically significant treatment related changes were observed in clinical chemistry parameters when compared to vehicle control group. However, the following statistically significant (p<0.05) variations were noted (↓: Decrease; ↑: Increase; HD: In-house (Bioneeds) Historical Data):
Main Group
GLU (mg/dL): G4 M ↓  Mild decrease within HD range from 74 to 126, considered incidental
Urea (mg/dL): G3M↑ & G4 M ↑  No dose relationship. No histopathological change in kidneys at high dose, considered incidental
BUN (mg/dL):G3M ↑ & G4 M ↑  No dose relationship. No histopathological change in kidneys at high dose, considered incidental
PHO (mg/dL): G3M ↓ & G4 M ↑  Not a consistent and dose dependent change, considered incidental
PHO (mg/dL): G3 F ↑  No dose dependency; within HD range from 3.3 to 5.8, considered incidental
K (mmol/L):G3M ↑  Mild increase. Within HD range from 3.05 to 5.17, considered incidental
Na (mmol/L): G3 F ↓  Minimal decrease. No dose dependency, considered incidental
CLO (mmol/L): G3 F ↓ & G4 F ↓  Minimal decrease. No dose dependency. Within HD range from103.1 to 113.7, considered incidental
Recovery group
GLU (mg/dL): G4R M ↓  Mild decrease. Appeared artefactual. Could be due to slight increase in control values in recovery group. Not of biological significance, considered incidental.
CAL (mg/dL): G4R M ↑  Minimal increase. Within HD range from 8.2 to 10.6, considered incidental
K (mmol/L): G4R M ↑  Mild increase Within HD range from 3.05 to 5.17, considered incidental
For details refer to Table 12.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no toxicologically significant variations noted in urinalysis, however, statistically significant (P<0.05) changes in pH (decrease) and specific gravity (increase) in G3 females were observed. The observed variations noted were considered incidental because of their isolated occurrences without any dose responsiveness.
Statistically significant (P<0.05) change in pH in G4R females (increase) was considered incidental as the change was minimal and since all other parameters were comparable to concurrent control values, this was considered an isolated case and hence not of toxicological significance; furthermore, as it was not observed in G4R males. For details refer to Table 13.
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
No adverse treatment related neurological/functional examination changes were noted. However, following statistically significant variations were noted such as decreased rearing (G4M), increased movement counts (G4M) and increased body temperature (G4F).
Decrease in rearing in G4M is considered incidental and not of toxicological significance. Increase in movement counts (G4M: 7%) and body temperature (G4F: 1.3%) is minimal when compared to respective control and hence not considered toxicologically significant and is considered incidental. In the absence of changes in all other neuromuscular parameters, the noted changes are considered incidental. For details refer to Table 5, 6, 7 & 8.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
The following statistically significant (p<0.05) changes were noted in organ weight and its ratios (relative to fasting body weight) when compared to vehicle control group.
In main group males, increase in absolute weight of liver and kidneys in G3 and brain weight in G2 and decrease in spleen weight in G2; increase in relative weight of heart, kidneys and liver in G3 and decrease in relative spleen weight in G2 were noted.
In main group females, increase in absolute weight of adrenals, kidneys and liver in G2 & G3 females and G2 thyroid and parathyroid; increase in relative weight of adrenals, kidneys and liver in G2 & G3 was noted.
In recovery males, decrease in absolute lungs weight (G4R) and females increase in spleen (G4R); increase in relative weight of spleen (G4R) was noted.
In main group when compared with brain weight, in males, increase in relative weight of kidneys, liver (G3M) and decrease in spleen weight in G2M was noted. In female, increase in relative weight of kidneys, liver and adrenals in G2 & G3 was noted.
In recovery group when compared with brain weight, increase in relative weight of lungs (G4RM) in males and spleen weight in females was noted.
All the variations noted in the main groups and recovery groups were not dose dependent also not associated with any macroscopic findings and hence considered incidental. For details refer to Tables 14, 15 & 16.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no gross pathological changes observed at any of the tested doses in either sex during gross pathological examination. Details are summarized in a Pathology Report.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related histopathological findings in the study. Few microscopic findings observed in this study such as ultimobranchial cyst in thyroid gland, accessory cortical tissue in adrenal glands, and all other findings were considered incidental as they occurred randomly across the dose groups including concurrent controls and/or were expected for laboratory rats of this age.
Histopathological findings: neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
See above!
Other effects:
not specified
Details on results:
The data recorded for all exposure days relating to the chamber conditions like temperature, relative humidity, oxygen concentration and carbon dioxide concentration determined during the exposure period are were found within the specified range. For details refer to Table 17 and 18.
Under the experimental conditions and doses employed, it is concluded that the No Observed Adverse Effect Concentration (NOAEC) of test item, Diethyl Ether [CAS No. 60-29-7] was found to be 1500 ppm when exposed for 6 hours for 5 days/week for 13 weeks by flow-past nose-only inhalation route to Sprague Dawley rats.

Effect levels

Dose descriptor:
NOAEC
Effect level:
1 500 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: absence of adverse effects
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity

Critical effects observed:
no

Any other information on results incl. tables

Table no. 1: SUMMARY OF CLINICAL SIGNS AND MORTALITY RECORD

Group, Sex & Dose (ppm)

No. of Animals

Clinical Signs of Toxicity

Mortality
(No. of Incidences/No. of Animals)

G1, M & 0

10

N

0/10

G2, M & 500

10

N

0/10

G3, M & 1000

10

N

0/10

G4, M & 1500

10

N

0/10

G1R, M & 0

5

N

0/5

G4R, M & 1500

5

N

0/5

G1, F & 0

10

N

0/10

G2, F & 500

10

N

0/10

G3, F & 1000

10

N

0/10

G4, F & 1500

10

N

0/10

G1R, F & 0

5

N

0/5

G4R, F & 1500

5

N

0/5

 M: Male; F: Female; N: Normal; R: Recovery

 

Table no. 2: SUMMARY OF BODY WEIGHTS (g) RECORD

Group, Sex & Dose (ppm)

Body Weight (g) on Days

1

5

8

12

15

19

22

26

29

36

43

50

57

64

71

78

85

89

G1, M & 0

Mean

182.2

177.72

200.79

200.35

220.21

223.84

245.78

242.42

264.24

283.91

302.42

318.6

333.64

341.78

350.98

358.22

366.64

365.81

±SD

9.66

10.02

10.16

11.14

11.88

13.02

13.65

14.52

15.74

17.04

20.28

21.09

23.36

23.59

25.19

27

27.22

25.9

n

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

G2, M & 500

Mean

177.37

172.83

194.04

195.06

216.38

222.66

247.46

250.09

274.55

296.64

314.94

331.28

342.86

352

359.26

367.04

376.54

377.02

±SD

11.4

12.25

13.27

11.65

10.32

10.49

11.2

13.86

15.23

11.48

11.97

11.52

12.41

11.9

12.23

13.31

13.88

13.97

n

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

G3, M & 1000

Mean

175.33

170.93

193.03

195.07

220.16

226.98

250.65

256.69

277.91

297.46

314.85

328.87

340.2

347.64

355.07

362.73

372.31

372.29

±SD

10.65

10

8.67

8.86

8.93

9.93

9.34

8.4

11.74

10.22

11.51

13.02

13.8

15.54

15.81

15.14

17.46

17.82

n

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

G4, M & 1500

Mean

186.48

184.13

204.32

205.48

226.86

230.29

251.94

253.2

278.5

300.38

320.73

340.1

349.23

355.33

363.24

373.35

382.81

384.22

±SD

12.72

11.63

11.21

12.87

13.32

17.38

18.58

24.41

26.69

28.38

29.55

31.14

31.07

33.24

35.26

36.63

36.83

35.42

n

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

G1, F & 0

Mean

160

156.32

168.62

168.86

181.76

180.29

192.26

189.67

201.31

210.13

222.25

228.95

236.22

241.47

245.99

250.96

254.24

253.83

±SD

9.36

8.81

6.79

9.38

9.42

12.4

14.07

15.88

16.13

15.31

17.09

17.99

19.06

19.71

19.62

19.27

19.68

19.24

n

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

G2, F & 500

Mean

156.61

152.53

163.83

167.08

180.86

183.66

189.1

197.87

208.92

219.05

227.93

235.79

242.99

249.37

254.82

258.66

262.48

262.59

±SD

7.72

7.86

7.33

8.03

8.1

9.18

30.88

14.92

15.96

17.99

18.81

22.62

25.06

28.37

29.9

30.33

29.83

29.56

n

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

G3, F & 1000

Mean

153.13

149.95

161.74

164.94

179.43

180.16

191.27

192.73

205.36

214.16

223.62

229.96

236.91

243.66

248.25

254.03

259.73

259.89

±SD

6.22

6.22

6.26

7.7

9.26

11.13

10.68

10.94

10.5

7.8

10.05

11.17

12.43

14.13

14.27

15.11

14.87

15.22

n

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

G4, F & 1500

Mean

161.95

156.68

168.84

169.45

181.37

177.13

190.95

189.68

202.6

211.37

219.73

224.31

230.49

236.94

242.17

247.82

252.08

252.28

±SD

10.36

9.75

9.83

9.12

8.2

9.73

9.22

8.86

10.34

11.6

11.78

11.76

11.86

12.04

12.3

12.28

12.2

12.87

n

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

Recovery Group, Sex & Dose (ppm)

Body Weight (g) on Days

1

5

8

12

15

19

22

26

29

36

43

50

57

64

71

78

85

92

99

106

113

119

G1R, M & 0

Mean

189.11

186.08

207.86

207.37

228.54

233.58

258.59

265.92

289.97

313.04

332.99

349.66

362.71

372.67

380

389.06

398.76

406.29

417.82

433.09

446.65

458.71

±SD

10.55

10.66

10.02

9.07

9.83

12.07

13.79

13.69

12.87

15.94

12.74

14.79

15.32

17.79

19.75

21.96

23.06

21.53

20.14

21.92

19.94

18.3

n

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

G4R, M & 1500

Mean

188.32

185.22

209.26

207.29

227.7

229.98

254.52

261.57

282.08

299.98

319.02

333.91

349.84

360.1

369.97

381.4

391.09

397.79

409.97

423.56

437.89

449.48

±SD

10.87

11.29

13.19

14.09

11.83

13.4

13.7

14.05

12.19

11.6

14.91

16.93

16.27

18.2

18.82

20.23

17.23

16.43

14.8

14.14

14.5

14.57

n

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

G1R, F & 0

Mean

179.86

175.39

186.25

182.86

193.09

186.83

197.6

195.37

206.07

216.41

224.79

231.85

240.56

245.38

250.2

255.09

258.44

262.67

272.47

279.31

287.92

294.05

±SD

11.69

10.87

10.44

10.35

10.23

9.9

8.81

7.53

6.49

5.28

8.01

10.34

10.22

10.26

10.85

12.1

11.56

11.95

11.62

10.84

10.19

10.88

n

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

G4R, F & 1500

Mean

178.38

173.55

185.26

183.14

193.57

189.67

201.21

199.24

209.53

219.55

226.75

232.47

239.74

243.87

249.36

255

259.23

263.31

272.17

278.09

285.57

291.53

±SD

12.53

12.79

11.29

14.44

16.04

18.61

20.53

17.98

16.77

17.08

16.63

18.66

17.18

17.2

15.44

16.84

16.06

15.79

15.53

14.38

15.5

14.39

n

5

5

5

5

5

5

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 no. 3: SUMMARY OF PERCENT CHANGE IN BODY WEIGHT WITH RESPECT TO DAY 1 RECORD

Group, Sex & Dose (ppm)

Percent Change in Body Weight (%) during Days

1 - 5

1 - 8

1 - 12

1 - 15

1 - 19

1 - 22

1 - 26

1 - 29

1 - 36

1 - 43

1 - 50

1 - 57

1 - 64

1 - 71

1 - 78

1 - 85

1 - 89

G1, M & 0

Mean

-2.47

10.22

9.97

20.9

22.93

35.03

33.22

45.22

56.01

66.17

75.07

83.36

87.87

92.9

96.92

101.57

101.17

±SD

0.96

0.82

2.3

3.48

5.15

6.53

8.06

8.77

8.91

10.56

10.87

12.6

13.17

13.64

15.15

15.55

15.86

n

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

G2, M & 500

Mean

-2.6

9.37

10.03

22.15

25.85

39.94

41.61

55.59

68.09

78.51

87.75

94.33

99.5

103.61

107.99

113.39

113.66

±SD

0.78

1

2.33

3.6

7.32

9.21

12.77

15.68

15.47

17.18

17.53

18.52

18.75

19.05

19.25

20.01

20.19

n

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

G3, M & 1000

Mean

-2.5

10.19

11.39

25.74*

29.7

43.24

46.74*

58.83*

70.02*

79.96

87.95

94.42

98.61

102.88

107.33

112.82

112.8

±SD

0.55

2.4

3.69

4.24

6.34

6.42

7.48

8.17

8.26

9

9.1

9.63

9.03

9.75

11.19

12.65

12.77

n

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

G4, M & 1500

Mean

-1.21

9.68

10.27

21.78

23.6

35.3

35.83

49.4

61.18

72.13

82.53

87.47

90.73

94.97

100.4

105.51

106.3

±SD

2.02

2.66

3.3

4.09

6.97

8.71

10.37

11.21

12.39

13.15

13.99

14.32

15.4

16.57

17.25

17.66

17.16

n

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

G1, F & 0

Mean

-2.28

5.5

5.58

13.68

12.71

20.17

18.53

25.83

31.41

38.91

43.08

47.61

50.9

53.72

56.83

58.88

58.63

±SD

0.68

2.29

2.51

3.17

4.89

5.42

6.79

7.21

7.4

6.9

7.21

7.65

8.29

7.91

7.27

7.7

7.37

n

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

G2, F & 500

Mean

-2.62

4.63

6.7

15.53

17.33

20.52

26.39*

33.44*

39.91*

45.58

50.58

55.18

59.23

62.76

65.21

67.65

67.72

±SD

0.63

0.83

1.75

2.43

4.1

17.94

8.15

8.71

10

10.52

12.81

14.47

16.4

17.98

18.18

17.84

17.62

n

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

G3, F & 1000

Mean

-2.08

5.64

7.72

17.16

17.63

24.88

25.87*

34.17*

39.94*

46.1

50.24

54.79

59.19

62.2

65.97

69.71

69.81

±SD

0.63

1.31

2.97

3.5

4.77

4.06

5.54

6.11

4.67

5.57

6.59

7.58

8.57

8.84

9.35

9.29

9.47

n

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

G4, F & 1500

Mean

-3.24*

4.3

4.72

12.16

9.55

18.15

17.31

25.27

30.66

35.88

38.74

42.56

46.53

49.75

53.23

55.88

55.98

±SD

0.47

1.31

2.77

3.84

5.25

6.05

4.74

4.64

4.4

6.11

6.57

6.68

6.29

5.98

5.43

5.89

5.88

n

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

Recovery Group, Sex & Dose (ppm)

Body Weight (g) on Days

1 - 5

1 - 8

1 - 12

1 - 15

1 - 19

1 - 22

1 - 26

1 - 29

1 - 36

1 - 43

1 - 50

1 - 57

1 - 64

1 - 71

1 - 78

1 - 85

1 - 92

1 - 99

1 - 106

1 - 113

1 - 119

G1R, M & 0

Mean

-1.61

9.96

9.74

20.95

23.59

36.82

40.71

53.46

65.63

76.31

85.08

91.99

97.19

101.02

105.79

110.93

114.96

121.09

129.17

136.39

142.79

±SD

0.57

1.28

2.71

3.35

3.97

4.65

4.89

4.81

5.42

7.22

6.23

6.56

5.41

4.82

5.56

6.98

7.06

6.9

7.9

7.6

7.15

n

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

G4R, M & 1500

Mean

-1.66

11.09

10.03

20.95

22.15

35.21

38.95

49.93

59.48

69.6

77.52

86

91.49

96.72

102.83

108.05

111.65

118.18

125.42

133.05

139.21

±SD

0.62

0.84

1.59

0.73

2.76

3.41

3.22

4.41

5.26

6.87

7.87

8.09

9.89

9.86

11.35

11.62

12.11

12.79

13.13

13.54

13.63

n

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

G1R, F & 0

Mean

-2.47

3.61

1.72

7.43

3.95

9.99

8.82

14.8

20.62

25.23

29.11

34

36.7

39.43

42.14

43.99

46.37

51.81

55.65

60.45

63.86

±SD

0.77

1.7

1.72

2.07

2.15

2.73

4.67

4.99

6.05

5.42

5.33

6.13

6.76

8.01

8.19

7.71

8.52

7.98

8.52

8.56

8.66

n

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

G4R, F & 1500

Mean

-2.73

3.91

2.63

8.47

6.22

12.69

11.68

17.52

23.2

27.3

30.56

34.69

37

40.09

43.24

45.61

47.93

52.89

56.23

60.38

63.78

±SD

0.58

1.4

2.1

3.12

4.58

5.73

5.78

5.85

6.98

7.75

9.73

9.84

9.76

8.9

9.11

8.77

9.18

8.72

8.47

8.07

8.67

n

5

5

5

5

5

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 *: Statistically significant (p<0.05)

 

Table no. 4: SUMMARY OF AVERAGE FEED CONSUMPTION (g/rat/day) RECORD

Group, Sex & Dose (ppm)

Week 1

Week 2

Week 3

Week 4

Week 5

Week 6

Week 7

Week 8

Week 9

Week 10

Week 11

Week 12

Week 13

G1, M & 0

Mean

18.92

17.92

20.08

19.74

21.57

20.51

20.56

20.36

20.69

20.24

20.32

20.45

28.76

±SD

0.28

1.44

0.51

0.54

2.24

1.16

0.76

0.76

1.03

0.76

0.55

0.81

0.94

n

5

5

5

5

5

5

5

5

5

5

5

5

5

G2, M & 500

Mean

18.87

18.36

19.65

19.85

20.55

20.4

20.32

20.47

20.65

20.29

20.16

20.44

28.67

±SD

0.31

0.69

0.73

0.67

2.16

1.34

0.83

1.15

1.1

0.66

0.56

0.83

1.24

n

5

5

5

5

5

5

5

5

5

5

5

5

5

G3, M & 1000

Mean

18.52

18.03

19.94

19.65

21.47

20.29

20.25

20.41

20.56

20.27

20.14

20.64

28.88

±SD

0.26

0.66

0.29

0.58

1.81

0.99

0.85

0.99

1.12

1.15

0.55

0.9

1.18

n

5

5

5

5

5

5

5

5

5

5

5

5

5

G4, M & 1500

Mean

18.65

18.25

19.7

20.06

21.38

20.18

20.41

20.39

20.51

20.01

20.15

20.55

29.09

±SD

0.52

0.67

0.93

0.67

0.94

1

1.08

1.02

1.05

0.55

0.57

1.05

1.02

n

5

5

5

5

5

5

5

5

5

5

5

5

5

G1, F & 0

Mean

16.7

14.88

15.49

15.19

15.11

15.16

14.92

14.75

15.01

14.97

14.54

14.81

17.11

±SD

0.19

0.23

0.36

0.44

0.28

0.36

0.38

0.41

0.54

0.56

0.3

0.25

0.64

n

5

5

5

5

5

5

5

5

5

5

5

5

5

G2, F & 500

Mean

16.61

15.27

16.60*

15.55

14.98

15.2

14.97

14.67

15.09

14.87

14.62

14.96

16.15

±SD

0.13

0.39

0.3

0.48

0.44

0.51

0.28

0.48

0.52

0.26

0.48

0.4

1.19

n

5

5

5

5

5

5

5

5

5

5

5

5

5

G3, F & 1000

Mean

16.2

15.31

16.06

15.7

15.47

15.31

15.3

14.9

15.04

15.15

14.72

14.95

16.32

±SD

0.43

0.27

0.48

0.63

0.62

0.47

0.64

0.49

0.53

0.62

0.38

0.4

0.95

n

5

5

5

5

5

5

5

5

5

5

5

5

5

G4, F & 1500

Mean

15.75*

15.01

15.59

15.34

15.32

15.41

15.27

14.82

15.19

15.19

14.71

15.04

16.06

±SD

0.45

0.43

0.31

0.54

0.43

0.36

0.44

0.6

0.44

0.43

0.26

0.31

0.93

n

5

5

5

5

5

5

5

5

5

5

5

5

5

Recovery Group, Sex & Dose (ppm)

Week 1

Week 2

Week 3

Week 4

Week 5

Week 6

Week 7

Week 8

Week 9

Week 10

Week 11

Week 12

Week 13

Week 14

Week 15

Week 16

Week 17

G1R, M & 0

Mean

19.19

18.59

20.56

21.1

21.37

21.38

22.44

21.24

21.1

20.8

21.15

20.83

20.65

20.8

20.93

20.89

23.6

±SD

0.76

0.28

1.1

1.79

1.3

1.94

2.85

1.02

1.48

0.92

1.69

1.06

0.55

1.06

1.16

0.73

0.2

n

3

3

3

3

3

3

3

3

3

3

3

3

3

3

3

3

3

G4R, M & 1500

Mean

16.53*

17.73

19.05

19.31

21.06

20.35

21.96

20.59

20.95

20.07

20.76

20.98

20.33

20.96

21.25

21.04

23.51

±SD

0.28

3.42

1.28

0.63

0.99

0.33

2.43

0.8

1.22

0.64

1.05

1.58

0.46

1.36

1.33

1.06

0.41

n

3

3

3

3

3

3

3

3

3

3

3

3

3

3

3

3

3

G1R, F & 0

Mean

16.9

15.67

16.34

15.93

15.58

15.97

15.8

15.26

15

14.9

14.79

15.05

15.69

15.59

15.71

15.88

17.58

±SD

0.45

1.4

1.77

1.21

0.77

1.82

1.8

0.49

0.46

0.3

0.48

0.05

0.08

0.76

0.62

0.27

0.28

n

3

3

3

3

3

3

3

3

3

3

3

3

3

3

3

3

3

G4R, F & 1500

Mean

16.3

16.44

16.69

16.19

15.45

15.98

15.91

14.74

15.19

14.66

14.96

14.76

15.55

15.65

15.53

16.06

17.5

±SD

1.82

3.13

1.37

0.81

0.51

1.56

2.07

0.28

0.61

0.33

0.39

0.42

0.09

0.94

0.49

0.33

0.26

n

3

3

3

3

3

3

3

3

3

3

3

3

3

3

3

3

3

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

 

Table no. 5: SUMMARY OF NEUROLOGICAL/FUNCTIONAL OBSERVATION BATTERY (FOB) RECORD

Week 13
Parameters↓

Group & Sex

G1 & M

G4 & M

G1 & F

G4 & F

Dose (ppm)

0

1500

0

1500

Number of Animals

10

10

10

10

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

Open field Observation

Mobility

1

1

1

1

Gait

1

1

1

1

Arousal

3

3

3

3

Number of Rearing

Mean

7.9

7.1*

7.2

6.7

±SD

0.9

0.7

1

0.9

Numbers of Urination

Mean

1.3

1.4

1.1

1.5

±SD

0.9

1

0.7

0.8

Number of Defecation

Mean

1.5

1.6

1.6

1.5

±SD

0.8

1.1

1

0.8

Clonic involuntary movement

1

1

1

1

Tonic involuntary movement

1

1

1

1

Stereotype Behaviour

1

1

1

1

Excessive Grooming

Mean

3.1

2.7

2.7

2.7

±SD

0.9

0.8

0.8

0.7

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

97.4

97.3

97.3

98.6*

±SD

0.6

0.6

0.5

0.6

Recovery Group
Week 17
Parameters↓

Group & Sex

G1R & M

G4R & M

G1R & F

G4R & F

Dose (ppm)

0

1500

0

1500

Number of Animals

10

10

10

10

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

Open field Observation

Mobility

1

1

1

1

Gait

1

1

1

1

Arousal

3

3

3

3

Number of Rearing

Mean

7.2

7.6

7.2

7

±SD

0.8

1.1

0.8

1

Numbers of Urination

Mean

1.6

1

1.2

1.2

±SD

1.1

0.7

0.8

0.8

Number of Defecation

Mean

1.2

1.6

1.6

1.8

±SD

0.4

0.5

0.5

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

2.8

2.8

2.6

±SD

0.7

0.8

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

97.9

97.4

98.8

99

±SD

0.6

0.8

0.7

0.6

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; Statistically significant (p<0.05)
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 no. 6: SUMMARY OF HIND LIMB FOOT SPLAY (cm) RECORD

Group, Sex & Dose (ppm)

Hind Limb Foot Splay (cm)

G1, M & 0

Mean

9.1

±SD

1.3

n

10

G4, M & 1500

Mean

8

±SD

1.5

n

10

G1, F & 0

Mean

6.9

±SD

2

n

10

G4, F & 1500

Mean

8.2

±SD

1.4

n

10

G1R, M & 0

Mean

9.3

±SD

0.6

n

5

G4R, M & 1500

Mean

9.5

±SD

0.4

n

5

G1R, F & 0

Mean

6.6

±SD

0.7

n

5

G4R, F & 1500

Mean

6.3

±SD

0.4

n

5

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

Week 13 for main group animals and week 17 for recovery group animals.

 

Table no. 7: SUMMARY OF ACTIMETER READING RECORD

Group, Sex &Dose (ppm)

Actimeter Readings Movement Counts (no.)

G1, M & 0

Mean

1672.6

±SD

42.2

n

10

G4, M & 1500

Mean

1790.1*

±SD

55.9

n

10

G1, F & 0

Mean

2075.5

±SD

228

n

10

G4, F & 1500

Mean

2054.1

±SD

138.6

n

10

G1R, M & 0

Mean

1849

±SD

28.7

n

5

G4R, M & 1500

Mean

1834.4

±SD

6.5

n

5

G1R, F & 0

Mean

2191.6

±SD

50.8

n

5

G4R, F & 1500

Mean

2172.6

±SD

46.1

n

5

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

Week 13 for main group animals and week 17 for recovery group animals.

 

Table no. 8 SUMMARY OF GRIP STRENGTH (kgf) MEASUREMENT RECORD

Group, Sex & Dose (ppm)

Grip Strength (kgf)

Fore limb

Hind limb

G1, M & 0

Mean

1.496

0.507

±SD

0.05

0.054

n

10

10

G4, M & 1500

Mean

1.48

0.489

±SD

0.048

0.045

n

10

10

G1, F & 0

Mean

1.399

0.491

±SD

0.06

0.056

n

10

10

G4, F & 1500

Mean

1.375

0.469

±SD

0.054

0.059

n

10

10

G1R, M & 0

Mean

1.543

0.496

±SD

0.017

0.033

n

5

5

G4R, M & 1500

Mean

1.521

0.481

±SD

0.014

0.043

n

5

5

G1R, F & 0

Mean

1.43

0.43

±SD

0.025

0.042

n

5

5

G4R, F & 1500

Mean

1.421

0.417

±SD

0.028

0.038

n

5

5

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

# Week 13 for main group animals and week 17 for recovery group animals.

 

Table no. 9: SUMMARY OF OPHTHALMOSCOPIC EXAMINATION RECORD

Week

Week 13

Week 17

Group, Sex & Dose (ppm)

G1, M & 0

G4, M & 1500

G1R, M & 0

G4R, M & 1500

Number of Animals

10

10

5

5

Observations

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

Week

Week 13

Week 17

Group, Sex & Dose (ppm)

G1, F & 0

G4, F & 1500

G1R, F & 0

G4R, F & 1500

Number of Animals

10

10

5

5

Observations

Eye

L

R

L

R

L

R

L

R

Eye Lids

N

N

N

N

N

N

N

N

Cornea

N

N

N

N

N

N

N

N

Iris

N

N

N

N

N

N

N

N

Aqueous Humour

N

N

N

N

N

N

N

N

Lens

N

N

N

N

N

N

N

N

Vitreous Humour

N

N

N

N

N

N

N

N

Retina/Optic disc

N

N

N

N

N

N

N

N

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

 

Table no. 10: SUMMARY OF HAEMATOLOGY RECORD

Group,Sex & Dose (ppm)

(WBC)

(RBC)

(HGB)

(HCT)

(MCV)

(MCH)

(MCHC)

(PLT)

(MPV)

(Retic)

(Neut)

(Lymph)

(Mono)

(Eos)

(Baso)

(Retic)

(Neut)

(Lymph)

(Mono)

(Eos)

(Baso)

(PT)

(APTT)

(103cells/µL)

(106cells/µL)

(g/dL)

(%)

(fL)

(pg)

(g/dL)

(103cells/µL)

(fL)

(%)

(%)

(%)

(%)

(%)

(%)

(109cells/L)

(103cells/µL)

(103cells/µL)

(103cells/µL)

(103cells/µL)

(103cells/µL)

(Seconds)

(Seconds)

G1, M & 0

Mean

8.8

8.16

14.61

44.52

54.58

17.9

32.82

773.3

5.6

1.38

22.99

71.24

2.97

1.22

0.25

111.77

1.99

6.3

0.26

0.11

0.02

23.94

30.8

±SD

1.68

0.2

0.4

1.16

1.81

0.58

0.71

90.78

0.28

0.37

4.67

4.65

0.59

0.47

0.07

27.98

0.42

1.43

0.06

0.04

0.01

0.92

3.48

n

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

G2, M & 500

Mean

8.3

7.72

13.59

42.68

55.96

17.7

31.78

880.1

6.18*

2.04

25.18

68.36

3.54

1.34

0.29

147.23

2.04

5.72

0.29

0.12

0.02

21.39*

31.81

±SD

2.3

1

1.5

2.88

5.92

0.8

1.89

183.33

0.69

1.39

7.84

8.47

1.43

0.4

0.07

67.91

0.67

1.92

0.12

0.06

0.01

1.29

9.23

n

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

G3, M & 1000

Mean

9.65

8.24

14.69

44.6

54.16

17.83

32.93

793.7

5.48

1.36

25.44

68.16

3.14

1.45

0.31

111.45

2.45

6.58

0.31

0.14

0.03

20.86*

26.22

±SD

2.24

0.28

0.7

1.99

2.08

0.63

0.49

131.75

0.21

0.66

7.83

8.33

0.67

0.47

0.1

52.64

0.9

1.69

0.12

0.07

0.02

2.28

3.5

n

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

G4, M & 1500

Mean

9.02

7.78

14

43.56

56.86

18.07

32.06

837.1

5.9

3.87

21.8

71.87

3.4

1.35

0.32

228.66

1.88

6.58

0.3

0.12

0.03

21.24*

26.66

±SD

2.05

1.06

1.67

3.01

8.05

1.27

2.18

93.04

0.53

7.91

5

5.5

0.89

0.55

0.11

377.62

0.11

2.01

0.09

0.05

0.01

2.09

2.85

n

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

G1R, M & 0

Mean

9.61

8.27

15.06

45.26

54.76

18.24

33.28

1074.4

6.22

1.38

21.4

71.64

3.86

1.88

0.32

114.44

2.05

6.89

0.38

0.18

0.03

20.64

25.5

±SD

1.32

0.3

0.17

0.94

1.39

0.58

0.82

83.66

0.19

0.31

6.51

7.46

1.57

0.34

0.08

27.72

0.72

1.26

0.18

0.01

0.01

1.77

2.72

n

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

G4R, M & 1500

Mean

11.46*

7.68*

14.66

45.28

59.28

19.18

32.42

829.80*

7.38

2.85

19.96

73.48

3.94

1.28

0.4

213.4

2.28

8.43

0.45

0.14

0.05

23.08

27.16

±SD

0.85

0.49

0.59

2.07

5.5

1.79

1.31

78.27

1.53

1.55

1.81

3.33

1.72

0.57

0.14

100.3

0.12

0.92

0.2

0.06

0.02

2.84

5.95

n

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

G1, F & 0

Mean

8.37

7.41

13.53

40.42

54.66

18.29

33.47

854

5.88

1.67

20.59

74.51

2.25

1.1

0.28

122.85

1.64

6.32

0.19

0.09

0.02

21.42

26.23

±SD

3.1

0.4

0.56

1.69

1.21

0.73

1.02

138.33

0.46

0.39

6.2

6.46

0.47

0.19

0.08

26.76

0.47

2.76

0.09

0.03

0.02

5.45

3.03

n

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

G2, F & 500

Mean

6.73

7.36

13.18

40.02

54.4

17.88

32.91

855.2

6.07

1.92

21.42

72.64

3.06

1.41

0.23

141.62

1.4

4.93

0.21

0.1

0.02

22.67

25.15

±SD

1.75

0.45

1

2.42

1.44

0.63

0.54

178.33

0.38

0.36

5.19

5.5

0.85

0.64

0.05

27.97

0.4

1.47

0.08

0.05

0.01

1.63

1.54

n

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

G3, F & 1000

Mean

11.40*

7.6

13.9

41.97

55.28

18.31

33.1

899.7

5.89

1.87

22.07

72.38

3.11*

1.02

0.25

140.79

2.53

8.25

0.34*

0.11

0.03

22.48

25.37

±SD

3.43

0.32

0.96

2.28

2.56

1.13

0.57

169.74

0.29

0.66

4.76

4.77

0.74

0.47

0.07

46.15

1.01

2.53

0.09

0.05

0.02

1.92

1.78

n

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

G4, F & 1500

Mean

8.14

7.42

13.49

40.48

54.61

18.22

33.37

838.1

6.67*

1.89

22.26

72.16

2.81

1.1

0.27

139.58

1.88

5.81

0.23

0.09

0.03

21.38

28.23

±SD

2.18

0.35

0.48

1.24

1.87

0.79

0.9

110.61

0.62

0.41

8.73

9.12

0.87

0.55

0.12

26.93

1.21

1.44

0.09

0.04

0.01

1.59

2.05

n

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

G1R, F & 0

Mean

6.9

7.2

13.3

39.76

55.3

18.48

33.44

944.4

6.22

1.42

21.24

73.42

2.82

1.42

0.22

102.22

1.44

5.1

0.19

0.1

0.02

23.4

24.28

±SD

0.94

0.4

0.1

0.63

2.73

1.04

0.44

111.01

0.25

0.14

4.73

5.13

0.44

0.49

0.04

10.07

0.22

1.01

0.04

0.03

0.01

2.56

1.22

n

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

G4R, F & 1500

Mean

8.34

7.33

14.12

41.78

57.26

19.32

33.74

1018.2

6.32

1.7

25.44

68.38

3.22

1.48

0.3

120.62

2.12

5.71

0.27

0.11

0.03

25.02

24.58

±SD

2.9

0.83

0.9

3.47

3.12

1.17

0.89

105.42

0.33

0.69

8.85

9.06

0.33

0.87

0.1

32.19

1.17

2.18

0.11

0.04

0.01

2.56

2.06

n

5

5

5

5

5

5

5

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; *: Statistically significant (p<0.05)

 

Table no. 11: SUMMARY OF BALF RECORD

Group, Sex & Dose (ppm)

(WBC)

(Neut)

(Lymph)

(Mono)

(Eos)

(Baso)

(Neut)

(Lymph)

(Mono)

(Eos)

(Baso)

(LDH)

(TPR)

(103cells/µL)

(%)

(%)

(%)

(%)

(%)

(103cells/µL)

(103cells/µL)

(103cells/µL)

(103cells/µL)

(103cells/µL)

(u/L)

(g/dL)

G1, M & 0

Mean

3.24

19.2

63.82

4.53

0.59

4.44

0.72

1.59

0.13

0.01

0.09

365.19

0.6

±SD

3.87

9.83

20

8.57

0.36

2.16

0.94

1.39

0.26

0.01

0.05

184.44

0.23

n

10

10

10

10

10

10

10

10

10

10

10

10

10

G2, M & 500

Mean

1.46

18.26

63.78

2.77

0.73

5.89

0.27

0.92

0.05

0.01

0.08

339.83

0.49

±SD

0.86

5.49

8.47

2.62

0.46

2.4

0.21

0.53

0.08

0.01

0.06

229.8

0.17

n

10

10

10

10

10

10

10

10

10

10

10

10

10

G3, M & 1000

Mean

1.35

15.34

70.73

2.6

0.66

6.16

0.19

0.99

0.03

0.01

0.09

332.34

0.41*

±SD

0.72

6.92

11.56

2.2

0.42

3.48

0.08

0.61

0.02

0

0.07

211.75

0.1

n

10

10

10

10

10

10

10

10

10

10

10

10

10

G4, M & 1500

Mean

2.06

24.14

58.78

6.78

0.96

6.02

0.66

0.88

0.29

0.01

0.14

501.08

0.40*

±SD

1.99

11.02

20.91

9.93

0.62

2.46

0.88

0.38

0.74

0

0.2

390.78

0.1

n

10

10

10

10

10

10

10

10

10

10

10

10

10

G1R, M & 0

Mean

3.24

69.84

16.78

6.26

4.22

4.52

2.47

0.34

0.11

0.25

0.15

896.98

0.4

±SD

2.79

25.79

25.76

5.77

5.52

2.15

2.64

0.54

0.09

0.45

0.15

502.54

0.17

n

5

5

5

5

5

5

5

5

5

5

5

5

5

G4R, M & 1500

Mean

1.62

53.74

38.5

4.64

1.88

7.44

0.82

0.68

0.08

0.02

0.13

562.5

0.25

±SD

0.64

28.23

31.42

6.5

3.05

2.1

0.44

0.65

0.13

0.03

0.07

805.8

0.15

n

5

5

5

5

5

5

5

5

5

5

5

5

5

G1, F & 0

Mean

0.96

20.72

62.11

1.84

0.85

6.74

0.2

0.59

0.02

0.01

0.07

188.72

0.28

±SD

0.25

6.11

10.42

0.53

0.25

3.79

0.09

0.16

0.01

0

0.05

88.88

0.07

n

10

10

10

10

10

10

10

10

10

10

10

10

10

G2, F & 500

Mean

2.15

20.47

67.42

2.63

1.59

5.38

0.61

1.22

0.09

0.01

0.09

931.59*

0.48

±SD

2.75

11.88

16.39

1.7

2.19

2.52

1.12

1.27

0.18

0.01

0.11

755.39

0.31

n

10

10

10

10

10

10

10

10

10

10

10

10

10

G3, F & 1000

Mean

1.43

15.79

72.6

4.12*

0.55

7.01

0.24

1.02

0.07

0.01

0.1

1242.71*

0.56*

±SD

1.07

5.21

8.02

1.9

0.36

2.29

0.18

0.76

0.07

0.01

0.09

490.39

0.23

n

10

10

10

10

10

10

10

10

10

10

10

10

10

G4, F & 1500

Mean

2.13

28.66

55.78

2.71

0.69

5.75

1.06

0.72

0.06

0.01

0.1

232.98

0.36

±SD

3.32

18.51

20.19

1.13

0.32

2.82

2.54

0.35

0.1

0.01

0.12

124.87

0.08

n

10

10

10

10

10

10

10

10

10

10

10

10

10

G1R, F & 0

Mean

3.12

19.7

73.22

2.7

0.34

6.12

0.52

2.37

0.07

0.01

0.17

268.7

0.32

±SD

3.15

12.79

16.12

1.84

0.29

1.82

0.4

2.68

0.06

0

0.16

91.1

0.13

n

5

5

5

5

5

5

5

5

5

5

5

5

5

G4R, F & 1500

Mean

1.91

38.3

51.14

3.96

1.7

8.44

0.53

1.17

0.06

0.02

0.17

970.24

0.63

±SD

1.14

25.46

27.21

3.34

2.05

3.36

0.12

0.99

0.04

0.01

0.16

943.45

0.29

n

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; *: Statistically significant (p<0.05)

 

Table no. 12: SUMMARY OF CLINICAL CHEMISTRY RECORD

Group, Sex & Dose (ppm)

(GLU)

Urea

(CRE)

(CHO)

(TRI)

(TPR)

(ALB)

(ALT)

(AST)

(ALP)

(BIT)

(CAL)

(PHO)

(GLO)

(BUN)

(Na)

(K)

(CLO)

(mg/dL)

(mg/dL)

(mg/dL)

(mg/dL)

(mg/dL)

(g/dL)

(g/dL)

(U/L)

(U/L)

(U/L)

(mg/dL)

(mg/dL)

(mg/dL)

(g/dL)

(mg/dL)

(mmol/L)

(mmol/L)

(mmol/L)

G1, M & 0

Mean

135.8

32.41

0.53

42.7

63

6.36

3.16

71.1

115.9

171.1

0

9.27

4.46

3.2

15.12

145.01

3.97

111.79

±SD

9.19

3.88

0.03

7.32

25.93

0.37

0.14

8.09

11.75

50.46

0

0.21

0.32

0.4

1.81

1.14

0.24

1.7

n

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

G2, M & 500

Mean

131.2

37.12

0.48

40

50.2

6.39

3.05

68.8

114.8

156.8

0

9.22

4.42

3.34

17.32

144.92

4.01

112.02

±SD

11.06

4.14

0.03

5.31

11.16

0.37

0.19

13.86

16.81

30.81

0

0.27

0.43

0.34

1.93

1.51

0.34

1.98

n

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

G3, M & 1000

Mean

133.3

43.64*

0.48

39.4

69

6.2

3.1

66.8

105.8

170.6

0.05

9.13

3.82*

3.1

20.37*

144.56

4.38*

112.03

±SD

13.28

5.44

0.11

3.53

17.31

0.33

0.17

19.31

30.74

52.84

0.13

0.27

0.28

0.2

2.54

1.27

0.21

2.28

n

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

G4, M & 1500

Mean

119.90*

37.97*

0.56

41.1

59.6

6.45

3.1

64

114

162.1

0.01

9.21

4.91*

3.35

17.72*

145.89

3.93

112.35

±SD

11.21

4.1

0.08

5.99

15.55

0.39

0.1

14.02

27.45

67.9

0.03

0.28

0.47

0.35

1.91

1.32

0.3

1.96

n

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

G1R, M & 0

Mean

146

39.26

0.57

41.6

53.2

6.4

3.39

52.2

89.4

107.6

0.21

8.92

4.58

3.01

18.32

144.78

3.55

109.14

±SD

7.38

4.41

0.04

8.26

8.04

0.37

0.08

6.06

4.93

14.31

0.04

0.04

0.29

0.31

2.06

0.97

0.2

1.37

n

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

G4R, M & 1500

Mean

129.20*

42.28

0.57

44.6

66.2

6.4

3.4

54.4

95.8

117

0.27

9.28*

4.54

3

19.73

144.78

3.96*

109.62

±SD

13.75

4.71

0.02

7.44

17.6

0.43

0.19

3.65

6.76

28.83

0.06

0.23

0.55

0.3

2.2

0.43

0.28

0.7

n

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

G1, F & 0

Mean

121.7

44.64

0.65

49.8

48.9

6.81

3.5

40.9

93.4

86

0

9.65

3.79

3.31

20.83

141.43

3.61

107.32

±SD

10.01

14.89

0.15

6.78

11.16

0.24

0.19

17.23

21.08

35.65

0.01

0.29

0.51

0.18

6.95

1.14

0.27

0.92

n

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

G2, F & 500

Mean

121.1

44.34

0.63

48

41.7

6.68

3.51

48.2

133.9

83.2

0

9.48

3.47

3.18

20.69

140.35

3.83

106.16

±SD

9.64

5.68

0.08

9.68

12.11

0.34

0.16

12

101.55

44.89

0

0.27

0.38

0.28

2.65

0.96

0.28

1.83

n

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

G3, F & 1000

Mean

124.4

48.44

0.51

55.4

50.4

6.94

3.38

54.2

101.4

114.8

0.02

9.64

4.53*

3.56

22.61

139.51*

3.54

104.95*

±SD

13.53

7.85

0.18

9.2

29.1

0.42

0.26

17.78

27.24

41.79

0.05

0.27

0.88

0.35

3.67

1.61

0.34

2.15

n

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

G4, F & 1500

Mean

114.5

45.97

0.55

52.2

43.9

6.92

3.45

45.2

96.3

87.3

0

9.76

4.03

3.48

21.45

141.01

3.74

104.94*

±SD

8.9

9.52

0.15

7.3

13.08

0.26

0.17

17.52

19.35

26.94

0

0.23

0.35

0.29

4.44

1.51

0.2

1.08

n

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

10

G1R, F & 0

Mean

127.8

44.58

0.59

46.8

51.2

6.62

3.74

45

89.6

91.4

0.19

9.46

3.76

2.88

20.81

144.52

3.76

110.18

±SD

11.65

7

0.04

8.87

11.3

0.26

0.2

14.28

24.13

38.02

0.11

0.3

0.78

0.19

3.27

1.47

0.15

1.51

n

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

5

G4R, F & 1500

Mean

134.6

49.66

0.59

53.4

51.6

6.76

3.66

41

83.2

103

0.22

9.42

4.12

3.1

23.17

144.5

3.81

110.14

±SD

6.02

6.68

0.09

18.6

25.77

0.25

0.32

13.04

17.4

76.36

0.02

0.58

1.66

0.42

3.12

0.63

0.21

2.63

n

5

5

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; *: Statistically significant (p<0.05)

 

Table no. 13: SUMMARY OF URINALYSIS RECORD

Examination

Group & Sex

G1 & M

G2 & M

G3 & M

G4 & M

G1R & M

G4R & M

Dose (ppm)

0

500

1000

1500

0

1500

Number of Animals

10

10

10

10

5

5

Physical

Colour

Pale Yellow

4

7

3

5

5

5

Pale Red

-

-

1

-

-

-

Yellow

6

3

6

5

-

-

Appearance

Clear

9

8

6

8

5

4

Turbid

1

2

4

2

-

1

Volume (mL)

Mean

7.6

7.8

7.7

7.4

10.3

7.8

±SD

1.8

2.1

1.8

1.7

1.5

3.2

Chemical

pH

Mean

7.8

7.3

8.2

7.7

8.3

8.4

±SD

0.4

0.3

0.5

0.6

0.3

0.2

Specific Gravity

Mean

1.01

1.01

1.01

1.012

1.006

1.006

±SD

0

0.006

0.003

0.003

0.002

0.002

Urobilinogen (mg/dL)

Mean

0.2

0.2

0.3

0.2

0.2

0.9

±SD

0

0

0.3

0

0

1

Bilirubin (mg/dL)

Neg

10

10

9

10

5

4

1

-

-

1

-

-

1

Ketones (mg/dL)

Neg

10

9

3

6

3

5

5

-

1

7

4

2

-

Blood (Ery/µL)

Neg

5

-

1

2

4

2

Ca10

-

-

-

-

1

2

Ca25

1

2

4

-

-

1

Ca80

4

7

5

5

-

-

>=Ca200

-

1

-

3

-

-

Proteins (mg/dL)

Trace

5

2

3

4

1

2

Neg

5

7

1

3

3

1

30

-

-

6

3

1

1

100

-

1

-

-

-

-

>=300

-

-

-

-

-

1

Nitrite

Neg

9

6

5

7

4

1

Pos

1

4

5

3

1

4

Leucocytes (Leu/µL)

Neg

10

9

9

10

5

4

Ca15

-

1

1

-

-

-

Ca70

-

-

-

-

-

1

Glucose (mg/dL)

Neg

10

10

10

10

5

5

Microalbumin (MALB) (mg/dL)

Neg

-

5

1

-

3

1

>15

5

3

9

7

2

4

15

5

2

-

3

-

-

Microscopic

Epithelial cells

0

8

8

7

8

3

3

0-1

1

1

2

1

2

1

01-02.

1

1

1

1

-

1

Casts

Absent

10

10

10

10

5

5

Crystals

Present

10

10

10

10

5

5

Examination

Group & Sex

G1 & F

G2 & F

G3 & F

G4 & F

G1R & F

G4R & F

Dose (ppm)

0

500

1000

1500

0

1500

Number of Animals

10

10

10

10

5

5

Physical

Colour

Pale Yellow

8

7

3

6

5

5

Yellow

2

3

7

4

-

-

Appearance

Clear

9

10

9

7

5

4

Turbid

1

-

1

3

-

1

Volume (mL)

Mean

6.6

6.5

6.1

7.7

9.5

7

±SD

1.9

1.1

1.6

1.9

2.2

0.9

Chemical

pH

Mean

7.7

7.5

7.2*

7.8

7.9

8.5*

±SD

0.3

0.3

0.3

0.5

0.4

0

Specific Gravity

Mean

1.01

1.013

1.014*

1.012

1.005

1.005

±SD

0

0.004

0.003

0.003

0

0

Urobilinogen (mg/dL)

Mean

0.2

0.2

0.2

0.2

0.2

0.2

±SD

0

0

0

0

0

0

Bilirubin (mg/dL)

Neg

10

10

10

10

5

5

Ketones (mg/dL)

Neg

10

10

10

10

5

5

Blood (Ery/µL)

Neg

6

1

1

4

-

5

Ca10

2

1

3

4

2

-

Ca25

2

8

4

-

3

-

Ca80

-

-

2

2

-

-

Proteins (mg/dL)

Trace

7

9

9

10

-

1

Neg

3

1

1

-

5

-

30

-

-

-

-

-

2

100

-

-

-

-

-

1

>=300

-

-

-

-

-

1

Nitrite

Neg

4

9

9

6

2

1

Pos

6

1

1

4

3

4

Leucocytes (Leu/µL)

Neg

10

10

10

10

5

4

Ca15

-

-

-

-

-

1

Glucose (mg/dL)

Neg

10

10

10

10

5

5

Microalbumin (MALB) (mg/dL)

Neg

1

-

-

-

4

-

>15

7

9

9

10

-

5

15

2

1

1

-

1

-

Microscopic

Epithelial cells

0

4

5

5

3

4

4

0-1

4

3

2

4

-

1

1-2.

1

1

2

2

1

-

2-3.

1

1

1

1

-

-

Casts

Absent

10

10

10

10

5

5

Crystals

Present

10

10

10

10

5

5

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

 

Table no. 14: SUMMARY OF ABSOLUTE ORGAN WEIGHTS (g) RECORD

Group, Sex & Dose (ppm)

Adrenals

Thymus

Spleen

Testes

Epididymes

Heart

Kidneys

Brain

Liver

Lungs

Thyroid along
with parathyroid

G1, M & 0

Mean

0.0563

0.2757

0.798

3.1308

1.3177

1.1358

2.3026

1.9577

9.5264

0.6754

0.0307

±SD

0.0142

0.0727

0.1301

0.3549

0.1484

0.1642

0.3824

0.1129

1.1438

0.0893

0.0055

n

10

10

10

10

10

10

10

10

10

10

10

G2, M & 500

Mean

0.0629

0.3064

0.6103*

3.3173

1.355

1.1996

2.57

2.0908*

10.1868

0.6563

0.0331

±SD

0.0093

0.0929

0.1042

0.405

0.1698

0.1201

0.3674

0.1393

1.444

0.0908

0.0028

n

10

10

10

10

10

10

10

10

10

10

10

G3, M & 1000

Mean

0.07

0.2908

0.7541

3.3246

1.3603

1.2703

2.7420*

2.0401

11.2034*

0.6297

0.0312

±SD

0.0242

0.0728

0.1726

0.3509

0.1362

0.1002

0.3489

0.101

1.1264

0.1134

0.0059

n

10

10

10

10

10

10

10

10

10

10

10

G4, M & 1500

Mean

0.0636

0.2485

0.8508

3.4128

1.3435

1.2551

2.4937

2.0307

10.158

0.6708

0.0332

±SD

0.0143

0.066

0.2337

0.3466

0.134

0.1113

0.2665

0.0918

0.7166

0.0935

0.0037

n

10

10

10

10

10

10

10

10

10

10

10

G1R, M & 0

Mean

0.0638

0.2993

0.7004

3.5165

1.5073

1.3288

2.897

2.1264

11.3052

0.683

0.0367

±SD

0.0155

0.0799

0.0853

0.3277

0.1158

0.1441

0.2846

0.152

1.6587

0.0826

0.0014

n

5

5

5

5

5

5

5

5

5

5

5

G4R, M & 1500

Mean

0.0485

0.271

0.7358

3.2861

1.3645

1.2279

2.4728

2.0786

10.1867

0.5939*

0.0367

±SD

0.0049

0.0647

0.075

0.4565

0.1766

0.0971

0.6658

0.117

1.6125

0.0231

0.0024

n

5

5

5

5

5

5

5

5

5

5

5

G1, F & 0

Mean

0.0693

0.3195

0.5032

0.1744

0.5306

0.8655

1.6102

1.8929

7.1787

0.5274

0.0237

±SD

0.0119

0.0654

0.0632

0.0344

0.148

0.0935

0.2085

0.0653

0.9993

0.0375

0.0036

n

10

10

10

10

10

10

10

10

10

10

10

G2, F & 500

Mean

0.0953*

0.2908

0.5239

0.1811

0.6875

0.9613

2.0083*

1.9387

8.5808*

0.5892

0.0274*

±SD

0.0278

0.0884

0.0972

0.0478

0.196

0.1056

0.3283

0.1073

1.4086

0.1185

0.0027

n

10

10

10

10

10

10

10

10

10

10

10

G3, F & 1000

Mean

0.0922*

0.3607

0.5568

0.1639

0.5842

0.898

1.9441*

1.9376

8.5525*

0.6284

0.0243

±SD

0.0187

0.0815

0.107

0.0253

0.0891

0.1064

0.2833

0.0798

0.8217

0.1181

0.0031

n

10

10

10

10

10

10

10

10

10

10

10

G4, F & 1500

Mean

0.0847

0.3312

0.5461

0.1653

0.6294

0.9073

1.7317

1.9398

7.6437

0.551

0.0226

±SD

0.0178

0.0646

0.0685

0.0337

0.2189

0.0751

0.279

0.0918

0.5878

0.0828

0.0036

n

10

10

10

10

10

10

10

10

10

10

10

G1R, F & 0

Mean

0.0645

0.2597

0.4534

0.1311

0.7806

0.8756

1.7557

2.0155

7.1945

0.5336

0.0238

±SD

0.0051

0.0752

0.0357

0.0169

0.1429

0.0645

0.1894

0.0761

0.7171

0.0454

0.0021

n

5

5

5

5

5

5

5

5

5

5

5

G4R, F & 1500

Mean

0.0614

0.295

0.5241*

0.1374

0.7021

0.8827

1.7024

1.9944

7.7587

0.6293

0.0272

±SD

0.0092

0.0216

0.0434

0.0405

0.1522

0.0956

0.2265

0.1196

0.8329

0.0898

0.0033

n

5

5

5

5

5

5

5

5

5

5

5

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

 

Table no. 15: SUMMARY OF ORGAN WEIGHT RELATIVE TO BODY WEIGHT (%) RECORD

Group, Sex & Dose (ppm)

Fasting Body
Weight (g)

Adrenals

Thymus

Spleen

Testes

Epididymes

Heart

Kidneys

Brain

Liver

Lungs

Thyroid along
with parathyroid

G1, M & 0

Mean

357.2

0.0158

0.0773

0.2242

0.8761

0.3691

0.3173

0.6436

0.549

2.6708

0.1905

0.0086

±SD

23.98

0.0042

0.0205

0.0395

0.074

0.036

0.033

0.0873

0.0288

0.3032

0.0332

0.0016

n

10

10

10

10

10

10

10

10

10

10

10

10

G2, M & 500

Mean

367.91

0.0171

0.083

0.1657*

0.902

0.3689

0.3261

0.6977

0.5691

2.7676

0.1785

0.009

±SD

13.35

0.0024

0.0236

0.0265

0.1083

0.0493

0.0303

0.0887

0.0457

0.3604

0.0246

0.0008

n

10

10

10

10

10

10

10

10

10

10

10

10

G3, M & 1000

Mean

362.43

0.0192

0.0801

0.2089

0.9167

0.3752

0.3505*

0.7555*

0.5636

3.0928*

0.1732

0.0086

±SD

16.09

0.0062

0.0188

0.0507

0.0773

0.0309

0.0229

0.0813

0.0313

0.3001

0.0264

0.0017

n

10

10

10

10

10

10

10

10

10

10

10

10

G4, M & 1500

Mean

373.21

0.0171

0.0675

0.2322

0.9176

0.3606

0.3374

0.6679

0.5476

2.7335

0.1795

0.0089

±SD

33.57

0.0034

0.0206

0.0773

0.0883

0.028

0.0281

0.0335

0.0494

0.2157

0.016

0.0012

n

10

10

10

10

10

10

10

10

10

10

10

10

G1R, M & 0

Mean

440.28

0.0145

0.0678

0.1588

0.7982

0.3426

0.3018

0.6585

0.4841

2.5656

0.1556

0.0083

±SD

17.2

0.0036

0.0175

0.0155

0.0598

0.0267

0.0303

0.0656

0.0462

0.3504

0.0225

0.0002

n

5

5

5

5

5

5

5

5

5

5

5

5

G4R, M & 1500

Mean

429.5

0.0113

0.0628

0.1711

0.7637

0.3173

0.2856

0.5744

0.4838

2.3655

0.1383

0.0085

±SD

13.95

0.001

0.0131

0.0138

0.0891

0.0358

0.0134

0.1499

0.0169

0.3067

0.0056

0.0004

n

5

5

5

5

5

5

5

5

5

5

5

5

G1, F & 0

Mean

249.43

0.0278

0.1279

0.2018

0.0705

0.214

0.3465

0.6439

0.7618

2.8714

0.2129

0.0095

±SD

18.66

0.0045

0.0222

0.0209

0.0167

0.0642

0.0182

0.0494

0.0473

0.2603

0.0256

0.0014

n

10

10

10

10

10

10

10

10

10

10

10

10

G2, F & 500

Mean

258.24

0.0366*

0.1113

0.2021

0.0696

0.2666

0.3734

0.7807*

0.7561

3.3309*

0.2308

0.0107

±SD

28.19

0.0085

0.0229

0.0242

0.0135

0.0776

0.0343

0.124

0.0633

0.4732

0.0548

0.0013

n

10

10

10

10

10

10

10

10

10

10

10

10

G3, F & 1000

Mean

255.29

0.0360*

0.1422

0.2177

0.0644

0.2283

0.3514

0.7598*

0.7607

3.3507*

0.2464

0.0095

±SD

14.1

0.0065

0.0379

0.0378

0.0104

0.0276

0.0322

0.085

0.0457

0.2691

0.0464

0.001

n

10

10

10

10

10

10

10

10

10

10

10

10

G4, F & 1500

Mean

248.22

0.034

0.1329

0.2198

0.0667

0.2536

0.3661

0.6953

0.7827

3.0768

0.2222

0.0091

±SD

12.02

0.0068

0.0224

0.0232

0.0136

0.0874

0.0327

0.0847

0.0453

0.1222

0.0327

0.0017

n

10

10

10

10

10

10

10

10

10

10

10

10

G1R, F & 0

Mean

282.37

0.0229

0.0918

0.1605

0.0466

0.2769

0.3106

0.6229

0.7146

2.5478

0.1891

0.0084

±SD

10.55

0.0027

0.0256

0.0092

0.0072

0.0524

0.028

0.0759

0.0386

0.2333

0.0158

0.0007

n

5

5

5

5

5

5

5

5

5

5

5

5

G4R, F & 1500

Mean

282.01

0.0218

0.1047

0.1858*

0.0484

0.2481

0.3127

0.6029

0.7069

2.7524

0.2232

0.0097

±SD

12.58

0.003

0.0069

0.0115

0.0124

0.0476

0.0267

0.0666

0.0178

0.2893

0.0307

0.0012

n

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; *: Statistically significant (p<0.05)

 

Table no. 16: SUMMARY OF ORGAN WEIGHT RELATIVE TO BRAIN WEIGHT (%) RECORD

Group, Sex & Dose (ppm)

Adrenals

Thymus

Spleen

Testes

Epididymes

Heart

Kidneys

Liver

Lungs

Thyroid along
with parathyroid

G1, M & 0

Mean

2.8718

14.043

40.7664

160.0845

67.3353

57.929

117.4008

487.1191

34.6474

1.5725

±SD

0.6544

3.44

6.3355

17.157

6.7679

6.5537

16.1513

56.8664

5.3982

0.2961

n

10

10

10

10

10

10

10

10

10

10

G2, M & 500

Mean

3.0026

14.5832

29.1486*

158.5088

64.769

57.5627

122.8963

488.5127

31.5859

1.5909

±SD

0.3565

4.0449

4.4575

15.6084

6.8791

6.6324

15.0415

70.5308

5.4031

0.2048

n

10

10

10

10

10

10

10

10

10

10

G3, M & 1000

Mean

3.4371

14.221

37.1578

162.9228

66.694

62.373

134.6529*

550.4004*

30.9247

1.5272

±SD

1.2025

3.3009

9.2386

14.329

5.8759

5.4514

18.4868

62.149

5.6337

0.2689

n

10

10

10

10

10

10

10

10

10

10

G4, M & 1500

Mean

3.1474

12.2208

41.9375

167.9976

66.2463

61.8966

122.7665

500.0963

33.02

1.6311

±SD

0.7889

3.0644

11.5316

14.0681

6.9128

5.8944

11.7118

24.3033

4.1196

0.1433

n

10

10

10

10

10

10

10

10

10

10

G1R, M & 0

Mean

2.9852

14.0726

33.0705

166.4988

71.0131

62.9212

136.4633

533.5598

32.0759

1.7343

±SD

0.5857

3.6037

4.7936

23.3739

5.3501

9.6241

13.2338

85.6333

2.5035

0.1355

n

5

5

5

5

5

5

5

5

5

5

G4R, M & 1500

Mean

2.3325

12.9743

35.3393

157.6031

65.4467

59.0438

118.0343

488.1477

28.6162*

1.7638

±SD

0.2014

2.5534

1.9305

14.3307

5.2093

2.4223

26.748

52.146

1.3391

0.0625

n

5

5

5

5

5

5

5

5

5

5

G1, F & 0

Mean

3.6589

16.89

26.5681

9.2434

28.0784

45.7186

84.9245

379.0235

27.9123

1.255

±SD

0.6076

3.4652

3.036

1.973

7.9477

4.6895

9.404

49.3978

2.473

0.1933

n

10

10

10

10

10

10

10

10

10

10

G2, F & 500

Mean

4.9087*

14.9709

26.9071

9.3383

35.4037

49.5814

103.3160*

442.2538*

30.5652

1.4157

±SD

1.377

4.2321

3.9922

2.3448

10.102

4.8076

14.2569

66.0204

6.8288

0.1444

n

10

10

10

10

10

10

10

10

10

10

G3, F & 1000

Mean

4.7580*

18.6552

28.7621

8.4646

30.1827

46.3306

100.2389*

441.7216*

32.5077

1.2517

±SD

0.9269

4.357

5.6529

1.2668

4.6212

4.994

12.8392

41.721

6.4472

0.148

n

10

10

10

10

10

10

10

10

10

10

G4, F & 1500

Mean

4.3449

17.0433

28.2296

8.5147

32.1526

46.9111

89.3484

394.4546

28.4151

1.1688

±SD

0.7681

3.1313

3.9239

1.6412

9.7088

4.9007

14.4414

30.9773

4.2119

0.2108

n

10

10

10

10

10

10

10

10

10

10

G1R, F & 0

Mean

3.204

12.839

22.558

6.5002

38.6043

43.4299

87.2618

358.0398

26.5416

1.187

±SD

0.2741

3.4775

2.4548

0.7592

6.0984

2.5737

10.6446

45.8426

2.89

0.1463

n

5

5

5

5

5

5

5

5

5

5

G4R, F & 1500

Mean

3.072

14.8071

26.3016*

6.8233

35.0169

44.1893

85.4146

389.9831

31.5356

1.372

±SD

0.3627

0.9646

1.9215

1.6405

6.0694

2.8934

10.6219

47.6312

3.9592

0.2071

n

5

5

5

5

5

5

5

5

5

5

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

 

Table no. 17: SUMMARY OF CHAMBER (EXPOSURE) CONDITIONS

Group & Dose

(ppm)

Temp.

Rh

O2

CO2

Air inlet flow rate

BZC

(°C)

(%)

(%)

(ppm)

(L/min)*

(ppm)

G1/G1R & 0

Mean

22.93

55.67

20.3

619.7

20

-

±SD

0.39

0.66

0.2

3.74

0

-

G2 & 500

Mean

22.72

55.77

20.27

618

20

514

±SD

0.27

0.53

0.21

3.71

0

5.61

G3 & 1000

Mean

22.77

55.99

20.25

619.25

20

1021

±SD

0.3

0.43

0.17

2.86

0

8.97

G4/G4R & 1500

Mean

22.78

55.81

20.37

620.15

22.78

1518

±SD

0.3

0.64

0.23

2.91

0.3

8.77

 

Table no. 18: SUMMARY OF NOMINAL CONCENTRATION

Week

Group

Test item Used (mg) (a)

Air flow rate (L/minute) (b)

Minute (min) (c)

Nominal Concentration (mg/L) (a) / (b) × (c)

1

G1 and G1R (Air Only)

-

20

360

-

G2, G3, G4 and G4R (Test Item)

16052

20

360

2.23

32104

4.46

47992

6.67

2

G1 and G1R (Air Only)

-

20

360

-

G2, G3, G4 and G4R (Test Item)

16055

20

360

2.23

32111

4.46

48206

6.7

3

G1 and G1R (Air Only)

-

20

360

-

G2, G3, G4 and G4R (Test Item)

16114

20

360

2.24

32228

4.48

48282

6.71

4

G1 and G1R (Air Only)

-

20

360

-

G2, G3, G4 and G4R (Test Item)

16224

20

360

2.25

32448

4.51

48360

6.72

5

G1 and G1R (Air Only)

-

20

360

-

G2, G3, G4 and G4R (Test Item)

16913

20

360

2.35

33825

4.7

50420

7

6

G1 and G1R (Air Only)

-

20

360

-

G2, G3, G4 and G4R (Test Item)

17443

20

360

2.42

34885

4.85

51276

7.12

7

G1 and G1R (Air Only)

-

20

360

-

G2, G3, G4 and G4R (Test Item)

17242

20

360

2.39

34484

4.79

51230

7.12

8

G1 and G1R (Air Only)

-

20

360

-

G2, G3, G4 and G4R (Test Item)

17129

20

360

2.38

34259

4.76

51512

7.15

9

G1 and G1R (Air Only)

-

20

360

-

G2, G3, G4 and G4R (Test Item)

17466

20

360

2.43

34932

4.85

51936

7.21

10

G1 and G1R (Air Only)

-

20

360

-

G2, G3, G4 and G4R (Test Item)

17703

20

360

2.46

35407

4.92

53184

7.39

11

G1 and G1R (Air Only)

-

20

360

-

G2, G3, G4 and G4R (Test Item)

17647

20

360

2.45

35293

4.9

52788

7.33

12

G1 and G1R (Air Only)

-

20

360

-

G2, G3, G4 and G4R (Test Item)

17620

20

360

2.45

35240

4.89

52676

7.32

13

G1 and G1R (Air Only)

-

20

360

-

G2, G3, G4 and G4R (Test Item)

17658

20

360

2.45

35316

4.91

52784

7.33

 

 

Applicant's summary and conclusion

Conclusions:
Under the experimental conditions and doses employed, it is concluded that the No Observed Adverse Effect Concentration (NOAEC) of test item, Diethyl Ether [CAS No. 60-29-7] was found to be 1500 ppm (high dose group) when exposed for 6 hours for 5 days/week for 13 weeks by flow-past nose-only inhalation route to Sprague Dawley rats.
Executive summary:

The objective of this study was to determine the toxic potential of test item, Diethyl Ether [CAS No. 60-29-7] when exposed for 6 hours/day for 5 days per week for 13 weeks by flow-past nose-only inhalation route to Sprague Dawley rats. This study provides information on major toxic effects, target organs, possibility of cumulative effects and also the reversibility of effects (after 28 days recovery period) and an estimate of the No Observed Adverse Effects Concentration (NOAEC).

A total of 100 (50 males and 50 females) healthy young Sprague Dawley rats were distributed to four main (G1, G2, G3 and G4) and two recovery groups (G1R and G4R). Each main group comprised of 20 animals (10 males and 10 females) and the recovery groups comprised of 10 animals (5 males and 5 females) each. Animals allocated to Groups G2, G3 and G4/G4R were exposed to vapours of test item (Diethyl Ether [CAS No. 60-29-7]) for 6 hours per day, 5 days per week for 13 consecutive weeks. Animals of the control group (G1/GR) received air only inhalation for 6 hours per week, 5 days per week for 13 consecutive weeks. The inhalation exposure of test item/air in animal was achieved by a flow-past, nose-only dynamic inhalation exposure system supplied by CH Technologies, USA.

The actual aerosol concentration was determined in ppm by using Gray Wolf Sensing Solution probe, supplied by CH Technologies, USA. It was carried out four times during exposure, initially before keeping the animals into the chamber and once in two hours further [i.e. 120 minutes (±15 minutes), 240 minutes (±15 minutes) and 360 minutes (±15 minutes)] after equilibration period for each exposure day.

All the animals were observed for clinical signs before exposure and at 1.5 h (±10 mins), 3 h (±10 mins), 4.5 h (±10 mins) and 6 h (±10 mins) during exposure period. The animals were observed for clinical signs and mortality at 30 to 40 minutes and 1.5 h (±10 mins) following exposure to the test item on day 1, thereafter once a day for clinical signs and twice daily for mortality. Detailed clinical examination was carried out weekly. Body weight was recorded twice weekly up to 4 weeks for all groups and once weekly from week 5 onwards. Food consumption was measured weekly. Ophthalmoscopic examination was performed during acclimatization for all the groups and during week 13 for main groups (G1 and G4) and during week 17 for recovery groups (G1R and G4R). Neurological/Functional observational battery was performed during week 13 for G1 and G4 groups and during week 17 for recovery group animals.

At the end of treatment and recovery period, all animals were fasted overnight (water was provided ad libitum) and the next day, blood, urine and broncho-alveolar lavage fluid (BALF) samples were collected and analysed. Subsequently, the animals were sacrificed and subjected to gross pathological examination and the organs were collected, weighed and preserved. The tissues/organs in vehicle control (G1) and high dose (G4) group animals were subjected to histopathological examination.

The chamber conditions like temperature, relative humidity, oxygen and carbon dioxide concentrations determined during the exposure period were found within the specified range.

No clinical signs of toxicity and mortality was noted in any of the doses.

No treatment related changes in body weight, percent change in body weight with respect to day 1, feed consumption and ophthalmoscopic examination were noted. No adverse variations were noted in neurological/functional examination test. No adverse treatment related changes were noted in haematology, clinical chemistry, coagulation, BALF analysis and urinalysis parameters. No toxicologically significant changes were noted in fasting body weights, organ weights and its ratios. No treatment related gross and histopathology changes were noted in any organ and/or tissues at the high dose group animals.

Thus, under the experimental conditions and doses employed, it is concluded that the No Observed Adverse Effect Concentration (NOAEC) of the test item Diethyl Ether [CAS No. 60-29-7] was found to be 1500 ppm when exposed for 6 h for 5 days/week for 13 weeks by flow-past nose-only inhalation route to Sprague Dawley rats.