N-(2,3-dihydro-2-oxo-1H-benzimidazol-5-yl)-2-[(4-nitrophenyl)azo]-3-oxobutyramide

Repeated dose toxicity: inhalation

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

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)

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

252-650-1

EC Name:

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

Cas Number:

35636-63-6

Molecular formula:

C21H19N5O7

IUPAC Name:

dimethyl 2-({2-oxo-1-[(2-oxo-2,3-dihydro-1H-benzimidazol-5-yl)carbamoyl]propyl}diazenyl)terephthalate

Test material form:

solid: nanoform

Test animals

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

Administration / exposure

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

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/L air (nominal)

Remarks:

Air Only

Doses / concentrations 2

Dose / conc.:

0.001 mg/L air (nominal)

Remarks:

Low Dose

Doses / concentrations 3

Dose / conc.:

0.003 mg/L air (nominal)

Remarks:

Mid Dose

Doses / concentrations 4

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

Examinations

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).

Results and discussion

Results of examinations

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.

Effect levels

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

Target system / organ toxicity

Key result

Critical effects observed:

no

Any other information on results incl. tables

TABLE 1.   SUMMARY OF CLINICAL SIGNS AND MORTALITY RECORD

Group, Sex & Dose (mg/L)	No. of Animals	Clinical Signs of Toxicity	Mortality (No. of Incidences/ No. of Animals)
G1, M & 0	5	N	0/5
G2, M & 0.001	5	N	0/5
G3, M & 0.003	5	N	0/5
G4, M & 0.03	5	N	0/5
G1R, M & 0	5	N	0/5
G4R, M & 0.03	5	N	0/5

M: Male; N: None.

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

Group, Sex & Dose (mg/L)	No. of Animals	Clinical Signs of Toxicity	Mortality (No. of Incidence/ No. of Animals)
G1, F & 0	5	N	0/5
G2, F & 0.001	5	N	0/5
G3, F & 0.003	5	N	0/5
G4, F & 0.03	5	N	0/5
G1R, F & 0	5	N	0/5
G4R, F & 0.03	5	N	0/5

F: Female; N: None.

TABLE 2.  SUMMARY OF BODY WEIGHTS (g) RECORD

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

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

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

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

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

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

Group, Sex & Dose (mg/L)		Percent Change in Body Weight (%) during Days
		1 to 5	1 to 8	1 to 12	1 to 15	1 to 22	1 to 26
G1, M & 0	Mean	2.16	9.19	11.44	17.71	25.05	28.59
	±SD	0.52	1.38	1.95	2.09	2.93	3.31
	n	5	5	5	5	5	5
G2, M & 0.001	Mean	2.10	8.58	10.93	16.97	24.38	28.62
	±SD	0.42	1.46	1.86	2.19	2.28	2.25
	n	5	5	5	5	5	5
G3, M & 0.003	Mean	1.89	8.50	10.98	17.87	25.84	29.76
	±SD	0.53	1.67	2.17	3.16	3.61	4.10
	n	5	5	5	5	5	5
G4, M & 0.03	Mean	2.18	9.25	11.86	18.72	26.75	31.25
	±SD	0.62	1.77	2.42	3.76	4.74	5.28
	n	5	5	5	5	5	5
G1, F & 0	Mean	1.99	6.19	7.75	13.25	20.30	24.59
	±SD	0.53	1.70	2.28	2.33	2.66	3.28
	n	5	5	5	5	5	5
G2, F & 0.001	Mean	2.40	6.41	8.15	13.90	20.15	24.06
	±SD	0.28	0.53	1.01	1.18	2.05	1.85
	n	5	5	5	5	5	5
G3, F & 0.003	Mean	2.13	7.12	8.54	13.96	20.34	24.89
	±SD	0.72	1.16	1.75	2.21	3.07	2.90
	n	5	5	5	5	5	5
G4, F & 0.03	Mean	1.78	5.48	7.22	12.39	18.86	22.74
	±SD	0.29	0.59	1.35	1.44	0.90	1.30
	n	5	5	5	5	5	5

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

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

Group, Sex & Dose (mg/L)		Percent Change in Body Weight (%) during Days
		1 to 5	1 to 8	1 to 12	1 to 15	1 to 22	1 to 26	1 to 29	1 to 36	1 to 43	1 to 50	1 to 57	1 to 64	1 to 71	1 to 78	1 to 84
G1R, M & 0	Mean	2.35	9.31	11.59	18.14	25.84	31.73	36.01	47.54	59.58	71.99	84.25	96.85	109.61	123.32	134.68
	±SD	0.30	0.61	1.51	1.92	2.46	3.09	3.31	3.90	3.97	4.48	5.40	5.85	6.55	7.11	7.80
	n	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5
G4R, M & 0.03	Mean	2.06	8.94	11.20	17.86	25.92	33.48	37.82	49.42	61.60	74.08	86.54	99.24	111.99	125.80	137.28
	±SD	0.45	0.81	0.61	0.99	1.38	3.24	3.40	4.36	4.67	5.39	5.93	6.81	7.54	8.13	8.78
	n	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5
G1R, F & 0	Mean	1.84	5.48	7.11	12.75	19.33	24.13	27.57	37.71	48.14	58.41	68.75	79.81	90.99	102.65	112.97
	±SD	0.41	1.23	1.71	2.42	4.43	4.95	4.92	5.32	6.27	6.72	7.66	8.36	9.11	10.22	11.06
	n	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5
G4R, F & 0.03	Mean	1.87	5.81	7.39	14.41	20.25	25.20	28.63	38.75	49.05	59.39	69.67	80.81	92.06	103.64	113.99
	±SD	0.23	1.11	1.55	2.31	2.97	3.11	3.53	4.27	4.67	5.22	6.01	6.67	7.64	8.66	9.19
	n	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5

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

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

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

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

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

Group, Sex & Dose (mg/L)		Week 1	Week 2	Week 3	Week 4	Week 5	Week 6	Week 7	Week 8	Week 9	Week 10	Week 11	Week 12
G1R, M & 0	Mean	18.58	18.64	18.42	18.94	18.81	19.28	19.29	19.31	19.34	19.25	19.39	22.32
	±SD	2.54	2.78	2.63	3.14	3.13	3.09	3.30	3.52	3.64	3.55	3.65	4.15
	n	2	2	2	2	2	2	2	2	2	2	2	2
G4R, M & 0.03	Mean	18.63	18.68	18.50	18.63	18.66	19.20	19.21	19.32	19.45	19.33	19.46	22.34
	±SD	2.65	2.89	2.76	2.78	2.98	3.14	3.30	3.21	3.30	3.24	3.29	3.79
	n	2	2	2	2	2	2	2	2	2	2	2	2
G1R, F & 0	Mean	14.24	14.26	14.25	14.15	14.30	15.14	15.26	15.30	15.57	15.58	15.67	18.02
	±SD	1.57	1.28	1.39	1.84	1.88	1.82	1.84	2.09	2.21	2.01	1.97	2.33
	n	2	2	2	2	2	2	2	2	2	2	2	2
G4R, F & 0.03	Mean	14.07	14.19	14.19	14.15	14.36	14.74	14.93	15.16	15.33	15.27	15.43	17.69
	±SD	1.48	1.88	1.67	1.78	1.94	2.25	2.19	2.27	2.43	2.11	2.13	2.41
	n	2	2	2	2	2	2	2	2	2	2	2	2

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

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

Week 4
Parameters↓	Group & Sex		G1 & M	G4 & M	G1 & F	G4 & F
	Dose (mg/L)		0	0.03	0	0.03
	Number of Animals		5	5	5	5
Home cage observations
Home Cage posture			1	1	1	1
Respiratory pattern			1	1	1	1
Clonic involuntary movements			1	1	1	1
Tonic involuntary movements			1	1	1	1
Vocalization			1	1	1	1
Palpebral Closure			1	1	1	1
Handling observation
Ease of removal from the cage			2	2	2	2
Ease of handling animal in hand			2	2	2	2
Red or crusty deposits		Eyes	1	1	1	1
		Nose	1	1	1	1
		Mouth	1	1	1	1
Lacrimation			1	1	1	1
Salivation			1	1	1	1
Fur Appearance			1	1	1	1
Piloerection			1	1	1	1
Eye Prominence			1	1	1	1
Muscle Tone			1	1	1	1
Home cage observations: Home cage posture- 1=Normal,Respiratory Pattern -1=Normal,Clonic involuntary movements -1=None/Normal,Tonic involuntary movements -1=None/Normal,Vocalization -1=Absent (Normal),Palpebral closure -1=Eyelids wide open (Normal),Handling observation : Ease of removal from the cage -2=Normal,Ease of handling animal in hand -2=Normal,Red/Crusty deposits -1=Absent,Lacrimation- 1=None,Salivation-1=Absent (Normal),Fur Appearance -1=Normal,Piloerection -1=None, Eye Prominence -1=Normal,Muscle tone -1=Normal

M: Male; F: Female.

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

Week 4
Parameters↓	Group & Sex	G1 & M	G4 & M	G1 & F	G4 & F
	Dose (mg/L)	0	0.03	0	0.03
	Number of Animals	5	5	5	5
Open field Observation
Mobility		1	1	1	1
Gait		1	1	1	1
Arousal		3	3	3	3
Number of Rearing	Mean	7.4	6.6	7.8	7.6
	±SD	1.5	0.9	1.9	1.1
Numbers of Urination	Mean	1.4	1.4	1.4	1.6
	±SD	0.5	0.5	0.5	0.5
Number of Defecation	Mean	1.0	1.0	1.0	0.8
	±SD	0.7	0.7	0.7	0.4
Clonic involuntary movement		1	1	1	1
Tonic involuntary movement		1	1	1	1
Stereotype Behaviour		1	1	1	1
Excessive Grooming	Mean	3.8	4.0	4.2	4.4
	±SD	0.4	1.0	0.8	0.5
Sensory Observations
Approach Response		2	2	2	2
Auditory Response		2	2	2	2
Touch Response		2	2	2	2
Pupil Reflex		2	2	2	2
Tail Pinch Response		2	2	2	2
Righting Reflex		1	1	1	1
Physiological observation
Body temperature (°F)	Mean	98.2	98.2	98.5	98.5
	±SD	0.2	0.2	0.1	0.1
Open field Observation: Mobility -1=Normal,Gait -1=Normal,Arousal -3=Normal,Clonic involuntary movements -1=None/Normal,Tonic involuntary movements -1=None/Normal,Stereotype Behaviour -1=Absent, Sensory Observations: Approach response -2=Normal, Auditory Response -2=Normal,Touch Response -2=Normal,Pupil Reflex -2=Normal,Tail Pinch Response -2=Normal,Righting Reflex -1=Present

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

Week 12
Parameters↓	Group & Sex		G1R & M	G4R & M	G1R & F	G4R & F
	Dose (mg/L)		0	0.03	0	0.03
	Number of Animals		5	5	5	5
Home cage observations
Home Cage posture			1	1	1	1
Respiratory pattern			1	1	1	1
Clonic involuntary movements			1	1	1	1
Tonic involuntary movements			1	1	1	1
Vocalization			1	1	1	1
Palpebral Closure			1	1	1	1
Handling observation
Ease of removal from the cage			2	2	2	2
Ease of handling animal in hand			2	2	2	2
Red or crusty deposits		Eyes	1	1	1	1
		Nose	1	1	1	1
		Mouth	1	1	1	1
Lacrimation			1	1	1	1
Salivation			1	1	1	1
Fur Appearance			1	1	1	1
Piloerection			1	1	1	1
Eye Prominence			1	1	1	1
Muscle Tone			1	1	1	1
Home cage observations: Home cage posture- 1=Normal,Respiratory Pattern -1=Normal,Clonic involuntary movements -1=None/Normal,Tonic involuntary movements -1=None/Normal,Vocalization -1=Absent (Normal),Palpebral closure -1=Eyelids wide open (Normal),Handling observation : Ease of removal from the cage -2=Normal,Ease of handling animal in hand -2=Normal,Red/Crusty deposits -1=Absent,Lacrimation- 1=None,Salivation-1=Absent (Normal),Fur Appearance -1=Normal,Piloerection -1=None, Eye Prominence -1=Normal,Muscle tone -1=Normal

M: Male; F: Female; R: Recovery.

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

Week 12
Parameters↓	Group & Sex	G1R & M	G4R & M	G1R & F	G4R & F
	Dose (mg/L)	0	0.03	0	0.03
	Number of Animals	5	5	5	5
Open field Observation
Mobility		1	1	1	1
Gait		1	1	1	1
Arousal		3	3	3	3
Number of Rearing	Mean	7.8	7.6	8.8	9.0
	±SD	0.8	1.7	0.8	1.0
Numbers of Urination	Mean	1.2	1.2	1.0	1.4
	±SD	0.8	0.4	0.7	0.5
Number of Defecation	Mean	1.0	1.0	1.2	1.0
	±SD	0.7	0.7	0.4	0.7
Clonic involuntary movement		1	1	1	1
Tonic involuntary movement		1	1	1	1
Stereotype Behaviour		1	1	1	1
Excessive Grooming	Mean	4.2	3.4	4.0	5.4*
	±SD	0.8	0.9	0.7	0.5
Sensory Observations
Approach Response		2	2	2	2
Auditory Response		2	2	2	2
Touch Response		2	2	2	2
Pupil Reflex		2	2	2	2
Tail Pinch Response		2	2	2	2
Righting Reflex		1	1	1	1
Physiological observation
Body temperature (°F)	Mean	97.9	97.9	98.7	98.7
	±SD	0.2	0.2	0.2	0.2
Open field Observation: Mobility -1=Normal,Gait -1=Normal,Arousal -3=Normal,Clonic involuntary movements -1=None/Normal,Tonic involuntary movements -1=None/Normal,Stereotype Behaviour -1=Absent, Sensory Observations: Approach response -2=Normal, Auditory Response -2=Normal,Touch Response -2=Normal,Pupil Reflex -2=Normal,Tail Pinch Response -2=Normal,Righting Reflex -1=Present

M: Male; F: Female; R: Recovery; *: Statistically significant (p<0.05).

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

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

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

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

TABLE 7. SUMMARY OF ACTIMETER READING RECORD

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

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

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

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

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

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

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

TABLE 9.     SUMMARY OF OPHTHALMOSCOPIC EXAMINATION RECORD

Week 4					Week 12
Group, Sex & Dose (mg/L)	G1, M & 0		G4, M & 0.03		G1R, M & 0		G4R, M & 0.03
Number of Animals	05		05		05		05
Observations
Eye Parameters ↓
Eye	L	R	L	R	L	R	L	R
Eye Lids	N	N	N	N	N	N	N	N
Cornea	N	N	N	N	N	N	N	N
Iris	N	N	N	N	N	N	N	N
Aqueous Humour	N	N	N	N	N	N	N	N
Lens	N	N	N	N	N	N	N	N
Vitreous Humour	N	N	N	N	N	N	N	N
Retina/Optic disc	N	N	N	N	N	N	N	N
	N	N	N	N	N	N	N	N
	N	N	N	N	N	N	N	N
	N	N	N	N	N	N	N	N
	N	N	N	N	N	N	N	N

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

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

Week 4					Week 12
Group, Sex & Dose (mg/L)	G1, F & 0		G4, F & 0.03		G1R, F & 0		G4R, F & 0.03
Number of Animals	05		05		05		05
Observations
Eye Parameters ↓
Eye	L	R	L	R	L	R	L	R
Eye Lids	N	N	N	N	N	N	N	N
Cornea	N	N	N	N	N	N	N	N
Iris	N	N	N	N	N	N	N	N
Aqueous Humour	N	N	N	N	N	N	N	N
Lens	N	N	N	N	N	N	N	N
Vitreous Humour	N	N	N	N	N	N	N	N
Retina/Optic disc	N	N	N	N	N	N	N	N
	N	N	N	N	N	N	N	N
	N	N	N	N	N	N	N	N
	N	N	N	N	N	N	N	N
	N	N	N	N	N	N	N	N

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

TABLE 10.     SUMMARY OF HAEMATOLOGY RECORD

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

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

TABLE 10 (Contd…). SUMMARY OF HAEMATOLOGY RECORD

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

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

TABLE 10 (Contd…). SUMMARY OF HAEMATOLOGY RECORD

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

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

TABLE 10 (Contd…). SUMMARY OF HAEMATOLOGY RECORD

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

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

TABLE 10 (Contd…). SUMMARY OF HAEMATOLOGY RECORD

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

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

TABLE 10 (Contd…). SUMMARY OF HAEMATOLOGY RECORD

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

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

TABLE 11. SUMMARY OF BALF RECORD

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

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

TABLE 11 (Contd…). SUMMARY OF BALF RECORD

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

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

TABLE 11 (Contd…). SUMMARY OF BALF RECORD

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

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

TABLE 11 (Contd…). SUMMARY OF BALF RECORD

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

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

 TABLE 12. SUMMARY OF CLINICAL CHEMISTRY RECORD

Group, Sex & Dose (mg/L)		Glucose	Urea	Creatinine	Total Cholesterol	Triglycerides	Total Protein	Albumin	Alanine aminotransferase	Aspartate aminotransferase
		(GLU)		(CRE)	(CHO)	(TRI)	(TPR)	(ALB)	(ALT)	(AST)
		(mg/dL)	(mg/dL)	(mg/dL)	(mg/dL)	(mg/dL)	(g/dL)	(g/dL)	(U/L)	(U/L)
G1, M & 0	Mean	91.60	31.90	0.41	42.00	44.60	6.82	3.09	53.20	94.20
	±SD	8.88	2.88	0.03	8.40	8.85	0.48	0.07	13.94	10.66
	n	5	5	5	5	5	5	5	5	5
G2, M & 0.001	Mean	85.00	31.50	0.40	49.60	41.20	7.00	3.15	57.40	103.20
	±SD	8.51	1.64	0.01	6.43	12.76	0.20	0.15	7.57	12.77
	n	5	5	5	5	5	5	5	5	5
G3, M & 0.003	Mean	97.00	32.28	0.39	47.60	49.20	6.90	3.10	53.40	108.20
	±SD	15.70	4.40	0.02	11.01	8.23	0.30	0.11	6.19	7.76
	n	5	5	5	5	5	5	5	5	5
G4, M & 0.03	Mean	86.60	35.32	0.38	50.40	48.00	6.64	2.99	71.00	109.00
	±SD	9.71	11.73	0.03	12.30	13.67	0.43	0.17	15.89	21.51
	n	5	5	5	5	5	5	5	5	5
G1R, M & 0	Mean	77.20	34.62	0.52	53.60	29.80	7.52	3.37	62.20	106.60
	±SD	10.08	6.83	0.05	7.47	4.71	0.29	0.18	7.26	17.56
	n	5	5	5	5	5	5	5	5	5
G4R, M & 0.03	Mean	89.00	36.06	0.53	52.60	33.60	7.66	3.50	62.00	104.40
	±SD	6.71	1.99	0.05	8.68	9.02	0.15	0.33	13.62	14.06
	n	5	5	5	5	5	5	5	5	5

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

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

Group, Sex & Dose (mg/L)		Alkaline phosphatase	Total Bilirubin	Calcium	Phosphorous	Globulin	Blood Urea Nitrogen	Sodium	Potassium	Chloride
		(ALP)	(BIT)	(CAL)	(PHO)	(GLO)	(BUN)	(Na)	(K)	(CLO)
		(U/L)	(mg/dL)	(mg/dL)	(mg/dL)	(g/dL)	(mg/dL)	(mmol/L)	(mmol/L)	(mmol/L)
G1, M & 0	Mean	202.00	0.01	9.78	7.30	3.73	14.89	142.10	3.87	109.74
	±SD	79.50	0.02	0.33	0.63	0.45	1.34	0.83	0.18	0.91
	n	5	5	5	5	5	5	5	5	5
G2, M & 0.001	Mean	178.80	0.04	9.92	7.24	3.85	14.70	142.12	3.97	110.44
	±SD	21.75	0.02	0.29	0.43	0.15	0.76	0.89	0.16	0.73
	n	5	5	5	5	5	5	5	5	5
G3, M & 0.003	Mean	184.40	0.04	9.94	7.14	3.80	15.06	141.14	3.96	109.42
	±SD	47.26	0.05	0.21	0.55	0.33	2.05	0.91	0.29	1.27
	n	5	5	5	5	5	5	5	5	5
G4, M & 0.03	Mean	188.80	0.03	9.80	7.44	3.65	16.48	142.92	3.94	111.54*
	±SD	57.73	0.02	0.12	0.36	0.27	5.47	0.91	0.09	0.52
	n	5	5	5	5	5	5	5	5	5
G1R, M & 0	Mean	142.60	0.05	9.60	6.08	4.15	16.16	144.46	3.54	105.24
	±SD	31.91	0.01	0.28	0.47	0.26	3.19	0.27	0.34	1.42
	n	5	5	5	5	5	5	5	5	5
G4R, M & 0.03	Mean	163.80	0.05	9.52	5.36	4.16	16.83	144.52	3.55	106.68
	±SD	44.64	0.01	0.36	0.92	0.30	0.93	0.64	0.12	0.63
	n	5	5	5	5	5	5	5	5	5

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

TABLE 12 (Contd…). SUMMARY OF CLINICAL CHEMISTRY

Group, Sex & Dose (mg/L)		Glucose	Urea	Creatinine	Total Cholesterol	Triglycerides	Total Protein	Albumin	Alanine aminotransferase	Aspartate aminotransferase
		(GLU)		(CRE)	(CHO)	(TRI)	(TPR)	(ALB)	(ALT)	(AST)
		(mg/dL)	(mg/dL)	(mg/dL)	(mg/dL)	(mg/dL)	(g/dL)	(g/dL)	(U/L)	(U/L)
G1, F & 0	Mean	95.80	29.54	0.39	53.60	48.60	7.08	3.35	42.60	91.20
	±SD	9.86	4.95	0.04	11.74	5.50	0.36	0.25	7.44	8.76
	n	5	5	5	5	5	5	5	5	5
G2, F & 0.001	Mean	100.20	28.98	0.37	54.40	50.80	7.32	3.33	44.20	96.00
	±SD	2.68	2.77	0.01	6.62	14.79	0.22	0.13	6.61	6.63
	n	5	5	5	5	5	5	5	5	5
G3, F & 0.003	Mean	102.20	30.38	0.35	59.60	37.40	7.28	3.34	46.80	105.40
	±SD	5.45	5.89	0.03	14.59	5.94	0.28	0.11	12.19	10.31
	n	5	5	5	5	5	5	5	5	5
G4, F & 0.03	Mean	108.00	32.98	0.37	58.20	45.20	7.20	3.16	38.20	92.00
	±SD	11.98	7.09	0.07	14.75	14.13	0.61	0.18	9.98	27.06
	n	5	5	5	5	5	5	5	5	5
G1R, F & 0	Mean	90.80	40.48	0.59	73.60	37.40	8.14	3.92	41.60	88.60
	±SD	8.64	2.53	0.04	27.56	10.43	0.58	0.32	5.03	14.03
	n	5	5	5	5	5	5	5	5	5
G4R, F & 0.03	Mean	98.00	36.66	0.57	72.40	38.20	8.14	3.95	40.80	85.60
	±SD	13.69	4.61	0.06	19.49	5.36	0.42	0.26	5.89	15.04
	n	5	5	5	5	5	5	5	5	5

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

TABLE 12 (Contd…). SUMMARY OF CLINICAL CHEMISTRY

Group, Sex & Dose (mg/L)		Alkaline phosphatase	Total Bilirubin	Calcium	Phosphorous	Globulin	Blood Urea Nitrogen	Sodium	Potassium	Chloride
		(ALP)	(BIT)	(CAL)	(PHO)	(GLO)	(BUN)	(Na)	(K)	(CLO)
		(U/L)	(mg/dL)	(mg/dL)	(mg/dL)	(g/dL)	(mg/dL)	(mmol/L)	(mmol/L)	(mmol/L)
G1, F & 0	Mean	105.20	0.02	10.40	7.02	3.73	13.79	140.90	4.12	111.32
	±SD	23.99	0.02	0.31	0.54	0.24	2.31	0.99	0.29	2.77
	n	5	5	5	5	5	5	5	5	5
G2, F & 0.001	Mean	96.00	0.03	10.14	6.98	3.99	13.53	141.66	4.05	111.20
	±SD	12.41	0.01	0.32	0.87	0.34	1.29	1.60	0.70	1.14
	n	5	5	5	5	5	5	5	5	5
G3, F & 0.003	Mean	140.60	0.04	10.28	6.80	3.94	14.18	141.68	3.82	111.46
	±SD	71.25	0.01	0.36	0.94	0.33	2.75	1.44	0.23	1.94
	n	5	5	5	5	5	5	5	5	5
G4, F & 0.03	Mean	129.80	0.03	10.34	7.20	4.04	15.39	142.58	4.07	112.06
	±SD	26.81	0.02	0.30	0.35	0.46	3.31	1.00	0.20	0.86
	n	5	5	5	5	5	5	5	5	5
G1R, F & 0	Mean	80.00	0.06	10.02	5.12	4.22	18.89	143.94	3.56	106.40
	±SD	19.27	0.01	0.49	0.79	0.32	1.18	0.88	0.41	1.02
	n	5	5	5	5	5	5	5	5	5
G4R, F & 0.03	Mean	62.20	0.07	10.06	4.72	4.19	17.11	143.88	3.17	106.60
	±SD	10.69	0.02	0.26	0.85	0.47	2.15	1.39	0.29	1.04
	n	5	5	5	5	5	5	5	5	5

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

TABLE 13.  SUMMARY OF URINALYSIS

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

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

TABLE 13 (Contd…). SUMMARY OF URINALYSIS

Examination	Group, Sex & Dose (mg/L)		G1, F & 0	G2, F & 0.001	G3, F & 0.003	G4, F & 0.03	G1R, F & 0	G4R, F & 0.03
	Number of Animals		5	5	5	5	5	5
Physical	Colour	Pale Yellow	5	4	4	4	5	5
		Yellow	-	1	1	1	-	-
	Appearance	Clear	5	3	4	4	5	5
		Turbid	-	2	1	1	-	-
	Volume (mL)	Mean	6.1	6.6	7.6	6.6	6.6	6.9
		±SD	2.3	0.8	2.3	2.9	1.0	1.3
Chemical	pH	Mean	6.6	7.1	6.8	6.4	7.0	7.6
		±SD	1.7	1.3	1.2	1.0	0.4	0.7
	Specific Gravity	Mean	1.006	1.005	1.005	1.005	1.013	1.009*
		±SD	0.002	0.000	0.000	0.000	0.003	0.002
	Urobilinogen (mg/dL)	Mean	0.2	0.2	0.2	0.2	0.2	0.2
		±SD	0.0	0.0	0.0	0.0	0.0	0.0
	Bilirubin (mg/dL)	Neg	5	5	5	5	5	5
	Ketones (mg/dL)	Neg	5	5	5	5	5	5
	Blood (Ery/µL)	Neg	4	1	1	-	4	4
		Ca10	-	-	-	3	1	-
		Ca25	-	3	4	2	-	1
		Ca80	1	1	-	-	-	-
	Proteins (mg/dL)	Neg	1	3	2	4	5	3
		Trace	3	2	3	1	-	1
		30	1	-	-	-	-	-
		100	-	-	-	-	-	1
	Nitrite	Neg	4	4	4	5	3	5
		Pos	1	1	1	-	2	-
	Leucocytes (Leu/µL)	Neg	1	-	-	-	4	4
		Ca15	4	5	5	5	1	-
		Ca70	-	-	-	-	-	1
	Glucose (mg/dL)	Neg	5	5	5	5	5	5
	Micro albumin (MALB) (mg/dL)	Neg	1	3	2	4	3	3
		>15	4	2	3	1	-	2
		15	-	-	-		2	-
Microscopic	Epithelial cells	0	1	1	2	2	5	4
		0-1	3	1	1	3	-	1
		1-2	1	3	2	-	-	-
	Casts	Absent	4	5	4	5	5	5
		Present	1	-	1	-	-	-
	Crystals	Absent	2	1	-	3	-	-
		Present	3	4	5	2	5	5

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

*: Statistically significant (p<0.05).

TABLE 14.      SUMMARY OF ABSOLUTE ORGAN WEIGHTS (g)

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

TABLE 17.      SUMMARY OF CHAMBER (EXPOSURE) CONDITIONS

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

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

     CO₂: Carbon Dioxide Concentration;BZC: Breathing Zone Concentration:

     SD: Standard Deviation.

TABLE 18.    SUMMARY OF NOMINAL CONCENTRATION

Day	Group	Test item Used (mg) (a)	Air flow rate (L/minute)(b)	Minute (min) (c)	Nominal Concentration (mg/L) (a)    / (b) × (c)
1	G1/G1R  (Air Only)	-	20	360	-
	G2, G3 and G4/G4R  (Test Item)	1150	20	360	0.16
		4370			0.61
		5230			0.73
2	G1/G1R (Air Only)	-	20	360	-
	G2, G3 and G4/G4R  (Test Item)	1120	20	360	0.16
		4440			0.62
		5110			0.71
3	G1/G1R  (Air Only)	-	20	360	-
	G2, G3 and G4/G4R  (Test Item)	1200	20	360	0.17
		4380			0.61
		5140			0.71
4	G1/G1R  (Air Only)	-	20	360	-
	G2, G3 and G4/G4R  (Test Item)	1160	20	360	0.16
		4350			0.60
		5160			0.72
5	G1/G1R  (Air Only)	-	20	360	-
	G2, G3 and G4/G4R  (Test Item)	1180	20	360	0.16
		4430			0.62
		5200			0.72
6	G1/G1R  (Air Only)	-	20	360	-
	G2, G3 and G4/G4R  (Test Item)	1090	20	360	0.15
		4340			0.60
		5180			0.72
7	G1/G1R  (Air Only)	-	20	360	-
	G2, G3 and G4/G4R  (Test Item)	1170	20	360	0.16
		4430			0.62
		5190			0.72

TABLE 18 (Contd…). SUMMARY OF NOMINAL CONCENTRATION

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

TABLE 18 (Contd…). SUMMARY OF NOMINAL CONCENTRATION

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

TABLE 19.     SUMMARY OF GROSS PATHOLOGY FINDINGS

Sex	Male
Route of administration	Inhalation
Treatment	Air only	Low Dose	Mid Dose	High Dose	Air only	High Dose Recovery
Group	G1	G2	G3	G4	G1R	G4R
Nominal Target Concentration (mg/L)	0	0.001*	0.003*	0.03*	0	0.03*
Number of animals	5	5	5	5	5	5
No. of dead rats during treatment	-	-	-	-	-	-
No. of moribund sacrificed rats	-	-	-	-	-	-
No. of terminally sacrificed rats	5	5	5	5	5	5
No. of rats showing gross pathology	-	-	-	-	-	-

Sex	Female
Route of administration	Inhalation
Treatment	Air only	Low Dose	Mid Dose	High Dose	Air only	High Dose Recovery
Group	G1	G2	G3	G4	G1R	G4R
Nominal Target Concentration (mg/L)	0	0.001*	0.003*	0.03*	0	0.03*
Number of animals	5	5	5	5	5	5
No. of dead rats during treatment	-	-	-	-	-	-
No. of moribund sacrificed rats	-	-	-	-	-	-
No. of terminally sacrificed rats	5	5	5	5	5	5
No. of rats showing gross pathology	-	-	1	-	-	-
Uterus– small in size	-	-	1	-	-	-

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

TABLE 20.      SUMMARY OF HISTOPATHOLOGY FINDINGS – MAIN GROUP

Route of administration		Inhalation
Treatment		Air only		High Dose
Nominal Target Concentration (mg/L)		0		0.03
Group		G1		G4
Sex		M	F	M	F
Number of Animals		5	5	5	5
Adrenals	Number examined	5	5	5	5
	Within normal limits	5	5	5	5
Brain	Number examined	5	5	5	5
	Within normal limits	5	5	5	5
Cervical lymph nodes	Number examined	5	5	5	5
	Within normal limits	5	5	5	5
Esophagus	Number examined	5	5	5	5
	Within normal limits	5	5	5	5
Eyes with optic nerve and eye lids	Number examined	5	5	5	5
	Within normal limits	5	5	5	5
Heart	Number examined	5	5	5	5
	Within normal limits	5	5	5	5
Hilar lymph nodes	Number examined	5	5	5	5
	Within normal limits	5	5	5	5
Kidneys	Number examined	5	5	5	5
	Within normal limits	4	4	3	4
Cyst	Present	1	1	2	-
Cast	Minimal	1	-	-	-
Basophilia, tubules	Minimal	-	-	1	-
Dilatation, tubules	Minimal	-	-	-	1
Larynx	Number examined	5	5	5	5
	Within normal limits	5	5	5	5
Liver	Number examined	5	5	5	5
	Within normal limits	5	5	5	5
Lungs(left lobe)	Number examined	5	5	5	5
	Within normal limits	5	5	5	5
Mandibular lymph nodes	Number examined	5	5	5	5
	Within normal limits	5	5	5	5
Mesenteric lymph nodes	Number examined	5	5	5	5
	Within normal limits	5	5	5	5
Nasopharyngeal tissue	Number examined	5	5	5	5
	Within normal limits	5	5	5	5
Olfactory bulb	Number examined	5	5	5	5
	Within normal limits	5	5	5	5
Ovaries	Number examined	X	5	X	5
	Within normal limits	X	5	X	5
Parathyroid	Number examined	5	5	5	5
	Within normal limits	5	5	5	5

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

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

Route of administration		Inhalation
Treatment		Air only		High Dose
Nominal Target Concentration (mg/L)		0		0.03
Group		G1		G4
Sex		M	F	M		F
Number of Animals		5	5	5		5
Seminal vesicles	Number examined	5	X	5		X
	Within normal limits	5	X	4		X
Inflammation	Mild	-	X	1		X
Spinal cord	Number examined	5	5	5		5
	Within normal limits	5	5	5		5
Spleen	Number examined	5	5	5	5
	Within normal limits	5	5	5	5
Stomach	Number examined	5	5	5	5
	Within normal limits	5	5	5	5
Testes	Number examined	5	X	5	X
	Within normal limits	4	X	5	X
Atrophy, tubules	Minimal	1	X	-	X
Thymus	Number examined	5	5	5	5
	Within normal limits	5	3	5	4
Cyst, epithelial	Present	-	2	-	1
Thyroid	Number examined	5	5	5	5
	Within normal limits	5	3	3	5
Cyst, ultimobranchial	Present	-	1	2	-
Ectopic tissue, thymus	Present	-	1	-	-
Trachea	Number examined	5	5	5	5
	Within normal limits	5	5	5	5
Urinary bladder	Number examined	5	5	5	5
	Within normal limits	5	5	5	5
Uterus	Number examined	X	5	X	5
	Within normal limits	X	5	X	4
Dilatation, lumen	Minimal	X	-	X	1

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

TABLE 21. SUMMARY OF HISTOPATHOLOGY FINDINGS – RECOVERY GROUP

Route of administration		Inhalation
Treatment		Air only		High Dose Recovery
Nominal Target Concentration (mg/L)		0		0.03
Group		G1R		G4R
Sex		M	F	M	F
Number of Animals		5	5	5	5
Adrenals	Number examined	5	5	5	5
	Within normal limits	4	5	4	5
Ectopic tissue, adrenocortical	Present	1	-	1	-
Brain	Number examined	5	5	5	5
	Within normal limits	5	5	5	5
Cervical lymph nodes	Number examined	5	5	5	5
	Within normal limits	4	5	5	5
Cellularity, increased, lymphocytes	Mild	1	-	-	-
Esophagus	Number examined	5	5	5	5
	Within normal limits	5	5	5	5
Eyes with optic nerve and eye lids	Number examined	5	5	5	5
	Within normal limits	5	5	5	5
Heart	Number examined	5	5	5	5
	Within normal limits	5	5	5	5
Hilar lymph nodes	Number examined	5	5	5	5
	Within normal limits	5	5	5	5
Kidneys	Number examined	5	5	5	5
	Within normal limits	4	2	5	5
Infiltrate, mononuclear cells	Minimal	1	1	-	-
Cyst	Present	-	1	-	-
Dilatation, tubules	Minimal	-	1	-	-
Larynx	Number examined	5	5	5	5
	Within normal limits	5	5	5	5
Liver	Number examined	5	5	5	5
	Within normal limits	5	5	5	5
Lungs(left lobe)	Number examined	5	5	5	5
	Within normal limits	3	3	3	3
Pigment (background finding-likely hemosiderin)	Minimal	2	2	2	1
	Mild	-	-	-	1
Mandibular lymph nodes	Number examined	5	5	5	5
	Within normal limits	5	5	5	5
Mesenteric lymph nodes	Number examined	5	5	5	5
	Within normal limits	5	5	5	5
Nasopharyngeal tissue	Number examined	5	5	5	5
	Within normal limits	5	5	5	5
Olfactory bulb	Number examined	5	5	5	5
	Within normal limits	5	5	5	5
Ovaries	Number examined	X	5	X	5
	Within normal limits	X	5	X	5

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

TABLE 21 (Contd…). SUMMARY OF HISTOPATHOLOGY FINDINGS – RECOVERY GROUP

Route of administration		Inhalation
Treatment		Air only		High Dose Recovery
Nominal Target Concentration (mg/L)		0		0.03
Group		G1R		G4R
Sex		M	F	M		F
Number of Animals		5	5	5		5
Parathyroid	Number examined	5	5	5		5
	Within normal limits	5	5	5		5
Seminal vesicles	Number examined	5	X	5		X
	Within normal limits	5	X	5		X
Spinal cord	Number examined	5	5	5		5
	Within normal limits	5	5	5		5
Spleen	Number examined	5	5	5	5
	Within normal limits	5	5	5	5
Stomach	Number examined	5	5	5	5
	Within normal limits	5	5	5	5
Testes	Number examined	5	X	5	X
	Within normal limits	5	X	5	X
Thymus	Number examined	5	5	5	5
	Within normal limits	5	3	4	3
Cyst, epithelial	Present	-	2	1	2
Cellularity, increased, epithelial cells	Minimal	-	-	1	-
Thyroid	Number examined	5	5	5	5
	Within normal limits	4	4	3	4
Cyst, ultimobranchial	Present	1	1	1	1
Ectopic tissue, thymus	Present	-	-	1	-
Trachea	Number examined	5	5	5	5
	Within normal limits	5	5	5	5
Urinary bladder	Number examined	5	5	5	5
	Within normal limits	5	5	5	5
Uterus	Number examined	X	5	X	5
	Within normal limits	X	4	X	3
Dilatation, lumen	Minimal	X	1	X	-
	Mild	X	-	X	2

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

Applicant's summary and conclusion

Conclusions:

Based on the observed results, it is concluded that the No Observed Adverse Effect Concentration (NOAEC) of test item was found to be 0.03 mg/L when exposed for 6 hours/day, 5 days/week, for 04 weeks by flow-past nose-only inhalation route to Sprague Dawley rats. The nominal dose of 0.03 mg/L (6 hrs/day, 5 days/week) is the actual exposure concentration in males and females.

Executive summary:

The objective of this study was to determine the toxic potential of the test item when administered for 6 hours/day, 5 days per week, for 04 consecutive weeks by flow-past nose-only inhalation route to Sprague Dawley rats. This study provides information on toxic effects, target organs, the possibility of cumulative effects, the reversibility of effects (after 56 days recovery period), and an estimate of the No Observed Adverse Effects Concentration (NOAEC).

A total of 60 (30 males + 30 females) healthy young Sprague Dawley rats were distributed to four groups viz., control (G1), low dose (G2), mid dose (G3), high dose (G4) and two recovery groups [(G1R (Air only) and G4R (high dose)]. Each main and recovery group comprised of 10 animals (5 males and 5 females). Animals allocated to Groups G2, G3 and G4/G4R were exposed to test item Hostaperm Yellow H6G for 6 hours per day, 5 days per week, for 04 consecutive weeks, at a nominal target concentration of 0.001, 0.003 and 0.03 mg/L. Animals of the control group (G1/G1R) received air only inhalation for 6 hours/day for 04 consecutive weeks. The inhalation exposure of test item/air was achieved by a flow-past, nose-only dynamic inhalation exposure system supplied by CH Technologies, USA.

All animals were observed once daily for clinical signs and twice daily for mortality. Body weight was recorded before exposure (day 1) and weekly twice first two weeks during exposure period and weekly once for remaining two weeks during exposure period and recovery period. Feed consumption was recorded weekly. Ophthalmoscopic examinations were performed during the acclimatization period for all groups, and during Week4 for main groups (G1and G4), and duringWeek12 for recovery groups (G1Rand G4R). Neurological/Functional tests were performed during Week4 for G1 and G4 groups and during Week12 for recovery group animals.

At the end of treatment and recovery periods, all animals were fasted overnight (water was available 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 and high dose group animals including recovery animals were subjected to histopathological examinations.

The data recorded for all exposure days relating to the chamber conditions like particle size, temperature, relative humidity, oxygen, and carbon dioxide concentrations determined during the exposure period were found within the specified range.

No treatment-related changes in body weight, percent change in body weight with respect to day 1, feed consumption,or ophthalmology were noted. No adverse effects were observed in the neurological/functional examination tests. No adverse effects were observed in haematology, clinical chemistry, coagulation, BALF analysis,or urinalysis parameters. No toxicologically significant changes were observed infasting body weight, absolute organ weight, or relative organ weight.

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.