2,2'-[ethylenebis(oxyphenyl-2,1-eneazo)]bis[N-(2,3-dihydro-2-oxo-1H-benzimidazol-5-yl)-3-oxobutyramide

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29 March 2021 to 23 August 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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

2,2'-[ethylenebis(oxyphenyl-2,1-eneazo)]bis[N-(2,3-dihydro-2-oxo-1H-benzimidazol-5-yl)-3-oxobutyramide

EC Number:

278-770-4

EC Name:

2,2'-[ethylenebis(oxyphenyl-2,1-eneazo)]bis[N-(2,3-dihydro-2-oxo-1H-benzimidazol-5-yl)-3-oxobutyramide

Cas Number:

77804-81-0

Molecular formula:

C36H32N10O8

IUPAC Name:

2,2'-[Ethylenebis(oxyphenyl-2,1-eneazo)]bis[N-(2,3-dihydro-2-oxo-1H-benzimidazol-5-yl)-3-oxobutyramide

Test material form:

solid: nanoform

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient (21 to 29°C)

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: No
- Preliminary purification step (if any): No

FORM AS APPLIED IN THE TEST: As it is

Test animals

Species:

rat

Strain:

Sprague-Dawley

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: 7-8 weeks at the time of randomization
- Weight at study initiation: Males: 140.22 g to 146.05 g
Females: 120.13 g to 135.28 g

- Fasting period before study: minimum of 16 hours
- Housing: 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
- Diet (e.g. ad libitum): Yes except when fasting
- Water (e.g. ad libitum): Yes
- Acclimation period: 29 March 2021 to 04 April 2021


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.4°C to 22.9°C
- Humidity (%): 47% 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: 05 April 2021 To: 23 August 2021

Administration / exposure

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

air

Mass median aerodynamic diameter (MMAD):

> 1 - <= 2 µm

Geometric standard deviation (GSD):

3

Remarks on MMAD:

within the prescribed range

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Inhalation Exposure System, CH Technologies, USA
- Method of holding animals in test chamber: Restrainer tubes
- Source and rate of air: 20 lt/min
- Method of conditioning air: filtered air
- System of generating particulates/aerosols: dust generater RBG
- Temperature, humidity, pressure in air chamber: 22.1 to 23.2, 52.1% to 58.4%, 60 psi
- Air flow rate: 20 lt/min
- Air change rate: 12 air changes per hour
- Method of particle size determination: Gravimetric
- Treatment of exhaust air:NaoH
TEST ATMOSPHERE
- Samples taken from breathing zone: yes

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

G1 - Air only G2- 0.001 mg/L of air G3- 0.003 mg/L of air G3- 0.03 mg/L of air

Duration of treatment / exposure:

6 hours 5 days per week for 4 weeks

Frequency of treatment:

5 days per week for 4 weeks

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/m³ air (analytical)

Remarks:

Control

Doses / concentrations 2

Dose / conc.:

30 mg/m³ air (analytical)

Doses / concentrations 3

Dose / conc.:

3 mg/m³ air (analytical)

Doses / concentrations 4

Dose / conc.:

1 mg/m³ air

No. of animals per sex per dose:

05 Males+ 05 Females

Details on study design:

The selected animals were assigned to different treatment groups, as shown below:
Group Group Description Nominal Target Dose
mg/L No. of Animals / Group Duration of Exposure Animal No.
Male Female
G1 Air only 0 5 M + 5 F 6 hours Rf4001
to
Rf4005 Rf4031
to
Rf4035
G2 Low Dose 0.001 5 M + 5 F 6 hours Rf4006
to
Rf4010 Rf4036
to
Rf4040
G3 Mid Dose 0.003 5 M + 5 F 6 hours Rf4011
to
Rf4015 Rf4041
to
Rf4045
G4 High Dose 0.03 5 M + 5 F 6 hours Rf4016
to
Rf4020 Rf4046
to
Rf4050
G: Group; M: Male; F: Female; * Selected doses provided by the sponsor
Recovery Groups:
Group Group Description Nominal Target Dose
mg/L No. of Animals / Group Duration of Exposure Animal No.
Male Female
G1R Air only 0 5 M + 5 F 6 hours Rf4021
to
Rf4025 Rf4051
to
Rf4055
G4R High Dose Recovery 0.03 5 M + 5 F 6 hours Rf4026
to
Rf4030 Rf4056
to
Rf4060
G: Group; M: Male; F: Female;

Rational for dose selection: The mid dose was selected equal to the German general occupational exposure limit for dust (3 mg/m³). The high dose was chosen ten-fold higher.

- Rationale for selecting satellite groups: the animals in the recovery groups (G1R and G4R) were not given any treatment and 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 post treatment period

Positive control:

no

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes .

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly once

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

OPHTHALMOSCOPIC EXAMINATION: Yes / No / Not specified
- Time schedule for examinations: during randamization
- Dose groups that were examined: main group week 4 and recovery week 12

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: 40 for main group and 20 for recovery group
- Parameters checked in table [No.?] were examined.Table 10

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: lood 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: 40 for main group and 20 for recovery group
- Parameters checked in table [No.?] were examined. Table 12

URINALYSIS: Yes
- Time schedule for collection of urine: Urine was collected and analyzed from all surviving rats on day 27 and 85
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked in table [No.?] were examined. Table 13

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: week 4 for main group and week 12 for recovery.
- Dose groups that were examined: control and high dose
- Battery of functions tested: sensory activity / grip strength / motor activity

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Other examinations:

BALF Analysis

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 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.

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 consumption and compound intake (if feeding study):

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:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

No ocular abnormalities were noted.

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 MCH (G4) and APTT (G2, G3 and G4) was noted. In females, increase in percent and absolute neutrophils (G4); increase in Absolute monocytes and APTT (G3) was noted.
Decrease in APTT in main group males is not considered to be toxicologically significant and also similar variation was not noted in females. Variations in few differential counts in females could be secondary due to accumulation of test item particles in lungs which is considered not be an adverse effect.
In recovery males, increase in WBC, Platelets, percent basophil and absolute basophils (G4R) was noted. In recovery females, increase in Platelets and MPV (G4R); decrease in absolute eosinophils (G4R) was noted. Variations noted in few differential counts could be secondary due to accumulation of test item particles in lungs. All other variations were not noted at the end of treatment period and hence considered to be incidental.

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 glucose, urea, AST and BUN (G3); in females decrease in glucose (G3) and increase in calcium (G2) was noted. All the noted variations are considered incidental in the absence of dose responsiveness.
In recovery group, increase in Sodium (G4R) and in females increase in creatinine (G4R) was noted which is considered incidental as no such variation was noted at the end of treatment period or in the other sex at the end of recovery period.

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.
In Main group, males decrease in specific gravity (G3) was noted. In recovery males, decrease in pH (G4R) was noted.
The noted variations are considered incidental due to their isolated occurrences without any changes in any of the other parameters tested.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

There were no adverse effects observed during neurological/functional examinations in main group (G1 and G4) and recovery group (G1R and G4R), however in males decrease in rearing (G4) when compared with control group was noted. This isolated variation is considered incidental due to lack of dose responsiveness and also none of the other neuromuscular parameters were affected.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

The following statistically significant variations were noted in organ weights and its ratios.
In main group males, increase in relative weight of liver (G3); decrease in relative weight of heart (G3); in females increase in absolute weight of uterus and decrease in adrenals (G4); decrease in absolute and relative weight of spleen (G3 and G4) was noted.
In recovery group males, decrease in absolute and relative weight of lungs (G4R); in females, increase in absolute and relative weight of uterus (G4R) was noted.
Relative weight changes (relative to brain weight)
In main group males, decrease in relative weight of heart (G3) and lungs (G2); in females increase in relative weight of adrenal and uterus (G4); decrease in relative weight of spleen (G3 and G4), heart (G2) and lungs (G3) was noted.
In recovery group males, decrease in relative weight of lungs (G4R) was noted.
The variations noted in organ weight were not associated with any macroscopic examination and also no microscopic changes were noted at the high dose main group animals and hence considered incidental.

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.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

In lungs, minimal to moderate, diffuse/multifocal, variably sized, pigmented (golden yellow) granular material was observed throughout the parenchyma of lung at mid, high and high dose recovery groups of rats. This pigment was distributed both in alveolar and bronchiolar spaces with no accompanying inflammation or tissue destruction to the lung parenchyma. This pigment was suggestive of inhaled test item particles as the physical appearance of test item was also yellow coloured powder. In the absence of cellular changes to lung parenchyma, mere presence of pigment in lungs could be considered as non-adverse effect up to the dose level of 0.03 mg/L. (Nikula KJ et.al., 2014).
Few other microscopic findings observed in this study such as ultimobranchial cyst in thyroid gland, dilatation of lumen in uterus and all other findings were considered incidental as they occurred randomly across the dose groups including concurrent controls and/or were expected for laboratory rats of this age.

Histopathological findings: neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

The bronchio-alveolar lavage fluid collected during necropsy revealed the following statistically significant changes:
In main group males, decrease in absolute eosinophils (G2) was noted which is considered incidental due to lack of dose responsiveness.
In recovery group, males increase in total protein (G4R) was noted. In females decrease in WBC, LDH and absolute lymphocytes (G4R) was noted. Variations in WBC and lymphocytes could be secondary changes due to accumulation of test item particles in lungs. Other variations noted at the end of treatment period, hence considered incidental.

Details on results:

Based on the observed results, it is concluded that the No Observed Adverse Effect Concentration (NOAEC) of test item, PV Fast Yellow HG01 was found to be 0.03 mg/L when exposed for 6 hours/day, 5 days/week, for 4 weeks by flow-past nose-only inhalation route to Sprague Dawley rats. The nominal dose of 0.03 mg/L (6 hrs/day, 5 days/week) corresponded to an actual exposure concentration in males and females.

Effect levels

Key result

Dose descriptor:

NOAEC

Effect level:

>= 30 mg/m³ air

Sex:

male/female

Basis for effect level:

behaviour (functional findings)

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

gross pathology

haematology

histopathology: non-neoplastic

mortality

organ weights and organ / body weight ratios

serum/plasma biochemistry

urinalysis

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	166.93	176.04	187.88	198.42	206.01	216.66	227.71
	±SD	5.48	8.20	9.73	9.11	8.53	8.50	8.83
	n	5	5	5	5	5	5	5
G2, M & 0.001	Mean	170.04	176.84	188.36	200.45	209.60	220.77	232.55
	±SD	12.14	12.69	11.14	10.77	10.84	11.19	11.25
	n	5	5	5	5	5	5	5
G3, M & 0.003	Mean	167.42	173.49	185.53	197.31	206.57	217.52	228.51
	±SD	11.71	12.06	10.84	10.47	10.10	10.53	10.98
	n	5	5	5	5	5	5	5
G4, M & 0.03	Mean	170.74	176.44	189.01	200.50	210.27	221.09	232.36
	±SD	12.79	13.37	10.99	10.61	10.37	9.35	9.06
	n	5	5	5	5	5	5	5
G1, F & 0	Mean	149.53	155.48	162.61	169.69	176.05	182.41	188.66
	±SD	10.12	11.45	10.72	11.03	11.18	11.07	11.81
	n	5	5	5	5	5	5	5
G2, F & 0.001	Mean	148.83	154.95	161.85	169.10	174.92	181.71	189.69
	±SD	10.46	12.14	11.89	12.21	11.64	11.53	9.87
	n	5	5	5	5	5	5	5
G3, F & 0.003	Mean	148.31	155.11	162.11	168.69	174.43	181.88	188.11
	±SD	12.68	13.26	13.50	13.44	13.16	13.55	14.00
	n	5	5	5	5	5	5	5
G4, F & 0.03	Mean	152.00	155.46	163.76	170.17	176.51	183.48	190.21
	±SD	9.51	9.59	10.19	9.95	9.81	9.83	9.80
	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	162.99	173.88	183.86	192.52	202.49	212.93	224.04	236.11	258.81	279.54	303.39	328.87	356.03	383.60	417.81	430.99
	±SD	7.56	6.61	6.38	5.03	3.48	3.31	3.38	3.11	3.39	3.26	4.25	3.98	4.83	5.87	6.20	7.47
	n	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5
G4R, M & 0.03	Mean	164.50	174.85	182.82	193.53	202.18	212.57	224.04	237.28	259.48	281.93	305.47	330.41	358.96	387.58	418.97	432.89
	±SD	8.02	7.87	8.30	7.37	7.65	6.81	7.03	6.60	8.45	9.99	10.24	9.36	13.00	13.14	9.26	9.06
	n	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5
G1R, F & 0	Mean	148.49	154.13	162.59	169.09	174.76	182.15	189.84	196.77	211.40	223.53	237.19	251.73	269.03	288.36	309.32	317.96
	±SD	9.89	11.31	10.11	10.23	9.44	9.83	9.87	9.86	10.04	14.15	13.32	13.52	13.49	13.63	13.17	12.60
	n	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5
G4R, F & 0.03	Mean	153.17	158.57	166.90	173.75	179.85	187.04	192.54	199.27	213.28	227.25	241.48	255.80	274.97	293.35	314.56	322.29
	±SD	6.27	6.61	6.27	6.14	6.55	6.53	6.75	7.37	6.13	7.73	7.40	7.20	8.70	9.30	10.78	11.02
	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.

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

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	5.42	12.50	18.82	23.38	29.77	36.39
	±SD	1.62	2.64	1.82	1.38	1.41	1.58
	n	5	5	5	5	5	5
G2, M & 0.001	Mean	4.00	10.86	18.01	23.41	29.99	36.95
	±SD	1.71	2.03	2.70	2.83	3.08	3.52
	n	5	5	5	5	5	5
G3, M & 0.003	Mean	3.64	10.90	17.97	23.54	30.09	36.67
	±SD	1.73	2.36	2.55	2.96	3.31	3.38
	n	5	5	5	5	5	5
G4, M & 0.03	Mean	3.34	10.81	17.58	23.34	29.75	36.39
	±SD	1.40	2.13	2.65	3.36	4.49	5.22
	n	5	5	5	5	5	5
G1, F & 0	Mean	3.95	8.76	13.50	17.76	22.04	26.20
	±SD	0.83	0.24	0.59	0.67	1.19	1.08
	n	5	5	5	5	5	5
G2, F & 0.001	Mean	4.07	8.73	13.61	17.56	22.14	27.59
	±SD	1.27	0.71	0.46	0.96	1.16	2.49
	n	5	5	5	5	5	5
G3, F & 0.003	Mean	4.59	9.33	13.80	17.70	22.73	26.94
	±SD	1.39	1.63	1.67	1.87	2.07	1.86
	n	5	5	5	5	5	5
G4, F & 0.03	Mean	2.29	7.75	11.98	16.18	20.78	25.22
	±SD	1.78	2.38	2.16	2.59	2.98	2.76
	n	5	5	5	5	5	5

M: Male; F: Female; 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	6.71	12.86	18.20	24.37	30.80	37.63	45.04	58.99	71.74	86.37	102.04	118.71	135.62	156.70	164.77
	±SD	1.14	1.52	2.50	3.91	4.62	4.91	5.07	5.55	6.58	6.42	7.31	7.52	7.55	10.48	10.18
	n	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5
G4R, M & 0.03	Mean	6.31	11.16	17.71	22.97	29.32	36.29	44.37	57.85	71.51	85.84	101.06	118.38	135.83	155.04	163.50
	±SD	0.89	0.87	1.75	2.24	2.71	2.68	3.47	2.94	4.04	4.50	5.79	5.99	7.48	9.70	9.42
	n	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5
G1R, F & 0	Mean	3.76	9.53	13.91	17.78	22.76	27.95	32.63	42.52	50.60	59.85	69.68	81.37	94.45	108.63	114.50
	±SD	0.87	1.21	0.90	1.76	1.80	2.31	2.57	3.53	3.85	3.92	4.55	5.17	6.30	7.38	8.09
	n	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5
G4R, F & 0.03	Mean	3.53	8.98	13.46	17.44	22.14	25.73	30.12	39.31	48.41	57.72	67.09	79.58	91.59	105.42	110.47
	±SD	0.41	0.40	0.67	0.60	0.87	0.77	0.56	1.92	1.35	1.92	2.50	2.27	2.39	1.97	1.97
	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	18.66	18.64	18.67	32.70
	±SD	2.90	2.76	2.60	4.63
	n	2	2	2	2
G2, M & 0.001	Mean	18.65	18.64	18.65	32.46
	±SD	2.76	2.80	2.94	4.68
	n	2	2	2	2
G3, M & 0.003	Mean	18.59	18.69	18.59	32.81
	±SD	2.69	2.86	2.75	5.06
	n	2	2	2	2
G4, M & 0.03	Mean	18.52	18.63	18.53	32.95
	±SD	2.77	2.76	2.81	5.52
	n	2	2	2	2
G1, F & 0	Mean	14.59	14.57	14.83	25.66
	±SD	1.68	1.65	2.09	3.34
	n	2	2	2	2
G2, F & 0.001	Mean	14.44	14.56	14.41	25.28
	±SD	1.71	1.74	1.60	2.92
	n	2	2	2	2
G3, F & 0.003	Mean	14.46	14.45	14.48	25.26
	±SD	1.54	1.71	1.76	2.60
	n	2	2	2	2
G4, F & 0.03	Mean	14.40	14.54	14.36	24.97
	±SD	1.59	1.68	1.50	2.84
	n	2	2	2	2

 M: Male; F: Female; SD: Standard Devi

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	18.59	18.65	18.54	18.79	18.51	18.30	18.46	19.32	19.71	20.09	20.41	23.02
	±SD	2.96	2.75	2.89	2.79	2.71	2.63	2.93	3.74	3.87	3.90	3.91	4.47
	n	2	2	2	2	2	2	2	2	2	2	2	2
G4R, M & 0.03	Mean	18.46	18.58	18.50	18.63	18.53	18.35	18.26	19.22	19.97	20.15	20.49	23.25
	±SD	2.76	2.72	2.58	2.73	2.69	2.61	2.52	3.79	3.96	3.96	4.06	4.96
	n	2	2	2	2	2	2	2	2	2	2	2	2
G1R, F & 0	Mean	14.42	14.51	14.36	14.37	14.55	14.40	14.48	14.55	15.33	15.70	16.06	17.68
	±SD	1.70	1.79	1.73	1.28	1.20	1.18	1.46	1.42	1.61	1.83	2.01	1.79
	n	2	2	2	2	2	2	2	2	2	2	2	2
G4R, F & 0.03	Mean	14.37	14.47	14.36	14.54	14.59	14.39	14.44	14.42	15.29	15.75	16.00	17.64
	±SD	1.64	1.66	1.43	1.27	1.42	1.31	1.27	1.38	1.67	1.73	1.87	1.59
	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	8.0	6.0*	8.4	8.8
	±SD	1.4	1.0	1.1	0.8
Numbers of Urination	Mean	1.4	1.2	1.2	1.4
	±SD	0.5	0.4	0.8	0.5
Number of Defecation	Mean	1.0	1.0	1.0	1.0
	±SD	0.0	0.7	0.0	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	4.0	4.6	5.4	5.0
	±SD	0.7	0.5	1.1	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	97.9	97.9	98.5	98.5
	±SD	0.1	0.2	0.4	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

M: Male; F: Female; *: Statistically significant (p<0

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	7.0	6.2	7.0	6.4
	±SD	0.0	1.9	0.0	2.5
Numbers of Urination	Mean	1.0	1.0	1.0	1.0
	±SD	0.0	0.0	0.0	0.0
Number of Defecation	Mean	1.0	1.0	1.0	1.0
	±SD	0.0	0.0	0.0	0.0
Clonic involuntary movement		1	1	1	1
Tonic involuntary movement		1	1	1	1
Stereotype Behaviour		1	1	1	1
Excessive Grooming	Mean	4.0	4.2	5.0	5.4
	±SD	0.0	1.9	1.6	1.3
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	97.8	98.7	98.5
	±SD	0.3	0.2	0.2	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

M: Male; F: Female; R: Recovery

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		0.5
		n		5
G4, M & 0.03		Mean		5.9
		±SD		0.4
		n		5
G1, F & 0		Mean		5.0
		±SD		0.5
		n		5
G4, F & 0.03		Mean		5.8
		±SD		0.6
		n		5
G1R, M & 0		Mean		6.5
		±SD		1.3
		n		5
G4R, M & 0.03		Mean		5.6
		±SD		0.7
		n		5
G1R, F & 0		Mean		5.1
		±SD		0.3
		n		5
G4R, F & 0.03		Mean		4.5
		±SD		0.5
		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.

TABLE7. SUMMARY OF ACTIMETER READING RECORD 

# Week 4 & 12
Group, Sex & Dose (mg/L)				Movement Counts (no.)
G1, M & 0		Mean		2296.2
		±SD		32.0
		n		5
G4, M & 0.03		Mean		2309.8
		±SD		69.2
		n		5
G1, F & 0		Mean		2160.2
		±SD		24.2
		n		5
G4, F & 0.03		Mean		2167.8
		±SD		25.1
		n		5
G1R, M & 0		Mean		2223.0
		±SD		8.5
		n		5
G4R, M & 0.03		Mean		2220.2
		±SD		12.0
		n		5
G1R, F & 0		Mean		2339.4
		±SD		38.4
		n		5
G4R, F & 0.03		Mean		2417.2
		±SD		75.4
		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. SUMMARYOF GRIP STRENGTH (kgf) MEASUREMENT RECORD

# Week 4 & 12
Group, Sex & Dose (mg/L)				Fore limb		Hind limb
G1, M & 0		Mean		1.503		0.533
		±SD		0.050		0.088
		n		5		5
G4, M & 0.03		Mean		1.484		0.471
		±SD		0.049		0.020
		n		5		5
G1, F & 0		Mean		1.431		0.408
		±SD		0.038		0.047
		n		5		5
G4, F & 0.03		Mean		1.409		0.375
		±SD		0.022		0.056
		n		5		5
G1R, M & 0		Mean		1.502		0.531
		±SD		0.053		0.087
		n		5		5
G4R, M & 0.03		Mean		1.489		0.475
		±SD		0.049		0.021
		n		5		5
G1R, F & 0		Mean		1.427		0.404
		±SD		0.038		0.049
		n		5		5
G4R, F & 0.03		Mean		1.413		0.377
		±SD		0.023		0.054
		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 (ppm)	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: No Abnormality Detected; L: Left; R: Right; M: Male; R: Recovery.

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

 

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

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

 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	8.13	8.04	16.86	50.12	62.64	21.02	33.76	763.40	6.36
	±SD	1.13	0.95	1.67	4.20	5.22	1.40	3.66	383.67	0.34
	n	5	5	5	5	5	5	5	5	5
G2, M & 0.001	Mean	11.24	8.47	16.62	49.02	57.90	19.62	33.88	803.20	6.40
	±SD	3.78	0.26	0.37	0.89	2.08	0.61	0.52	254.95	0.48
	n	5	5	5	5	5	5	5	5	5
G3, M & 0.003	Mean	8.45	7.92	16.12	50.70	65.18	20.54	31.82	754.80	6.62
	±SD	1.65	1.17	1.43	3.22	10.83	1.53	2.61	82.93	0.41
	n	5	5	5	5	5	5	5	5	5
G4, M & 0.03	Mean	11.48	8.68	16.56	50.88	58.82	19.10*	32.54	1018.60	6.34
	±SD	2.43	0.62	0.68	0.74	4.45	0.73	1.20	142.27	0.38
	n	5	5	5	5	5	5	5	5	5
G1R, M & 0	Mean	8.32	9.40	16.84	51.08	54.36	17.94	32.92	615.80	6.86
	±SD	2.72	0.39	0.74	1.80	0.96	0.52	0.50	176.73	0.34
	n	5	5	5	5	5	5	5	5	5
G4R, M & 0.03	Mean	11.95*	9.35	17.18	50.60	54.12	18.40	34.04	871.60*	7.14
	±SD	2.17	0.31	0.36	3.14	2.08	0.50	1.85	51.70	0.36
	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	6.71	26.42	68.82	2.84	0.46	0.48	518.48
	±SD	4.14	12.96	12.05	0.72	0.25	0.30	286.55
	n	5	5	5	5	5	5	5
G2, M & 0.001	Mean	3.84	27.76	66.82	3.16	0.80	0.42	323.30
	±SD	1.19	5.66	5.64	0.85	0.46	0.16	95.02
	n	5	5	5	5	5	5	5
G3, M & 0.003	Mean	7.09	21.40	73.62	3.20	0.52	0.52	543.84
	±SD	2.51	6.16	7.56	1.21	0.36	0.19	124.46
	n	5	5	5	5	5	5	5
G4, M & 0.03	Mean	3.02	30.36	65.34	2.44	0.56	0.38	248.50
	±SD	2.75	8.97	8.50	1.02	0.29	0.08	197.64
	n	5	5	5	5	5	5	5
G1R, M & 0	Mean	1.90	23.10	71.50	2.78	1.68	0.30	178.90
	±SD	0.51	6.07	6.51	1.15	0.59	0.00	46.18
	n	5	5	5	5	5	5	5
G4R, M & 0.03	Mean	2.36	20.14	74.56	3.16	1.20	0.40*	221.88
	±SD	0.75	3.68	4.45	0.78	0.49	0.07	73.13
	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	2.15	5.59	0.24	0.04	0.04	15.96	37.72
	±SD	1.09	1.30	0.09	0.02	0.02	1.89	8.70
	n	5	5	5	5	5	5	5
G2, M & 0.001	Mean	3.22	7.40	0.36	0.08	0.05	15.78	26.12*
	±SD	1.53	2.11	0.15	0.03	0.04	1.56	2.12
	n	5	5	5	5	5	5	5
G3, M & 0.003	Mean	1.84	6.17	0.28	0.05	0.04	17.94	28.04*
	±SD	0.64	1.10	0.13	0.04	0.02	3.29	4.23
	n	5	5	5	5	5	5	5
G4, M & 0.03	Mean	3.57	7.44	0.26	0.06	0.04	16.54	22.92*
	±SD	1.58	1.59	0.06	0.03	0.02	1.33	4.74
	n	5	5	5	5	5	5	5
G1R, M & 0	Mean	1.83	6.04	0.24	0.14	0.02	15.36	22.38
	±SD	0.40	2.38	0.14	0.05	0.01	0.90	7.23
	n	5	5	5	5	5	5	5
G4R, M & 0.03	Mean	2.39	8.93	0.38	0.14	0.05*	13.80	23.04
	±SD	0.54	1.87	0.11	0.05	0.02	0.44	6.41
	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)		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	9.09	8.87	17.42	52.36	59.48	19.68	33.18	765.00	6.40
	±SD	3.10	1.59	2.90	6.79	4.00	0.31	1.81	289.24	0.41
	n	5	5	5	5	5	5	5	5	5
G2, F & 0.001	Mean	11.57	7.82	15.54	46.86	59.96	19.86	33.20	971.60	6.46
	±SD	3.29	0.47	1.24	3.24	3.48	0.72	1.37	179.05	0.38
	n	5	5	5	5	5	5	5	5	5
G3, F & 0.003	Mean	10.78	8.30	16.16	48.66	58.68	19.48	33.26	1020.20	6.40
	±SD	3.21	0.26	0.72	2.19	2.27	0.59	0.75	260.90	0.27
	n	5	5	5	5	5	5	5	5	5
G4, F & 0.03	Mean	10.40	8.56	15.94	47.50	55.48	18.66	33.58	999.60	6.38
	±SD	1.59	0.12	0.90	1.73	2.13	1.05	0.92	36.07	0.26
	n	5	5	5	5	5	5	5	5	5
G1R, F & 0	Mean	10.88	9.15	16.56	49.00	53.54	18.06	33.80	802.00	7.32
	±SD	3.32	0.22	0.38	1.20	0.71	0.09	0.41	210.84	0.19
	n	5	5	5	5	5	5	5	5	5
G4R, F & 0.03	Mean	10.74	8.05	15.14	47.00	59.16	18.92	32.12	1081.80*	7.62*
	±SD	4.51	1.35	2.06	3.99	5.86	0.72	2.03	129.00	0.19
	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	5.73	18.94	75.04	2.94	0.82	1.48	481.44
	±SD	4.27	3.53	6.00	1.18	0.25	1.59	291.29
	n	5	5	5	5	5	5	5
G2, F & 0.001	Mean	8.16	15.90	78.64	3.20	0.60	0.58	624.82
	±SD	3.91	4.59	5.81	1.02	0.32	0.08	279.36
	n	5	5	5	5	5	5	5
G3, F & 0.003	Mean	5.04	18.44	75.24	3.90	0.68	0.64	412.10
	±SD	4.12	4.85	4.91	1.20	0.49	0.19	332.55
	n	5	5	5	5	5	5	5
G4, F & 0.03	Mean	1.58	27.16*	68.36	2.50	0.66	0.48	135.46
	±SD	0.20	5.08	5.71	0.75	0.17	0.13	16.62
	n	5	5	5	5	5	5	5
G1R, F & 0	Mean	2.06	23.12	70.52	3.06	1.94	0.36	189.28
	±SD	0.56	10.54	11.02	0.89	0.81	0.13	51.78
	n	5	5	5	5	5	5	5
G4R, F & 0.03	Mean	7.54	18.50	76.64	2.36	1.26	0.30	521.70
	±SD	8.09	5.76	5.58	0.30	0.44	0.14	512.41
	n	5	5	5	5	5	5	5

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

TABLE 10 (Contd…). SUMMARY OF HAEMATOLOGY RECORD 

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	1.66	6.93	0.25	0.07	0.11	17.08	18.96
	±SD	0.44	2.77	0.06	0.02	0.09	1.41	2.39
	n	5	5	5	5	5	5	5
G2, F & 0.001	Mean	1.74	9.23	0.35	0.06	0.07	17.98	22.62
	±SD	0.33	3.40	0.04	0.02	0.01	0.79	1.57
	n	5	5	5	5	5	5	5
G3, F & 0.003	Mean	1.89	8.20	0.43*	0.06	0.07	18.08	28.98*
	±SD	0.36	2.85	0.18	0.03	0.04	0.67	5.47
	n	5	5	5	5	5	5	5
G4, F & 0.03	Mean	2.82*	7.11	0.25	0.07	0.06	18.30	22.66
	±SD	0.69	1.29	0.06	0.02	0.02	1.16	2.06
	n	5	5	5	5	5	5	5
G1R, F & 0	Mean	2.29	7.90	0.34	0.20	0.04	14.14	24.38
	±SD	0.63	3.37	0.14	0.05	0.03	0.91	4.28
	n	5	5	5	5	5	5	5
G4R, F & 0.03	Mean	1.79	8.42	0.26	0.12*	0.04	13.28	24.28
	±SD	0.25	4.19	0.15	0.03	0.03	0.72	7.74
	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	1.00	36.94	50.18	4.72	2.88	5.70	0.39
	±SD	0.48	16.19	16.69	2.01	3.57	3.10	0.34
	n	5	5	5	5	5	5	5
G2, M & 0.001	Mean	0.69	39.84	47.06	7.16	0.60	3.34	0.29
	±SD	0.31	15.58	18.35	4.00	0.39	1.74	0.17
	n	5	5	5	5	5	5	5
G3, M & 0.003	Mean	1.39	40.12	46.28	5.46	0.70	8.18	0.74
	±SD	1.20	19.91	22.20	4.07	0.46	8.59	1.00
	n	5	5	5	5	5	5	5
G4, M & 0.03	Mean	0.43	33.14	58.08	3.64	1.66	3.60	0.13
	±SD	0.43	9.56	11.37	2.79	1.34	2.82	0.11
	n	5	5	5	5	5	5	5
G1R, M & 0	Mean	0.30	22.82	43.30	1.66	2.90	4.84	0.07
	±SD	0.05	7.81	18.36	0.87	3.48	3.96	0.02
	n	5	5	5	5	5	5	5
G4R, M & 0.03	Mean	0.25	31.86	55.90	1.14	4.48	5.30	0.05
	±SD	0.20	22.56	30.90	1.20	7.01	2.49	0.05
	n	5	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)		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	0.49	0.04	0.02	0.05	124.26	416.40
	±SD	0.26	0.03	0.01	0.03	58.30	298.11
	n	5	5	5	5	5	5
G2, M & 0.001	Mean	0.30	0.05	0.00*	0.02	114.56	401.51
	±SD	0.16	0.04	0.01	0.02	48.40	178.21
	n	5	5	5	5	5	5
G3, M & 0.003	Mean	0.46	0.11	0.01	0.08	140.04	335.57
	±SD	0.07	0.16	0.01	0.07	53.24	114.83
	n	5	5	5	5	5	5
G4, M & 0.03	Mean	0.27	0.02	0.01	0.02	86.62	424.80
	±SD	0.30	0.02	0.01	0.02	49.01	301.39
	n	5	5	5	5	5	5
G1R, M & 0	Mean	0.14	0.00	0.01	0.02	39.02	140.14
	±SD	0.07	0.01	0.01	0.02	13.30	28.00
	n	5	5	5	5	5	5
G4R, M & 0.03	Mean	0.17	0.00	0.00	0.01	38.32	262.27*
	±SD	0.19	0.00	0.00	0.01	20.73	66.93
	n	5	5	5	5	5	5

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

TABLE 11 (Contd…). SUMMARY OF BALF RECORD

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	1.76	32.44	47.72	4.48	0.46	5.08	0.99
	±SD	2.80	21.86	30.61	6.40	0.53	5.73	1.91
	n	5	5	5	5	5	5	5
G2, F & 0.001	Mean	0.90	34.48	36.38	9.98	1.54	7.64	0.37
	±SD	0.57	18.48	27.75	10.31	1.62	7.07	0.45
	n	5	5	5	5	5	5	5
G3, F & 0.003	Mean	0.50	40.60	44.62	4.84	1.02	3.88	0.22
	±SD	0.30	22.25	23.19	5.09	0.74	2.65	0.22
	n	5	5	5	5	5	5	5
G4, F & 0.03	Mean	0.38	26.40	59.50	1.88	1.22	4.10	0.10
	±SD	0.15	7.14	7.08	0.41	0.43	2.81	0.06
	n	5	5	5	5	5	5	5
G1R, F & 0	Mean	0.28	17.26	67.44	2.92	0.42	4.20	0.05
	±SD	0.05	7.14	11.60	2.94	0.41	1.94	0.03
	n	5	5	5	5	5	5	5
G4R, F & 0.03	Mean	0.18*	21.66	66.08	2.46	0.70	7.14	0.04
	±SD	0.05	6.93	16.29	1.40	0.72	4.51	0.01
	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	0.40	0.22	0.00	0.04	123.70	338.64
	±SD	0.36	0.47	0.00	0.05	127.06	100.37
	n	5	5	5	5	5	5
G2, F & 0.001	Mean	0.24	0.14	0.01	0.09	120.70	252.80
	±SD	0.22	0.22	0.01	0.12	95.18	103.64
	n	5	5	5	5	5	5
G3, F & 0.003	Mean	0.20	0.03	0.00	0.02	90.34	426.73
	±SD	0.10	0.06	0.01	0.03	56.38	250.71
	n	5	5	5	5	5	5
G4, F & 0.03	Mean	0.23	0.01	0.00	0.01	59.54	321.44
	±SD	0.08	0.00	0.01	0.01	21.20	123.61
	n	5	5	5	5	5	5
G1R, F & 0	Mean	0.19	0.01	0.00	0.01	40.42	268.54
	±SD	0.04	0.01	0.00	0.00	4.12	40.71
	n	5	5	5	5	5	5
G4R, F & 0.03	Mean	0.12*	0.01	0.00	0.01	30.54*	221.60
	±SD	0.04	0.01	0.00	0.01	2.99	89.44
	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	89.60	34.46	0.53	40.80	43.80	7.38	3.05	49.80	94.60
	±SD	11.10	2.46	0.02	11.54	17.06	0.23	0.28	4.76	16.59
	n	5	5	5	5	5	5	5	5	5
G2, M & 0.001	Mean	92.20	36.88	0.52	44.20	44.00	7.42	3.11	52.80	100.00
	±SD	7.85	3.03	0.04	4.49	10.79	0.35	0.11	6.42	10.15
	n	5	5	5	5	5	5	5	5	5
G3, M & 0.003	Mean	110.60*	46.28*	0.53	45.00	33.00	7.54	3.09	61.20	125.60*
	±SD	19.48	13.14	0.04	5.34	13.29	0.49	0.17	11.08	24.00
	n	5	5	5	5	5	5	5	5	5
G4, M & 0.03	Mean	91.80	35.62	0.50	39.20	32.20	7.30	2.98	49.40	106.00
	±SD	4.60	1.42	0.02	5.07	8.41	0.33	0.13	8.85	12.19
	n	5	5	5	5	5	5	5	5	5
G1R, M & 0	Mean	105.40	31.62	0.54	46.00	36.20	6.64	3.20	67.60	106.40
	±SD	5.50	6.25	0.04	7.28	9.96	0.15	0.20	9.58	20.08
	n	5	5	5	5	5	5	5	5	5
G4R, M & 0.03	Mean	108.40	24.74	0.52	39.40	40.80	6.50	3.37	65.40	97.60
	±SD	11.24	4.43	0.03	5.73	9.58	0.24	0.18	18.08	13.09
	n	5	5	5	5	5	5	5	5	5

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

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

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	209.40	0.06	9.22	6.16	4.33	16.08	144.20	3.19	105.48
	±SD	40.17	0.07	0.28	0.46	0.06	1.15	1.25	0.32	1.03
	n	5	5	5	5	5	5	5	5	5
G2, M & 0.001	Mean	193.80	0.02	9.50	6.28	4.31	17.21	143.50	3.21	104.68
	±SD	47.54	0.03	0.41	0.38	0.34	1.42	0.69	0.36	1.21
	n	5	5	5	5	5	5	5	5	5
G3, M & 0.003	Mean	218.80	0.03	9.66	6.44	4.45	21.60*	143.62	3.49	104.60
	±SD	12.79	0.05	0.22	0.69	0.62	6.13	0.74	0.27	1.79
	n	5	5	5	5	5	5	5	5	5
G4, M & 0.03	Mean	219.20	0.02	9.22	6.38	4.32	16.62	145.00	3.44	107.02
	±SD	27.13	0.02	0.27	0.52	0.23	0.67	1.15	0.12	1.77
	n	5	5	5	5	5	5	5	5	5
G1R, M & 0	Mean	159.40	0.05	10.06	5.76	3.44	14.76	142.10	4.08	104.12
	±SD	26.19	0.01	0.23	0.36	0.23	2.92	0.42	0.28	1.37
	n	5	5	5	5	5	5	5	5	5
G4R, M & 0.03	Mean	142.00	0.06	10.30	6.20	3.13	11.54	143.28*	3.66	105.00
	±SD	38.87	0.01	0.25	0.35	0.39	2.07	0.74	0.35	1.39
	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	101.40	38.94	0.53	43.00	32.60	7.42	3.72	49.40	110.80
	±SD	5.68	5.98	0.02	10.27	5.41	0.13	1.08	14.40	21.71
	n	5	5	5	5	5	5	5	5	5
G2, F & 0.001	Mean	97.80	42.00	0.53	46.60	38.80	7.68	3.29	43.20	100.60
	±SD	9.58	9.38	0.03	7.33	5.63	0.23	0.05	11.19	16.29
	n	5	5	5	5	5	5	5	5	5
G3, F & 0.003	Mean	89.80*	41.10	0.53	46.00	38.80	7.88	3.15	52.40	110.80
	±SD	4.21	13.46	0.03	9.30	9.73	0.57	0.19	8.38	29.26
	n	5	5	5	5	5	5	5	5	5
G4, F & 0.03	Mean	103.80	41.06	0.54	41.60	37.80	7.58	3.18	41.60	101.20
	±SD	1.79	6.10	0.03	7.77	10.64	0.33	0.08	10.69	11.01
	n	5	5	5	5	5	5	5	5	5
G1R, F & 0	Mean	107.20	31.10	0.55	49.80	39.00	6.82	3.55	45.40	85.40
	±SD	3.70	7.19	0.01	13.70	10.22	0.26	0.17	8.71	14.26
	n	5	5	5	5	5	5	5	5	5
G4R, F & 0.03	Mean	104.00	33.28	0.60*	62.20	41.40	6.82	3.79	50.00	92.40
	±SD	8.12	7.26	0.04	15.16	9.34	0.23	0.29	15.89	21.20
	n	5	5	5	5	5	5	5	5	5

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

 TABLE 12 (Contd…). SUMMARY OF CLINICAL CHEMISTRY

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	111.40	0.08	9.62	6.30	3.70	18.17	143.22	3.50	105.46
	±SD	20.21	0.02	0.19	1.04	1.15	2.79	2.22	0.56	1.62
	n	5	5	5	5	5	5	5	5	5
G2, F & 0.001	Mean	113.40	0.06	9.98*	6.54	4.39	19.60	143.24	3.31	105.50
	±SD	26.66	0.02	0.16	0.51	0.24	4.38	0.78	0.30	0.58
	n	5	5	5	5	5	5	5	5	5
G3, F & 0.003	Mean	134.60	0.06	9.80	6.36	4.73	19.18	143.74	3.16	105.70
	±SD	37.39	0.05	0.14	0.26	0.71	6.28	0.60	0.14	0.42
	n	5	5	5	5	5	5	5	5	5
G4, F & 0.03	Mean	134.00	0.04	9.56	6.04	4.40	19.16	143.22	3.23	105.18
	±SD	39.18	0.02	0.21	0.34	0.32	2.85	1.24	0.12	1.05
	n	5	5	5	5	5	5	5	5	5
G1R, F & 0	Mean	76.40	0.08	10.42	5.20	3.27	14.51	141.16	3.60	103.94
	±SD	13.56	0.01	0.19	0.51	0.39	3.35	1.30	0.38	1.95
	n	5	5	5	5	5	5	5	5	5
G4R, F & 0.03	Mean	57.80	0.10	10.72	5.08	3.03	15.53	141.90	3.63	105.62
	±SD	15.77	0.05	0.36	0.63	0.13	3.39	1.66	0.27	2.00
	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	5	3	3	5	3	5
		Yellow	-	2	2	-	2	-
	Appearance	Clear	5	3	3	5	5	5
		Turbid	-	2	2	-	-	-
	Volume (mL)	Mean	6.4	5.4	5.9	6.3	8.0	7.5
		±SD	1.1	1.5	1.1	1.0	1.3	1.5
Chemical	pH	Mean	7.6	7.8	8.0	7.7	7.5	6.7*
		±SD	0.2	0.3	0.5	0.3	0.6	0.4
	Specific Gravity	Mean	1.010	1.010	1.007*	1.009	1.013	1.015
		±SD	0.000	0.000	0.003	0.002	0.003	0.004
	Urobilinogen (mg/dL)	Mean	0.2	0.2	0.5	0.2	0.2	0.2
		±SD	0.0	0.0	0.4	0.0	0.0	0.0
	Bilirubin (mg/dL)	Neg	5	4	2	5	5	5
		1	-	1	3	-	-	-
	Ketones (mg/dL)	Neg	4	4	5	4	1	-
		5	1	1	-	1	4	5
	Blood (Ery/µL)	Neg	-	-	3	-	1	3
		Ca10	-	1	-	-	-	-
		Ca25	3	2	1	3	1	2
		Ca80	2	2	1	2	2	-
		>=Ca200	-	-	-	-	1	-
	Proteins (mg/dL)	Neg	5	3	2	5	5	4
		Trace	-	1	-	-	-	1
		30	-	1	1	-	-	-
		>=300	-	-	2	-	-	-
	Nitrite	Neg	5	4	3	5	5	2
		Pos	-	1	2	-	-	3
	Leucocytes (Leu/µL)	Neg	-	1	-	-	-	-
		Ca15	3	-	2	4	2	3
		Ca70	2	4	2	1	2	1
		Ca125	-	-	1	-	1	1
	Glucose (mg/dL)	Neg	5	5	5	5	5	5
	Micro albumin (MALB) (mg/dL)	Neg	2	2	2	3	-	1
		>15	-	2	3	-	-	1
		15	3	1	-	2	5	3
Microscopic	Epithelial cells	0	2	1	1	1	5	5
		0-1	3	3	3	3	-	-
		1-2	-	1	1	1	-	-
	Casts	Absent	5	5	5	5	5	5
	Crystals	Present	5	5	5	5	5	5

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

        *: Statistically significant (p<0.05)

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	3	3	3	2	4	5
		Yellow	2	2	2	3	1	-
	Appearance	Clear	3	3	3	1	5	5
		Turbid	2	2	2	4	-	-
	Volume (mL)	Mean	6.3	7.1	6.0	6.2	7.7	8.0
		±SD	0.9	1.3	1.0	1.2	1.4	1.1
Chemical	pH	Mean	7.8	7.9	7.8	8.1	6.9	6.7
		±SD	0.3	0.2	0.3	0.4	0.4	0.7
	Specific Gravity	Mean	1.008	1.007	1.008	1.007	1.014	1.013
		±SD	0.003	0.003	0.003	0.003	0.002	0.003
	Urobilinogen (mg/dL)	Mean	0.2	0.2	0.2	0.4	0.2	0.2
		±SD	0.0	0.0	0.0	0.4	0.0	0.0
	Bilirubin (mg/dL)	Neg	5	3	3	4	5	5
		1	-	2	2	1	-	-
	Ketones (mg/dL)	Neg	5	5	5	5	5	5
	Blood (Ery/µL)	Neg	-	1	1	1	1	2
		Ca10	-	-	1	2	1	1
		Ca25	4	3	3	1	3	2
		Ca80	1	1	-	1	-	-
	Proteins (mg/dL)	Neg	5	2	3	2	5	5
		Trace	-	2	1	2	-	-
		30	-	-	1	-	-	-
		100	-	1	-	-	-	-
		>=300	-	-	-	1	-	-
	Nitrite	Neg	5	4	4	5	5	5
		Pos	-	1	1	-	-	-
	Leucocytes (Leu/µL)	Neg	-	1	2	-	5	5
		Ca15	5	1	1	1	-	-
		Ca70	-	3	2	4	-	-
	Glucose (mg/dL)	Neg	5	5	5	5	5	5
	Micro albumin (MALB) (mg/dL)	Neg	3	1	3	-	4	5
		>15	-	3	2	3	-	-
		15	2	1	-	2	1	-
Microscopic	Epithelial cells	0	1	1	1	1	4	3
		0-1	3	2	3	1	1	2
		1-2	1	2	1	3	-	-
	Casts	Absent	5	5	5	5	5	5
	Crystals	Present	5	5	5	5	5	5

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

      TABLE 14.      SUMMARY OF ABSOLUTE ORGAN WEIGHTS (g)

Group, Sex & Dose (mg/L)		Adrenals	Thymus	Spleen	Testes	Heart	Kidneys	Brain	Liver	Lungs
G1, M & 0	Mean	0.0527	0.2339	0.8565	2.8210	0.8361	1.6059	1.7274	5.5655	0.4897
	±SD	0.0083	0.0413	0.1945	0.0903	0.0688	0.1802	0.0728	0.7674	0.0412
	n	5	5	5	5	5	5	5	5	5
G2, M & 0.001	Mean	0.0511	0.2436	0.9547	2.8027	0.7959	1.5345	1.8076	6.0419	0.4387
	±SD	0.0068	0.0235	0.1372	0.1482	0.0516	0.1245	0.0931	0.5475	0.0213
	n	5	5	5	5	5	5	5	5	5
G3, M & 0.003	Mean	0.0501	0.2345	1.0145	2.8952	0.7489	1.5967	1.7830	6.3929	0.5007
	±SD	0.0022	0.0146	0.0126	0.1496	0.0707	0.1291	0.0377	0.5340	0.0589
	n	5	5	5	5	5	5	5	5	5
G4, M & 0.03	Mean	0.0551	0.2346	0.7923	2.9268	0.8487	1.6192	1.7770	6.2191	0.5150
	±SD	0.0055	0.0282	0.2265	0.0943	0.0613	0.0859	0.0561	0.3189	0.0313
	n	5	5	5	5	5	5	5	5	5
G1R, M & 0	Mean	0.0561	0.2687	0.7827	3.3216	1.2686	2.5481	2.0494	9.6626	0.7409
	±SD	0.0055	0.0450	0.0668	0.2269	0.1601	0.2306	0.1283	0.7918	0.0644
	n	5	5	5	5	5	5	5	5	5
G4R, M & 0.03	Mean	0.0645	0.2973	0.9437	3.4166	1.3296	2.5736	2.0387	9.4686	0.6325*
	±SD	0.0076	0.0446	0.1544	0.2035	0.1943	0.3079	0.0959	0.9268	0.0566
	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	0.0509	0.3158	0.9215	0.1068	0.4884	0.7359	1.4747	1.7259	5.8398	0.5190
	±SD	0.0015	0.0359	0.1064	0.0129	0.0253	0.0394	0.0692	0.0523	0.1397	0.0212
	n	5	5	5	5	5	5	5	5	5	5
G2, F & 0.001	Mean	0.0496	0.3163	0.9228	0.1083	0.5147	0.6604	1.4128	1.7701	5.8464	0.4930
	±SD	0.0012	0.0470	0.0693	0.0100	0.0171	0.0369	0.0541	0.0279	0.1122	0.0382
	n	5	5	5	5	5	5	5	5	5	5
G3, F & 0.003	Mean	0.0543	0.2917	0.6591*	0.1019	0.5289	0.7419	1.4251	1.7663	5.8692	0.4850
	±SD	0.0062	0.0170	0.0595	0.0085	0.0418	0.0380	0.0411	0.0364	0.2150	0.0222
	n	5	5	5	5	5	5	5	5	5	5
G4, F & 0.03	Mean	0.0615*	0.3242	0.5625*	0.1174	0.5928*	0.6597	1.4839	1.7217	5.9847	0.4922
	±SD	0.0047	0.0576	0.1001	0.0131	0.0975	0.0739	0.0806	0.0756	0.4976	0.0392
	n	5	5	5	5	5	5	5	5	5	5
G1R, F & 0	Mean	0.0826	0.3556	0.7772	0.1446	0.5627	0.9666	1.6459	1.9205	7.1936	0.6412
	±SD	0.0062	0.0377	0.1095	0.0197	0.1372	0.0603	0.0577	0.0687	0.1242	0.0612
	n	5	5	5	5	5	5	5	5	5	5
G4R, F & 0.03	Mean	0.0880	0.3618	0.6633	0.1480	0.7905*	0.9553	1.7126	2.0074	7.6419	0.5947
	±SD	0.0077	0.0161	0.1795	0.0170	0.1704	0.0922	0.0936	0.0504	0.4337	0.0817
	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	220.66	0.0240	0.1057	0.3871	1.2802	0.3787	0.7272	0.7833	2.5168	0.2221
	±SD	8.28	0.0047	0.0155	0.0789	0.0715	0.0229	0.0683	0.0323	0.2696	0.0193
	n	5	5	5	5	5	5	5	5	5	5
G2, M & 0.001	Mean	225.28	0.0226	0.1082	0.4232	1.2451	0.3534	0.6808	0.8032	2.6786	0.1950
	±SD	12.57	0.0022	0.0088	0.0507	0.0530	0.0144	0.0326	0.0372	0.1234	0.0110
	n	5	5	5	5	5	5	5	5	5	5
G3, M & 0.003	Mean	221.96	0.0226	0.1058	0.4581	1.3066	0.3373*	0.7192	0.8046	2.8835*	0.2263
	±SD	11.65	0.0017	0.0077	0.0251	0.0839	0.0246	0.0434	0.0344	0.2381	0.0313
	n	5	5	5	5	5	5	5	5	5	5
G4, M & 0.03	Mean	225.33	0.0245	0.1041	0.3512	1.3002	0.3765	0.7184	0.7896	2.7594	0.2292
	±SD	8.88	0.0028	0.0122	0.0984	0.0589	0.0203	0.0206	0.0403	0.0671	0.0219
	n	5	5	5	5	5	5	5	5	5	5
G1R, M & 0	Mean	423.55	0.0132	0.0633	0.1847	0.7850	0.2993	0.6013	0.4838	2.2811	0.1751
	±SD	7.40	0.0012	0.0097	0.0142	0.0643	0.0355	0.0476	0.0278	0.1774	0.0178
	n	5	5	5	5	5	5	5	5	5	5
G4R, M & 0.03	Mean	425.81	0.0152	0.0698	0.2223	0.8019	0.3121	0.6049	0.4792	2.2257	0.1485*
	±SD	8.97	0.0018	0.0101	0.0407	0.0327	0.0440	0.0774	0.0294	0.2397	0.0122
	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	180.16	0.0284	0.1754	0.5156	0.0599	0.2722	0.4091	0.8206	0.9628	3.2505	0.2891
	±SD	11.86	0.0026	0.0164	0.0885	0.0116	0.0250	0.0186	0.0523	0.0943	0.1813	0.0222
	n	5	5	5	5	5	5	5	5	5	5	5
G2, F & 0.001	Mean	181.99	0.0273	0.1749	0.5074	0.0595	0.2835	0.3643	0.7790	0.9750	3.2202	0.2709
	±SD	9.51	0.0019	0.0337	0.0336	0.0046	0.0200	0.0358	0.0672	0.0577	0.1922	0.0145
	n	5	5	5	5	5	5	5	5	5	5	5
G3, F & 0.003	Mean	180.08	0.0304	0.1625	0.3687*	0.0570	0.2942	0.4149	0.7944	0.9866	3.2817	0.2700
	±SD	13.82	0.0052	0.0113	0.0518	0.0085	0.0186	0.0495	0.0526	0.0954	0.3717	0.0142
	n	5	5	5	5	5	5	5	5	5	5	5
G4, F & 0.03	Mean	182.12	0.0339	0.1782	0.3092*	0.0648	0.3266	0.3632	0.8162	0.9473	3.2897	0.2708
	±SD	8.50	0.0033	0.0325	0.0555	0.0095	0.0587	0.0461	0.0576	0.0655	0.2848	0.0253
	n	5	5	5	5	5	5	5	5	5	5	5
G1R, F & 0	Mean	313.21	0.0264	0.1135	0.2491	0.0460	0.1790	0.3084	0.5265	0.6141	2.2997	0.2044
	±SD	12.85	0.0023	0.0099	0.0417	0.0048	0.0393	0.0097	0.0344	0.0357	0.0982	0.0122
	n	5	5	5	5	5	5	5	5	5	5	5
G4R, F & 0.03	Mean	317.76	0.0277	0.1139	0.2078	0.0466	0.2494*	0.3004	0.5390	0.6326	2.4045	0.1868
	±SD	10.94	0.0023	0.0063	0.0507	0.0056	0.0556	0.0224	0.0258	0.0351	0.0978	0.0209
	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	3.0514	13.5085	49.3486	163.5076	48.3791	92.7708	321.4914	28.3198
	±SD	0.4834	1.9822	9.4892	7.9700	2.9370	6.6096	33.1557	1.5500
	n	5	5	5	5	5	5	5	5
G2, M & 0.001	Mean	2.8241	13.4606	52.6892	155.1695	44.0264	84.8340	333.9034	24.2863*
	±SD	0.3111	0.7485	5.7958	7.2109	1.6664	4.0423	18.4618	0.9671
	n	5	5	5	5	5	5	5	5
G3, M & 0.003	Mean	2.8079	13.1454	56.9269	162.3513	41.9926*	89.4621	358.5755	28.1166
	±SD	0.1158	0.6335	1.8348	6.9304	3.7123	5.5684	29.2192	3.6111
	n	5	5	5	5	5	5	5	5
G4, M & 0.03	Mean	3.1064	13.2095	44.4020	164.8334	47.8082	91.2384	350.4051	28.9812
	±SD	0.3610	1.6046	11.7690	7.3621	3.9219	6.4342	23.6216	1.4860
	n	5	5	5	5	5	5	5	5
G1R, M & 0	Mean	2.7351	13.1122	38.2420	162.7398	62.0896	124.5000	471.9952	36.2346
	±SD	0.1876	2.0375	3.1809	16.7581	8.5065	10.4556	33.8733	3.5097
	n	5	5	5	5	5	5	5	5
G4R, M & 0.03	Mean	3.1709	14.6200	46.2381	168.0855	65.1907	126.1154	464.8150	31.0643*
	±SD	0.4121	2.4631	6.7987	15.8779	8.7206	12.1420	44.9406	2.9624
	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	2.9508	18.3267	53.3373	6.1771	28.3212	42.6865	85.5542	338.7184	30.0906
	±SD	0.0504	2.3458	5.3469	0.5573	1.7908	2.9480	5.7457	16.1824	1.5175
	n	5	5	5	5	5	5	5	5	5
G2, F & 0.001	Mean	2.8008	17.8769	52.1121	6.1151	29.0809	37.3223*	79.8356	330.4079	27.8453
	±SD	0.0369	2.7166	3.4286	0.5055	1.0510	2.2575	3.3902	10.4615	1.9887
	n	5	5	5	5	5	5	5	5	5
G3, F & 0.003	Mean	3.0709	16.5243	37.3415*	5.7634	29.9660	42.0043	80.7292	332.2571	27.4727*
	±SD	0.3019	1.0774	3.6538	0.3605	2.5865	2.0152	3.5143	8.8304	1.5142
	n	5	5	5	5	5	5	5	5	5
G4, F & 0.03	Mean	3.5751*	18.7785	32.5684*	6.8232	34.4187*	38.3535	86.2039	347.4261	28.5554
	±SD	0.2566	2.7133	4.6710	0.7113	5.2359	4.3975	3.3879	20.3936	1.1806
	n	5	5	5	5	5	5	5	5	5
G1R, F & 0	Mean	4.3094	18.5686	40.5984	7.5448	29.4614	50.4088	85.7230	374.8632	33.4283
	±SD	0.4044	2.4029	6.6361	1.1282	8.0893	4.0792	2.0529	11.7783	3.4948
	n	5	5	5	5	5	5	5	5	5
G4R, F & 0.03	Mean	4.3817	18.0309	33.0780	7.3741	39.2759	47.6125	85.3903	381.0484	29.6408
	±SD	0.3541	0.9481	9.1174	0.8720	7.8443	4.7027	5.8178	27.2735	4.1373
	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  (ppm)	Air inlet flow rate (L/min)*	BZC (mg/L)
G1/G1R & 0	Mean	22.59	56.16	20.43	622.36	20.00	-
	±SD	0.23	0.98	0.21	5.92	0.00	-
G2 & 0.003	Mean	22.67	56.49	20.53	624.48	20.00	0.0011
	±SD	0.25	0.40	0.20	4.29	0.00	0.0001
G3 & 0.01	Mean	22.68	56.52	20.51	623.78	20.00	0.0031
	±SD	0.25	0.85	0.22	5.80	0.00	0.0001
G4/G4R & 0.03	Mean	22.71	56.61	20.53	625.26	20.00	0.032
	±SD	0.26	0.40	0.20	4.36	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)	1850	20	360	0.26
		3240			0.45
		4990			0.69
2	G1/G1R (Air Only)	-	20	360	-
	G2, G3 and G4/G4R  (Test Item)	1910	20	360	0.27
		3350			0.47
		4890			0.68
3	G1/G1R  (Air Only)	-	20	360	-
	G2, G3 and G4/G4R  (Test Item)	1850	20	360	0.26
		3280			0.46
		4960			0.69
4	G1/G1R  (Air Only)	-	20	360	-
	G2, G3 and G4/G4R  (Test Item)	1880	20	360	0.26
		3270			0.45
		4910			0.68
5	G1/G1R  (Air Only)	-	20	360	-
	G2, G3 and G4/G4R  (Test Item)	1920	20	360	0.27
		3380			0.47
		4920			0.68
8	G1/G1R  (Air Only)	-	20	360	-
	G2, G3 and G4/G4R  (Test Item)	1890	20	360	0.26
		3450			0.48
		4880			0.68
9	G1/G1R  (Air Only)	-	20	360	-
	G2, G3 and G4/G4R  (Test Item)	1880	20	360	0.26
		3390			0.47
		4930			0.68

 TABLE 14 (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)
10	G1/G1R  (Air Only)	-	20	360	-
	G2, G3 and G4/G4R  (Test Item)	1930	20	360	0.27
		3380			0.47
		4940			0.69
11	G1/G1R  (Air Only)	-	20	360	-
	G2, G3 and G4/G4R  (Test Item)	1860	20	360	0.26
		3410			0.47
		4850			0.67
12	G1/G1R  (Air Only)	-	20	360	-
	G2, G3 and G4/G4R  (Test Item)	1830	20	360	0.25
		3550			0.49
		4910			0.68
15	G1/G1R  (Air Only)	-	20	360	-
	G2, G3 and G4/G4R  (Test Item)	1850	20	360	0.26
		3280			0.46
		4880			0.68
16	G1/G1R  (Air Only)	-	20	360	-
	G2, G3 and G4/G4R  (Test Item)	1880	20	360	0.26
		3550			0.49
		4930			0.68
17	G1/G1R  (Air Only)	-	20	360	-
	G2, G3 and G4/G4R  (Test Item)	1890	20	360	0.26
		3340			0.46
		4890			0.68
18	G1/G1R  (Air Only)	-	20	360	-
	G2, G3 and G4/G4R  (Test Item)	1940	20	360	0.27
		3420			0.47
		4810			0.67

 

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)
10	G1/G1R  (Air Only)	-	20	360	-
	G2, G3 and G4/G4R  (Test Item)	1930	20	360	0.27
		3380			0.47
		4940			0.69
11	G1/G1R  (Air Only)	-	20	360	-
	G2, G3 and G4/G4R  (Test Item)	1860	20	360	0.26
		3410			0.47
		4850			0.67
12	G1/G1R  (Air Only)	-	20	360	-
	G2, G3 and G4/G4R  (Test Item)	1830	20	360	0.25
		3550			0.49
		4910			0.68
15	G1/G1R  (Air Only)	-	20	360	-
	G2, G3 and G4/G4R  (Test Item)	1850	20	360	0.26
		3280			0.46
		4880			0.68
16	G1/G1R  (Air Only)	-	20	360	-
	G2, G3 and G4/G4R  (Test Item)	1880	20	360	0.26
		3550			0.49
		4930			0.68
17	G1/G1R  (Air Only)	-	20	360	-
	G2, G3 and G4/G4R  (Test Item)	1890	20	360	0.26
		3340			0.46
		4890			0.68
18	G1/G1R  (Air Only)	-	20	360	-
	G2, G3 and G4/G4R  (Test Item)	1940	20	360	0.27
		3420			0.47
		4810			0.67

 

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)
19	G1/G1R  (Air Only)	-	20	360	-
	G2, G3 and G4/G4R  (Test Item)	1880	20	360	0.26
		3560			0.49
		4910			0.68
22	G1/G1R  (Air Only)	-	20	360	-
	G2, G3 and G4/G4R  (Test Item)	1960	20	360	0.27
		3650			0.51
		4880			0.68
23	G1/G1R  (Air Only)	-	20	360	-
	G2, G3 and G4/G4R  (Test Item)	1910	20	360	0.27
		3410			0.47
		4880			0.68
24	G1/G1R  (Air Only)	-	20	360	-
	G2, G3 and G4/G4R  (Test Item)	1890	20	360	0.26
		3380			0.47
		4920			0.68
25	G1/G1R  (Air Only)	-	20	360	-
	G2, G3 and G4/G4R  (Test Item)	1940	20	360	0.27
		3420			0.47
		4860			0.68
26	G1/G1R  (Air Only)	-	20	360	-
	G2, G3 and G4/G4R  (Test Item)	1930	20	360	0.27
		3360			0.47
		4850			0.67

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
Group	G1	G2	G3	G4	G1R	G4R
Nominal Target Dose (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
Group	G1	G2	G3	G4	G1R	G4R
Nominal Target Dose (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	-	-	-	-	-	-

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 Dose (mg/L)					0		0.03
Group					G1		G4
Sex					M	F	M	F
Number of Animals					5	5	5	5
Adrenals	Number examined				5	5	5	5
	Within normal limits				5	5	5	5
Brain	Number examined				5	5	5	5
	Within normal limits				5	5	5	5
Cervical lymph nodes	Number examined				5	5	5	5
	Within normal limits				5	5	5	5
Esophagus	Number examined				5	5	5	5
	Within normal limits				5	5	5	5
Eyes with optic nerve and eye lids	Number examined				5	5	5	5
	Within normal limits				5	5	5	5
Heart	Number examined				5	5	5	5
	Within normal limits				5	5	5	5
Hilar lymph nodes	Number examined				5	5	5	5
	Within normal limits				5	5	5	5
Kidneys	Number examined				5	5	5	5
	Within normal limits				5	4	2	4
	Basophilia, tubule			Minimal	-	-	3	1
	Dilatation, tubule			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	3	5	3
	Infiltrate, mononuclear cell	Minimal			-	2	-	2
Lungs(left lobe)	Number examined				5	5	5	5
	Within normal limits				5	5	-	-
	Pigment, golden yellow		Mild		-	-	1	2
			Moderate		-	-	4	3
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

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 Dose (mg/L)				0		0.03
Group				G1		G4
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	4	5
	Cellularity, decreased, white pulp		Mild	-	-	1	-
Stomach	Number examined			5	5	5	5
	Within normal limits			5	5	5	5
Testes	Number examined			5	X	5	X
	Within normal limits			5	X	5	X
Thymus	Number examined			5	5	5	5
	Within normal limits			5	5	5	5
Thyroid	Number examined			5	5	5	5
	Within normal limits			5	3	4	5
	Cyst, ultimobranchial		Present	-	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	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
Nominal Target Dose (mg/L)					0		0.03
Group					G1R		G4R
Sex					M	F	M	F
Number of Animals					5	5	5	5
Adrenals	Number examined				5	5	5	5
	Within normal limits				5	4	5	5
	Ectopic tissue, adrenocortical	Present			-	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				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	4	5
	Infiltrate, mononuclear cell			Minimal	-	-	1	-
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	4	5
	Basophilia, tubule		Minimal		1	1	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	3	5	3
	Infiltrate, mononuclear cell		Minimal		-	2	-	2
Lungs(left lobe)	Number examined				5	5	5	5
	Within normal limits				5	5	-	-
	Pigment, golden yellow		Minimal		-	-	1	5
			Mild		-	-	4	-
			Moderate		-	-	-	-
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

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

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

Route of administration				Inhalation
Treatment				Air only		High Dose
Nominal Target Dose (mg/L)				0		0.03
Group				G1R		G4R
Sex				M	F	M	F
Number of Animals				5	5	5	5
Ovaries	Number examined			X	5	X	5
	Within normal limits			X	5	X	5
Parathyroid	Number examined			5	5	5	5
	Within normal limits			5	5	5	5
Seminal vesicles	Number examined			5	X	5	X
	Within normal limits			5	X	5	X
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	4	5
	Cellularity, decreased, white pulp		Mild	-	-	1	-
Stomach	Number examined			5	5	5	5
	Within normal limits			5	5	5	5
Testes	Number examined			5	X	5	X
	Within normal limits			5	X	5	X
Thymus	Number examined			5	5	5	5
	Within normal limits			5	5	5	5
Thyroid	Number examined			5	5	5	5
	Within normal limits			2	5	5	2
	Cyst, ultimobranchial		Present	2	-	-	3
	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	3	X	4
	Dilatation, lumen	Minimal		X	1	X	1
		Mild		X	1	X	-

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

 TABLE 22. SUMMARY OF HISTOPATHOLOGY FINDINGS – LOW DOSE AND MID DOSE GROUP

Route of administration			Inhalation
Treatment			Low Dose		Mid Dose
Nominal Target Dose (mg/L)			0.001		0.003
Group			G2		G3
Sex			M	F	M	F
Number of Animals			5	5	5	5
Lungs(left lobe)	Number examined		5	5	5	5
	Within normal limits		5	5	3	1
	Pigment, golden yellow	Minimal	-	-	2	4

M: Male; F: Female; -: no incidence

 

 

 

 

 

 

 

 

 

 

 

 

                                                                                                                                 

                                      

 

 

 

 

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 4 weeks by flow-past nose-only inhalation route to Sprague Dawley rats. The nominal dose of 0.03 mg/L (6 hrs/day, 5 days/week) corresponded to an actual exposure concentration in males and females.

Executive summary:

The objective of this study was to determine the toxic potential of the test item, when administered for 6 hours/day, 5 days per week, for 4 consecutive weeks by flow-past nose-only inhalation route to Sprague Dawley rats. This study provides information on toxic effects, target organs, the possibility of cumulative effects, the reversibility of effects (after 56 days recovery period), and an estimate of the No Observed Adverse Effects Concentration (NOAEC).
A total of 60 (30 males + 30 females) healthy young Sprague Dawley rats were distributed to four groups viz., control (G1), low dose (G2), mid dose (G3), high dose (G4) and two recovery groups [(G1R (Air only) and G4R (high dose)]. Each main and recovery group comprised of 10 animals (5 males and 5 females). Animals allocated to Groups G2, G3 and G4/G4R were exposed to test item for 6 hours per day, 5 days per week, for 4 consecutive weeks., at a nominal target concentration of 0.001, 0.003 and 0.03 mg/L. Animals of the control group (G1/G1R) received air only inhalation for 6 hours/5 days per week for 4 consecutive weeks. The inhalation exposure of test item/air was achieved by a flow-past, nose-only dynamic inhalation exposure system supplied by CH Technologies, USA.
All animals were observed once daily for clinical signs and twice daily for mortality. Body weight was recorded before first exposure (day 1), twice during first two weeks and weekly once thereafter. Feed consumption was recorded weekly. Ophthalmoscopic examination was performed during the acclimatization period for all groups, and during Week 4 for main groups (G1 and G4), and during Week 12 for recovery groups (G1R and G4R). Neurological/Functional test was performed during Week 4 for G1 and G4 groups and during Week 12 for recovery group animals.
At the end of treatment and recovery periods, 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 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 and ophthalmoscopic examination 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. However, few variations in differential counts in haematology and BALF fluid could be secondary effects due to accumulation of test item particles in lungs and considered nonadverse. No toxicologically significant changes were observed in fasting body weight, absolute organ weight, or relative organ weight.
No treatment related gross pathological lesions were observed in main group and recovery group animals.
During microscopic examination, in lungs, minimal to moderate, diffuse/multifocal, variably sized, pigmented (golden yellow) granular material was observed throughout the parenchyma of lung at mid, high and high dose recovery group rats. This pigment was distributed both in alveolar and bronchiolar spaces with no accompanying inflammation or tissue destruction to the lung parenchyma. This pigment was suggestive of inhaled test item particles as the physical appearance of test item was also yellow coloured powder. In the absence of cellular changes to lung parenchyma, mere presence of pigment in lungs could be considered as non-adverse effect up to the dose level of 0.03 mg/L. (Nikula KJ et.al., 2014).
Conclusion
Based on the observed results, it is concluded that the No Observed Adverse Effect Concentration (NOAEC) of test item was found to be 0.03 mg/L when exposed for 6 hours/day, 5 days/week, for 4 weeks by flow-past nose-only inhalation route to Sprague Dawley rats. The nominal dose of 0.03 mg/L (6 hrs/day, 5 days/week) corresponded to an actual exposure concentration in males and females.