N,N'-(2,5-dichloro-1,4-phenylene)bis[4-[[2-chloro-5-(trifluoromethyl)phenyl]azo]-3-hydroxynaphthalene-2-carboxamide]

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:

05 July 2022 to 19 August 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

“28-day (Sub-acute) inhalation toxicity study” adopted on 25 June 2018

Deviations:

no

GLP compliance:

no

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

N,N'-(2,5-dichloro-1,4-phenylene)bis[4-[[2-chloro-5-(trifluoromethyl)phenyl]azo]-3-hydroxynaphthalene-2-carboxamide]

EC Number:

257-776-0

EC Name:

N,N'-(2,5-dichloro-1,4-phenylene)bis[4-[[2-chloro-5-(trifluoromethyl)phenyl]azo]-3-hydroxynaphthalene-2-carboxamide]

Cas Number:

52238-92-3

Molecular formula:

C42H22Cl4F6N6O4

IUPAC Name:

N,N'-(2,5-dichloro-1,4-phenylene)bis(4-{[2-chloro-5-(trifluoromethyl)phenyl]diazenyl}-3-hydroxy-2-naphthamide)

Test material form:

solid: nanoform, no surface treatment

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and batch number of test material: Clariant Chemicals India Limited and INB3229979
- Purity, including information on contaminants, isomers, etc.: 99.7 % (w/w) C.I. Pigment Red 242

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 (e.g. warming, grinding): No
- Preliminary purification step (if any): No
- Final concentration of a dissolved solid, stock liquid or gel: No
- Final preparation of a solid (e.g. stock crystals ground to fine powder using a mortar and pestle): No

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

Sprague Dawley and 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 at the time of randomization
- Weight at study initiation: Males: 140.17 g to 159.83 g and Females: 120.19 g to 140.00 g
- Fasting period before study: No
- Housing: standard polycarbonate cage (size: L 430 x B 280 x H 210 mm)
- 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
- Acclimation period: 06 July 2022 to 11 July 2022


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.7°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: 06 July 2022 To: 19 August 2022

Administration / exposure

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose/head only

Vehicle:

clean air

Mass median aerodynamic diameter (MMAD):

> 1.82 - <= 1.99 µm

Geometric standard deviation (GSD):

2.52

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: flow-past, nose-only dynamic inhalation exposure system
- Method of holding animals in test chamber: restrainer
- Source and rate of air: filter air and 20 lt/min
- Method of conditioning air: purified filter air
- System of generating particulates/aerosols: RBG
- Temperature, humidity, pressure in air chamber: 22.1 to 22.9°C, 51.4 to 59.6%, 60 Psi
- Air flow rate: 20 liter/min
- Air change rate: 12 air changes per hour
- Method of particle size determination: Gravimetric method
- Treatment of exhaust air: NaOH

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

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

14 days of exposure (6 hours per day)

Frequency of treatment:

14 consecutive days

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0.1 mg/L air

Remarks:

G4

Doses / concentrations 2

Dose / conc.:

0.03 mg/L air

Remarks:

G3

Doses / concentrations 3

Dose / conc.:

0.01 mg/L air

Remarks:

G2

Doses / concentrations 4

Dose / conc.:

0 mg/L air

Remarks:

G1

No. of animals per sex per dose:

5 males and 5 females per group

Control animals:

yes, concurrent no treatment

Details on study design:

- Dose selection rationale: As per sponsor provided doses
- Rationale for animal assignment (if not random): randomization carried out
- Fasting period before blood sampling for clinical biochemistry: Yes
- Rationale for selecting satellite groups: No
- Post-exposure recovery period in satellite groups: No

Positive control:

No

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily
- Cage side observations checked in table [No.1] were included.

DETAILED CLINICAL OBSERVATIONS: No

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

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: on the day of randomization
- Dose groups that were examined: all animals

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Day 15
- Anaesthetic used for blood collection: Yes
- Animals fasted: Yes
- How many animals: 40
- Parameters checked in table [No.6] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Day 15
- Animals fasted: Yes
- How many animals: 40
- Parameters checked in table [No.8] were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: Day 15
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked in table [No.9] were examined.

NEUROBEHAVIOURAL EXAMINATION: No


BRONCHOALVEOLAR LAVAGE FLUID (BALF): Yes
- Time schedule for analysis: Day 15
- Dose groups that were examined: all groups
- Number of animals: 40
- Parameters checked in table [number 7] were examined.
LUNG BURDEN: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

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 27. Body weight, percent change in body weight with respect to Day 1, feed consumption, 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

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food efficiency:

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

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 males, decrease in triglycerides, total protein (G3) and albumin (G4) was noted.
The observed variations noted are considered incidental due to lack of dose responsiveness and/or could be due to random biological variation. The noted significance values were within the historical control data

Urinalysis findings:

no effects observed

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 Females, increase in relative weight of adrenals (G3), heart and kidneys (G4) was noted.
The variations noted in organ weights and its ratios were not associated with any macroscopic changes in all related organs in high dose group and also absolute weights were unaffected, hence considered not to be test item related and incidental

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Microscopically, test item-related changes were observed in lungs of all the test item administered animals and were characterized by presence of multifocal, minimal to moderate, variably sized, pigmented (reddish brown) granular material throughout parenchyma of lung. This pigmented granular material was distributed both in alveolar and bronchiolar spaces. In the absence of accompanying inflammation or tissue destruction to the lung parenchyma, this change could be considered as non-adverse from dose level of 0.01mg/L

Effect levels

Key result

Dose descriptor:

NOAEC

Effect level:

>= 100 mg/m³ air

Based on:

test mat.

Sex:

male/female

Basis for effect level:

behaviour (functional findings)

body weight and weight gain

clinical signs

food consumption and compound intake

haematology

immunology

mortality

ophthalmological examination

organ weights and organ / body weight ratios

serum/plasma biochemistry

serum/plasma hormone analyses

urinalysis

Target system / organ toxicity

Key result

Critical effects observed:

no

Any other information on results incl. tables

• SUMMARY OF CLINICAL SIGNS AND MORTALITY RECORD

                                                                                                                          Refer Appendix 1

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 & 0.01	5	N	0/5
G3, M & 0.03	5	N	0/5
G4, M & 0.1	5	N	0/5
G1, F & 0	5	N	0/5
G2, F & 0.01	5	N	0/5
G3, F & 0.03	5	N	0/5
G4, F & 0.1	5	N	0/5

M: Male; F: Female; N: None. The test item color was retained in snout region in test item exposure groups. 

                            

 

• SUMMARY OF BODY WEIGHTS (g) RECORD

  Refer Appendix 2

Group, Sex & Dose (mg/L)		Body Weight (g) on Days
		1	5	8	12	14
G1, M & 0	Mean	169.05	178.89	191.72	215.49	219.57
	±SD	7.68	12.14	10.83	19.16	19.92
	n	5	5	5	5	5
G2, M & 0.01	Mean	172.83	185.40	194.33	209.18	212.93
	±SD	8.54	8.85	13.71	14.49	14.72
	n	5	5	5	5	5
G3, M & 0.03	Mean	172.04	187.50	200.34	218.46	222.93
	±SD	5.55	5.06	5.96	7.26	6.61
	n	5	5	5	5	5
G4, M & 0.1	Mean	174.49	186.56	193.97	209.24	213.64
	±SD	5.90	7.36	10.80	12.98	13.45
	n	5	5	5	5	5
G1, F & 0	Mean	148.35	155.20	161.69	174.54	176.27
	±SD	6.67	6.42	9.32	16.60	16.41
	n	5	5	5	5	5
G2, F & 0.01	Mean	149.48	154.48	158.37	167.51	169.36
	±SD	7.38	6.65	6.59	6.47	6.23
	n	5	5	5	5	5
G3, F & 0.03	Mean	150.97	157.56	160.55	170.59	172.77
	±SD	8.08	10.01	9.28	8.24	7.74
	n	5	5	5	5	5
G4, F & 0.1	Mean	151.40	157.75	162.24	170.36	172.14
	±SD	7.60	5.51	7.97	6.31	5.86
	n	5	5	5	5	5

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

 

 

 

 

• SUMMARY OF PERCENT CHANGE IN BODY WEIGHT WITH RESPECT TO DAY 1 RECORD

Refer Appendix 3

Group, Sex & Dose (mg/L)		Percent Change in Body Weight (%) during Days
						1 to 5	1 to 8	1 to 12	1 to 14
		G1, M & 0	Mean	5.75	13.43	27.44	29.86
±SD	3.40		4.36	9.26	9.77
n	5		5	5	5
G2, M & 0.01	Mean	7.30	12.37	21.04	23.21
	±SD	1.82	3.41	6.27	6.46
	n	5	5	5	5
G3, M & 0.03	Mean	9.00	16.49	27.03	29.64
	±SD	0.85	3.32	4.15	4.15
	n	5	5	5	5
G4, M & 0.1	Mean	6.96	11.25	20.10	22.60
	±SD	3.85	7.09	9.63	9.60
	n	5	5	5	5
G1, F & 0	Mean	4.71	9.21	18.22	19.39
	±SD	4.68	8.68	16.63	16.59
	n	5	5	5	5
G2, F & 0.01	Mean	3.39	5.99	12.20	13.46
	±SD	2.43	2.20	5.27	5.63
	n	5	5	5	5
G3, F & 0.03	Mean	4.33	6.33	13.04	14.50
	±SD	2.03	1.83	2.43	2.63
	n	5	5	5	5
G4, F & 0.1	Mean	4.25	7.18	12.61	13.84
	±SD	1.63	2.53	3.23	4.84
	n	5	5	5	5

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

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

• SUMMARY OF AVERAGE FEED CONSUMPTION (g/rat/day) RECORD

                                                                                                                             Refer Appendix 4

Group, Sex & Dose (mg/L)		Week 1	Week 2
G1, M & 0	Mean	19.66	24.91
	±SD	1.43	3.52
	n	3	3
G2, M & 0.01	Mean	19.18	20.68
	±SD	2.29	0.41
	n	3	3
G3, M & 0.03	Mean	18.60	21.27
	±SD	1.08	7.35
	n	3	3
G4, M & 0.1	Mean	17.42	20.08
	±SD	2.24	6.05
	n	3	3
G1, F & 0	Mean	17.76	17.06
	±SD	4.02	2.69
	n	3	3
G2, F & 0.01	Mean	13.86	18.60
	±SD	1.84	1.45
	n	3	3
G3, F & 0.03	Mean	15.08	17.62
	±SD	0.34	0.92
	n	3	3
G4, F & 0.1	Mean	13.83	15.41
	±SD	0.17	4.05
	n	3	3

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

 

 

 

 

 

 

 

 

 

 

• SUMMARY OF OPHTHALMOSCOPIC EXAMINATION RECORD

Week 2                                                                                                                                            Refer Appendix 5

Group, Sex & Dose (mg/L)	G1, M & 0		G4, M & 0.1		G1, F & 0		G4 F & 0.1
Number of Animals	5		5		5		5
	Observations
Eye Parameters ↓	L	R	L	R	L	R	L	R
Eye	N	N	N	N	N	N	N	N
Eye Lids	N	N	N	N	N	N	N	N
Cornea	N	N	N	N	N	N	N	N
Iris	N	N	N	N	N	N	N	N
Aqueous Humour	N	N	N	N	N	N	N	N
Lens	N	N	N	N	N	N	N	N
Vitreous Humour	N	N	N	N	N	N	N	N
Retina/Optic disc	N	N	N	N	N	N	N	N

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

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

• SUMMARY OF HAEMATOLOGY RECORD

                                          Refer Appendix 6

Group, Sex & Dose (mg/L)		Total Leucocyte Count	Total Erythrocyte Count	Haemoglobin	Haematocrit	Mean Corpuscular Volume	Mean Corpuscular Haemoglobin	Mean Corpuscular Haemoglobin Concentration	Platelet Count	Mean Platelet Volume
		(WBC)	(RBC)	(HGB)	(HCT)	(MCV)	(MCH)	(MCHC)	(PLT)	(MPV)
		(10^3 cells/µL)	(10^6 cells/µL)	(g/dL)	(%)	(fL)	(pg)	(g/dL)	(10^3 cells/µL)	(fL)
G1, M & 0	Mean	8.64	7.20	15.90	50.40	70.40	22.16	31.54	820.00	8.60
	±SD	1.39	0.81	1.36	3.68	5.02	1.42	0.80	152.92	0.35
	n	5	5	5	5	5	5	5	5	5
G2, M & 0.01	Mean	6.76	6.81	15.38	50.98	75.48	22.70	30.18	1010.60	9.14
	±SD	2.32	0.76	1.00	2.40	7.30	1.66	2.18	320.35	0.61
	n	5	5	5	5	5	5	5	5	5
G3, M & 0.03	Mean	10.16	7.37	16.06	50.34	68.92	21.94	31.94	906.80	8.52
	±SD	2.67	0.68	0.93	2.96	8.85	2.15	1.41	103.97	0.73
	n	5	5	5	5	5	5	5	5	5
G4, M & 0.1	Mean	7.35	7.43	15.24	49.24	67.00	20.64	30.92	837.80	8.76
	±SD	1.54	1.01	1.40	2.76	7.53	1.30	1.57	217.28	0.51
	n	5	5	5	5	5	5	5	5	5
G1, F & 0	Mean	9.22	8.19	15.90	48.16	59.00	19.54	33.06	1027.80	8.40
	±SD	1.46	0.79	0.60	2.67	2.80	1.29	0.75	190.32	0.42
	n	5	5	5	5	5	5	5	5	5
G2, F & 0.01	Mean	7.07	7.77	15.72	49.14	64.18	20.60	32.04	1026.20	8.36
	±SD	1.52	1.15	0.69	2.38	8.33	3.01	0.67	192.39	0.17
	n	5	5	5	5	5	5	5	5	5
G3, F & 0.03	Mean	9.86	7.71	15.90	48.16	62.56	20.64	33.04	736.80	8.78
	±SD	1.52	0.52	1.10	2.40	3.75	0.98	1.17	278.81	1.21
	n	5	5	5	5	5	5	5	5	5
G4, F & 0.1	Mean	11.08	8.22	16.14	49.48	60.66	19.82	32.62	898.00	8.00
	±SD	4.98	0.82	0.40	1.22	6.07	1.87	0.48	150.67	0.47
	n	5	5	5	5	5	5	5	5	5

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

 

 

 

 

 

TABLE 6 (Contd…).  SUMMARY OF HAEMATOLOGY RECORD

                                                                                       Refer Appendix 6

Group, Sex & Dose (mg/L)		Reticulocyte Count	Neutrophils	Lymphocytes	Monocytes	Eosinophils	Basophils	Absolute Reticulocyte Count
		(Retic)	(Neut)	(Lymph)	(Mono)	(Eos)	(Baso)	(Retic)
		(%)	(%)	(%)	(%)	(%)	(%)	(10^9 cells/L)
G1, M & 0	Mean	9.43	20.74	71.22	5.74	0.60	0.36	660.60
	±SD	3.64	6.32	6.93	1.61	0.30	0.09	201.34
	n	5	5	5	5	5	5	5
G2, M & 0.01	Mean	14.73	19.42	75.08	3.74	0.42	0.36	975.80
	±SD	6.17	5.02	6.42	1.39	0.16	0.05	356.74
	n	5	5	5	5	5	5	5
G3, M & 0.03	Mean	6.23	25.68	68.06	4.36	0.34	0.34	441.56
	±SD	4.20	22.05	21.66	1.30	0.15	0.11	254.93
	n	5	5	5	5	5	5	5
G4, M & 0.1	Mean	8.46	25.02	68.30	4.48	0.46	0.38	574.28
	±SD	7.05	6.00	4.48	2.79	0.38	0.18	364.19
	n	5	5	5	5	5	5	5
G1, F & 0	Mean	5.88	15.22	76.56	4.06	2.36	0.36	467.00
	±SD	2.56	1.67	5.23	1.58	2.25	0.11	163.09
	n	5	5	5	5	5	5	5
G2, F & 0.01	Mean	7.65	21.04	73.20	3.80	0.40	0.32	591.80
	±SD	4.14	4.66	5.85	1.15	0.29	0.08	341.93
	n	5	5	5	5	5	5	5
G3, F & 0.03	Mean	6.14	18.66	74.82	3.38	1.46	0.40	466.74
	±SD	2.59	1.07	1.29	0.82	1.28	0.07	193.60
	n	5	5	5	5	5	5	5
G4, F & 0.1	Mean	3.78	17.88	76.20	3.30	0.84	0.42	298.06
	±SD	2.11	6.89	7.42	0.86	0.35	0.11	129.35
	n	5	5	5	5	5	5	5

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

 

                                       

 

 

TABLE 6 (Contd…). SUMMARY OF HAEMATOLOGY RECORD

    Refer Appendix 6

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)
		(10^3 cells/µL)	(10^3 cells/µL)	(10^3 cells/µL)	(10^3 cells/µL)	(10^3 cells/µL)	(Seconds)	(Seconds)
G1, M & 0	Mean	1.75	6.21	0.49	0.05	0.03	22.30	24.06
	±SD	0.46	1.55	0.11	0.02	0.01	5.38	4.86
	n	5	5	5	5	5	5	5
G2, M & 0.01	Mean	1.26	5.15	0.23	0.03	0.02	19.64	26.06
	±SD	0.33	2.14	0.05	0.01	0.01	1.64	7.49
	n	5	5	5	5	5	5	5
G3, M & 0.03	Mean	2.96	6.57	0.44	0.04	0.03	18.90	25.54
	±SD	3.29	1.96	0.16	0.02	0.02	2.16	4.90
	n	5	5	5	5	5	5	5
G4, M & 0.1	Mean	1.85	5.00	0.34	0.03	0.03	20.60	19.84
	±SD	0.67	1.02	0.26	0.02	0.01	1.60	3.55
	n	5	5	5	5	5	5	5
G1, F & 0	Mean	1.39	7.11	0.36	0.20	0.03	20.36	23.74
	±SD	0.08	1.55	0.11	0.17	0.01	0.13	5.39
	n	5	5	5	5	5	5	5
G2, F & 0.01	Mean	1.46	5.21	0.27	0.03	0.02	20.78	23.92
	±SD	0.33	1.34	0.10	0.03	0.01	0.53	7.42
	n	5	5	5	5	5	5	5
G3, F & 0.03	Mean	1.84	7.38	0.33	0.14	0.04	19.66	18.58
	±SD	0.30	1.17	0.08	0.13	0.00	2.76	1.38
	n	5	5	5	5	5	5	5
G4, F & 0.1	Mean	1.79	8.64	0.34	0.09	0.05	20.64	23.22
	±SD	0.49	4.50	0.11	0.05	0.04	3.51	4.39
	n	5	5	5	5	5	5	5

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

 

                                                                                                                                  

 

 

• SUMMARY OF BALF RECORD

                                                                                                                                                                                             Refer Appendix 7

Group, Sex & Dose (mg/L)		Total Leucocyte Count	Neutrophils	Lymphocytes	Monocytes	Eosinophils	Basophils	Absolute Neutrophils
		(10^3 cells/µL)	(%)	(%)	(%)	(%)	(%)	(10^3 cells/µL)
G1, M & 0	Mean	0.32	34.38	54.74	4.98	1.50	3.62	0.10
	±SD	0.17	16.38	20.69	3.27	1.47	2.43	0.08
	n	5	5	5	5	5	5	5
G2, M & 0.01	Mean	0.35	38.60	55.56	2.32	1.40	3.46	0.14
	±SD	0.16	4.64	6.40	0.85	0.41	0.99	0.08
	n	5	5	5	5	5	5	5
G3, M & 0.03	Mean	0.34	36.78	55.54	3.30	2.66	3.06	0.12
	±SD	0.10	8.71	9.43	1.19	0.67	2.24	0.04
	n	5	5	5	5	5	5	5
G4, M & 0.1	Mean	0.40	50.94	43.10	2.04	3.36*	2.52	0.19
	±SD	0.18	16.36	15.44	1.42	0.75	1.36	0.06
	n	5	5	5	5	5	5	5
G1, F & 0	Mean	0.24	31.92	58.22	2.44	1.76	3.08	0.07
	±SD	0.09	8.18	10.34	1.61	1.43	1.38	0.02
	n	5	5	5	5	5	5	5
G2, F & 0.01	Mean	0.28	40.10	52.32	2.52	1.56	5.20	0.10
	±SD	0.13	13.01	12.21	1.09	1.07	4.26	0.06
	n	5	5	5	5	5	5	5
G3, F & 0.03	Mean	0.30	40.38	52.90	1.80	2.82	1.74	0.13
	±SD	0.10	9.34	12.08	0.78	1.14	1.30	0.07
	n	5	5	5	5	5	5	5
G4, F & 0.1	Mean	0.38	46.38	47.52	1.64	3.78	1.54	0.17
	±SD	0.24	4.76	5.64	0.99	1.38	1.47	0.09
	n	5	5	5	5	5	5	5

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

 

       

 

TABLE 7 (Contd…). SUMMARY OF BALF RECORD

        Refer Appendix 7

Group, Sex & Dose (mg/L)		Absolute Lymphocytes	Absolute Monocytes	Absolute Eosinophils	Absolute Basophils	Lactate Dehydrogenase	Total Protein
		(Lymph)	(Mono)	(Eos)	(Baso)	(LDH)	(TPR)
		(10^3 cells/µL)	(10^3 cells/µL)	(10^3 cells/µL)	(10^3 cells/µL)	(u/L)	(μg/mL)
G1, M & 0	Mean	0.19	0.02	0.00	0.01	104.40	308.63
	±SD	0.12	0.02	0.00	0.02	59.75	61.25
	n	5	5	5	5	5	5
G2, M & 0.01	Mean	0.19	0.01	0.00	0.01	32.80*	268.80
	±SD	0.08	0.01	0.00	0.01	11.80	82.64
	n	5	5	5	5	5	5
G3, M & 0.03	Mean	0.19	0.01	0.01*	0.01	47.20*	257.40
	±SD	0.09	0.00	0.00	0.01	17.06	34.26
	n	5	5	5	5	5	5
G4, M & 0.1	Mean	0.18	0.01	0.01*	0.01	46.00*	235.45
	±SD	0.13	0.01	0.01	0.01	14.51	44.95
	n	5	5	5	5	5	5
G1, F & 0	Mean	0.15	0.00	0.00	0.01	36.40	271.14
	±SD	0.07	0.01	0.01	0.01	11.72	25.82
	n	5	5	5	5	5	5
G2, F & 0.01	Mean	0.15	0.01	0.00	0.01	30.80	260.63
	±SD	0.07	0.01	0.00	0.02	11.21	111.98
	n	5	5	5	5	5	5
G3, F & 0.03	Mean	0.15	0.00	0.01	0.00	37.80	209.58
	±SD	0.05	0.01	0.01	0.00	9.20	56.34
	n	5	5	5	5	5	5
G4, F & 0.1	Mean	0.19	0.01	0.01	0.00	47.80	257.45
	±SD	0.13	0.01	0.01	0.01	15.90	117.27
	n	5	5	5	5	5	5

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

 

 

 

• SUMMARY OF CLINICAL CHEMISTRY RECORD

                                                                                                                                                                                                                                                                   Refer Appendix 8

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	87.40	33.06	0.52	54.20	65.00	6.74	3.43	55.60	123.60
	±SD	12.20	6.98	0.04	15.47	22.53	0.32	0.11	15.47	17.40
	n	5	5	5	5	5	5	5	5	5
G2, M & 0.01	Mean	80.00	37.26	0.51	41.60	43.60	6.50	3.23	54.60	119.60
	±SD	16.08	5.79	0.02	5.86	19.97	0.34	0.19	15.34	14.03
	n	5	5	5	5	5	5	5	5	5
G3, M & 0.03	Mean	83.60	31.76	0.49	40.40	33.20*	6.14*	3.21	56.20	125.60
	±SD	12.10	4.85	0.03	2.79	9.28	0.25	0.14	5.40	15.04
	n	5	5	5	5	5	5	5	5	5
G4, M & 0.1	Mean	81.00	34.62	0.49	56.40	53.60	6.22	3.15*	53.80	124.00
	±SD	6.04	2.29	0.03	9.40	19.07	0.48	0.17	4.60	16.05
	n	5	5	5	5	5	5	5	5	5
G1, F & 0	Mean	93.80	37.62	0.54	53.00	32.80	6.74	3.32	44.40	115.80
	±SD	8.17	6.22	0.04	5.70	7.89	0.27	0.20	7.54	7.60
	n	5	5	5	5	5	5	5	5	5
G2, F & 0.01	Mean	87.80	35.41	0.53	50.60	38.20	6.68	3.29	40.40	125.00
	±SD	10.47	5.36	0.02	13.61	16.22	0.18	0.13	13.87	11.64
	n	5	5	5	5	5	5	5	5	5
G3, F & 0.03	Mean	99.80	35.18	0.54	50.20	37.20	6.60	3.35	37.40	115.20
	±SD	12.66	6.78	0.02	15.69	2.86	0.34	0.17	7.44	12.07
	n	5	5	5	5	5	5	5	5	5
G4, F & 0.1	Mean	101.60	35.42	0.52	60.80	41.60	6.90	3.33	43.60	120.80
	±SD	7.27	6.82	0.03	6.61	10.41	0.34	0.17	6.91	23.95
	n	5	5	5	5	5	5	5	5	5

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

 

 

 

TABLE 8 (Contd…). SUMMARY OF CLINICAL CHEMISTRY RECORD

Refer Appendix 8

Group, Sex & Dose (mg/L)		Alkaline phosphatase	Total Bilirubin	Calcium	Phosphorous	Globulin	Blood Urea Nitrogen	Sodium	Potassium	Chloride
		(ALP)	(BIT)	(CAL)	(PHO)	(GLB)	(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	243.00	0.08	10.40	3.68	3.31	15.45	141.58	3.25	107.32
	±SD	23.94	0.03	0.35	0.32	0.41	3.26	1.15	0.15	1.66
	n	5	5	5	5	5	5	5	5	5
G2, M & 0.01	Mean	301.80	0.07	10.42	3.74	3.27	17.41	143.08	3.53	106.78
	±SD	60.54	0.02	0.37	0.27	0.43	2.71	2.55	0.37	2.03
	n	5	5	5	5	5	5	5	5	5
G3, M & 0.03	Mean	230.60	0.06	10.40	3.35	2.93	14.84	142.98	3.58	107.18
	±SD	46.68	0.01	0.35	0.40	0.30	2.27	2.35	0.26	0.82
	n	5	5	5	5	5	5	5	5	5
G4, M & 0.1	Mean	216.20	0.07	10.24	3.46	3.07	16.18	141.48	3.60	108.68
	±SD	67.44	0.03	0.15	0.29	0.33	1.07	0.51	0.21	0.52
	n	5	5	5	5	5	5	5	5	5
G1, F & 0	Mean	162.00	0.05	10.28	3.26	3.42	17.58	141.08	3.60	107.60
	±SD	46.14	0.01	0.34	0.17	0.33	2.91	1.19	0.22	1.29
	n	5	5	5	5	5	5	5	5	5
G2, F & 0.01	Mean	173.20	0.06	10.18	3.36	3.39	16.54	140.86	3.70	108.10
	±SD	42.29	0.05	0.40	0.31	0.13	2.51	0.69	0.40	1.82
	n	5	5	5	5	5	5	5	5	5
G3, F & 0.03	Mean	173.80	0.03	10.26	3.17	3.25	16.44	141.02	3.56	108.88
	±SD	58.44	0.02	0.23	0.26	0.31	3.17	0.90	0.17	1.15
	n	5	5	5	5	5	5	5	5	5
G4, F & 0.1	Mean	174.20	0.04	10.18	3.49	3.57	16.55	140.94	3.58	107.54
	±SD	51.92	0.03	0.45	0.34	0.34	3.18	0.86	0.24	1.82
	n	5	5	5	5	5	5	5	5	5

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

 

 

 

 

• SUMMARY OF URINALYSIS RECORD

          Refer Appendix 9

Examination	Group, Sex & Dose (mg/L)		G1, M & 0	G2, M & 0.01	G3, M & 0.03	G4, M & 0.1
	Number of Animals		5	5	5	5
Physical Examination	Color	Pale Yellow	4	2	5	3
		Yellow	1	3	-	2
	Appearance	Clear	1	4	3	3
		Turbid	4	1	2	2
	Volume (mL)	Mean	4.7	6.4	6.2	5.5
		±SD	2.3	2.5	2.5	1.8
Chemical Examination	pH	Mean	7.7	7.5	7.4	7.8
		±SD	0.8	0.6	0.2	0.7
	Specific gravity (SG)	Mean	1.012	1.012	1.009	1.011
		±SD	0.008	0.004	0.007	0.007
	Urobilinogen (UBG)               (mg/dL)	Mean	0.2	0.2	0.2	0.2
		±SD	0.0	0.0	0.0	0.0
	Bilirubin (BIL)   (mg/dL)	Neg	5	5	5	5
	Ketones (KET) (mg/dL)	Neg	5	5	5	5
	Blood (BLD) (Ery/µL)	Neg	4	1	-	2
		Ca10	1	1	5	3
		Ca25	-	2	-	-
		>=Ca200	-	1	-	-
	Protein (PRO) (mg/dL)	Neg	5	5	5	5
	Nitrite (NIT)	Neg	2	2	3	-
		Pos	3	3	2	5
	Leucocytes (LEU) (Leu/µL)	Neg	5	5	5	2
		Ca15	-	-	-	2
		Ca70	-	-	-	1
	Glucose (GLU) (mg/dL)	Neg	5	5	5	5
	Micro Albumin (MALB) (mg/dL)	Neg	5	5	5	5
Microscopic Examination	Epi Cells	0	5	4	4	5
		0-1	-	1	1	-
	Casts	Absent	5	5	5	5
	Crystals	Absent	2	1	1	-
		Present	3	4	4	5

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

          -: Not applicable.

 

 

 

 

 

 

 

 

TABLE 9 (Contd…). SUMMARY OF URINALYSIS RECORD

Refer Appendix 9

Examination	Group, Sex & Dose (mg/L)		G1, F & 0	G2, F & 0.01	G3, F & 0.03	G4, F & 0.1
	Number of Animals		5	5	5	5
Physical Examination	Color	Pale Yellow	4	3	3	2
			1	2	2	3
	Appearance	Clear	4	2	2	2
		Turbid	1	3	3	3
	Volume (mL)	Mean	7.4	5.4	4.5	5.0
		±SD	2.5	2.1	1.9	1.9
Chemical Examination	pH	Mean	7.8	7.7	7.4	7.4
		±SD	0.4	0.4	0.4	0.2
	Specific gravity (SG)	Mean	1.007	1.008	1.009	1.012
		±SD	0.003	0.004	0.004	0.004
	Urobilinogen (UBG)               (mg/dL)	Mean	0.2	0.2	0.2	0.4
		±SD	0.0	0.0	0.0	0.4
	Bilirubin (BIL)   (mg/dL)	Neg	5	5	5	5
	Ketones (KET) (mg/dL)	Neg	4	4	5	4
		5	1	1	-	1
	Blood (BLD) (Ery/µL)	Neg	2	1	2	1
		Ca10	1	1	1	1
		Ca25	2	3	2	2
		Ca80	-	-	-	1
	Protein (PRO) (mg/dL)	Neg	3	5	2	2
		Trace	2	-	3	2
		>=300	-	-	-	1
	Nitrite (NIT)	Neg	2	2	2	1
		Pos	3	3	3	4
	Leucocytes (LEU) (Leu/µL)	Neg	-	1	-	-
		Ca15	5	3	-	-
		Ca70	-	1	4	4
		Ca125	-	-	1	1
	Glucose (GLU) (mg/dL)	Neg	5	5	5	5
	Micro Albumin (MALB) (mg/dL)	Neg	3	5	2	2
		>15	2	-	3	3
Microscopic Examination	Epi Cells	0	4	4	5	3
		0-1	1	1	-	2
	Casts	Absent	5	5	5	4
	Crystals	Absent	1	1	1	1
		Present	4	4	4	4

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

       -: Not applicable

      

 

• SUMMARY OF ABSOLUTE ORGAN WEIGHTS (g) RECORD

                                                                                                                                                                                                                                                                        Refer Appendix 10

Group, Sex & Dose (mg/L)		Adrenals	Thymus	Spleen	Testes	Epididymides	Heart	Kidneys	Brain	Liver	Lungs
G1, M & 0	Mean	0.0445	0.3048	1.2010	2.8675	0.6750	0.8806	1.6655	1.7737	7.2165	0.4442
	±SD	0.0060	0.0681	0.3913	0.2768	0.0632	0.1187	0.0979	0.0906	1.0042	0.0384
	n	5	5	5	5	5	5	5	5	5	5
G2, M & 0.01	Mean	0.0524	0.2878	1.1673	2.6393	0.5933	0.8193	1.6265	1.7227	6.5581	0.4633
	±SD	0.0026	0.0764	0.3451	0.3757	0.1527	0.0949	0.1034	0.0964	0.5737	0.0709
	n	5	5	5	5	5	5	5	5	5	5
G3, M & 0.03	Mean	0.0434	0.3658	0.8935	2.7219	0.6730	0.8264	1.6876	1.6954	6.5774	0.5038
	±SD	0.0083	0.0485	0.2797	0.1428	0.1308	0.0479	0.0647	0.0710	0.4937	0.2203
	n	5	5	5	5	5	5	5	5	5	5
G4, M & 0.1	Mean	0.0444	0.2754	0.9828	2.8671	0.5905	0.7924	1.5294	1.7530	6.4106	0.4777
	±SD	0.0087	0.0873	0.5711	0.1836	0.0216	0.0864	0.1144	0.0529	0.7913	0.0643
	n	5	5	5	5	5	5	5	5	5	5

 M: Male; SD: Standard Deviation, n: Number of animals

.

 

 

 

 

 

 

TABLE 10 (Contd…). SUMMARY OF ABSOLUTE ORGAN WEIGHTS (g) RECORD

Refer Appendix 10

Group, Sex & Dose (mg/L)		Adrenals	Thymus	Spleen	Ovaries	Uterus	Heart	Kidneys	Brain	Liver	Lungs
G1, F & 0	Mean	0.0565	0.3224	0.8817	0.1062	0.5510	0.6782	1.2103	1.6188	5.4854	0.4706
	±SD	0.0072	0.0497	0.3610	0.0166	0.1567	0.0425	0.1738	0.0815	0.8689	0.0517
	n	5	5	5	5	5	5	5	5	5	5
G2, F & 0.01	Mean	0.0580	0.3419	0.9006	0.0998	0.3195	0.6639	1.2385	1.6232	5.4225	0.4517
	±SD	0.0094	0.0730	0.3347	0.0196	0.0668	0.0348	0.1216	0.0625	0.2876	0.1088
	n	5	5	5	5	5	5	5	5	5	5
G3, F & 0.03	Mean	0.0635	0.3665	0.7147	0.1009	0.5523	0.6434	1.2780	1.8192	5.4926	0.3923
	±SD	0.0031	0.0348	0.1195	0.0171	0.2535	0.0341	0.0903	0.4821	0.3492	0.0364
	n	5	5	5	5	5	5	5	5	5	5
G4, F & 0.1	Mean	0.0551	0.3200	0.7168	0.1056	0.3859	0.7002	1.3219	1.6878	5.3210	0.5259
	±SD	0.0040	0.0686	0.2850	0.0081	0.1229	0.0180	0.1142	0.1161	0.4879	0.0708
	n	5	5	5	5	5	5	5	5	5	5

 F: Female; SD: Standard Deviation, n: Number of animals

 

 

•  

 

• SUMMARY OF ORGAN WEIGHT RELATIVE TO BODY WEIGHT (%) RECORD

Refer Appendix 11

Group, Sex & Dose (mg/L)		Fasting Body Weight (g)	Adrenals	Thymus	Spleen	Testes	Epididymides	Heart	Kidneys	Brain	Liver	Lungs
G1, M & 0	Mean	197.61	0.0227	0.1534	0.6153	1.4501	0.3431	0.4447	0.8446	0.9018	3.6414	0.2255
	±SD	15.35	0.0041	0.0263	0.2292	0.0574	0.0406	0.0377	0.0447	0.0835	0.2617	0.0220
	n	5	5	5	5	5	5	5	5	5	5	5
G2, M & 0.01	Mean	191.83	0.0273	0.1512	0.6127	1.3843	0.3094	0.4262	0.8487	0.9035	3.4173	0.2439
	±SD	12.90	0.0014	0.0443	0.1877	0.2429	0.0754	0.0272	0.0331	0.1100	0.1596	0.0524
	n	5	5	5	5	5	5	5	5	5	5	5
G3, M & 0.03	Mean	197.12	0.0221	0.1857	0.4564	1.3817	0.3414	0.4193	0.8567	0.8611	3.3372	0.2570
	±SD	7.16	0.0049	0.0253	0.1537	0.0763	0.0647	0.0229	0.0349	0.0489	0.2324	0.1179
	n	5	5	5	5	5	5	5	5	5	5	5
G4, M & 0.1	Mean	177.97	0.0258	0.1542	0.5745	1.6722	0.3439	0.4620	0.8839	1.0169	3.6754	0.2778
	±SD	31.94	0.0076	0.0362	0.3277	0.4301	0.0859	0.1298	0.1844	0.2242	0.6390	0.0755
	n	5	5	5	5	5	5	5	5	5	5	5

 M: Male; SD: Standard Deviation, n: Number of animals

 

 

TABLE 11 (Contd…). SUMMARY OF ORGAN WEIGHT RELATIVE TO BODY WEIGHT (%) RECORD

Refer Appendix 11

Group, Sex & Dose (mg/L)		Fasting Body Weight (g)	Adrenals	Thymus	Spleen	Ovaries	Uterus	Heart	Kidneys	Brain	Liver	Lungs
G1, F & 0	Mean	161.11	0.0351	0.1998	0.5414	0.0661	0.3432	0.4221	0.7489	1.0088	3.3926	0.2926
	±SD	15.45	0.0034	0.0219	0.1877	0.0091	0.1013	0.0166	0.0429	0.0613	0.2715	0.0274
	n	5	5	5	5	5	5	5	5	5	5	5
G2, F & 0.01	Mean	152.64	0.0379	0.2241	0.5938	0.0652	0.2082	0.4359	0.8109	1.0654	3.5523	0.2959
	±SD	7.16	0.0056	0.0469	0.2318	0.0105	0.0347	0.0326	0.0641	0.0657	0.0751	0.0704
	n	5	5	5	5	5	5	5	5	5	5	5
G3, F & 0.03	Mean	152.37	0.0417*	0.2415	0.4689	0.0662	0.3614	0.4225	0.8390	1.1988	3.6064	0.2575
	±SD	6.95	0.0020	0.0313	0.0755	0.0105	0.1619	0.0194	0.0516	0.3396	0.2045	0.0212
	n	5	5	5	5	5	5	5	5	5	5	5
G4, F & 0.1	Mean	151.18	0.0365	0.2118	0.4737	0.0699	0.2537	0.4635*	0.8741*	1.1170	3.5181	0.3487
	±SD	5.58	0.0019	0.0463	0.1836	0.0058	0.0744	0.0155	0.0655	0.0777	0.2763	0.0531
	n	5	5	5	5	5	5	5	5	5	5	5

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

 

 

• SUMMARY OF ORGAN WEIGHT RELATIVE TO BRAIN WEIGHT (%) RECORD

                                                                                                                                                                                                                                                         Refer Appendix 12

Group, Sex & Dose (mg/L)		Adrenals	Thymus	Spleen	Testes	Epididymides	Heart	Kidneys	Liver	Lungs
G1, M & 0	Mean	2.5033	17.1259	67.4063	162.0321	38.2382	49.7679	94.0002	407.7692	25.1065
	±SD	0.2830	3.4494	20.3471	17.3991	5.0531	7.1856	5.5530	60.3251	2.6547
	n	5	5	5	5	5	5	5	5	5
G2, M & 0.01	Mean	3.0494	16.6633	68.3240	153.0890	34.6489	47.8413	94.6904	382.7882	26.9255
	±SD	0.2609	4.2600	22.5065	19.9272	9.8429	7.3154	8.2349	50.5382	4.1169
	n	5	5	5	5	5	5	5	5	5
G3, M & 0.03	Mean	2.5655	21.6784	52.7006	161.0187	39.9951	48.8780	99.7918	389.2587	29.8785
	±SD	0.5277	3.5311	16.9064	14.8943	9.3704	4.4985	7.8113	43.0293	13.6400
	n	5	5	5	5	5	5	5	5	5
G4, M & 0.1	Mean	2.5450	15.7349	56.7751	163.7452	33.7253	45.2854	87.4396	366.3883	27.2410
	±SD	0.5528	5.0037	34.7609	12.7295	1.9116	5.4793	8.8662	49.8428	3.4646
	n	5	5	5	5	5	5	5	5	5

M: Male; SD: Standard Deviation, n: Number of animals

 

 

 

 

 

 

 

TABLE 12 (Contd…). SUMMARY OF ORGAN WEIGHT RELATIVE TO BRAIN WEIGHT (%) RECORD

Refer Appendix 12

Group, Sex & Dose (mg/L)		Adrenals	Thymus	Spleen	Ovaries	Uterus	Heart	Kidneys	Liver	Lungs
G1, F & 0	Mean	3.4853	19.8850	54.1090	6.5472	33.9043	41.9010	74.5850	337.5615	29.1192
	±SD	0.3725	2.6554	20.6012	0.8302	8.8902	1.5974	7.9652	38.0367	3.3758
	n	5	5	5	5	5	5	5	5	5
G2, F & 0.01	Mean	3.5857	21.1022	55.1615	6.1736	19.7611	40.9567	76.4331	334.4461	27.8514
	±SD	0.6850	4.7489	18.7195	1.3398	4.5076	2.8477	8.8241	21.7247	6.7452
	n	5	5	5	5	5	5	5	5	5
G3, F & 0.03	Mean	3.6548	21.0145	40.2693	5.8620	32.4794	36.8407	73.7372	313.2089	22.3524
	±SD	0.7972	4.6605	7.1752	1.7790	17.4461	7.2615	17.1155	56.1181	4.1577
	n	5	5	5	5	5	5	5	5	5
G4, F & 0.1	Mean	3.2700	19.1635	41.8617	6.2556	23.0893	41.6881	78.4275	315.0087	31.4645
	±SD	0.1666	4.9612	14.0226	0.1283	8.0301	3.7770	6.2328	13.6036	6.4251
	n	5	5	5	5	5	5	5	5	5

 F: Female; SD: Standard Deviation, n: Number of animals

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

• SUMMARY OF CHAMBER (EXPOSURE) CONDITIONS
  
  
  
  • Refer Appendix 13 and 14
  
  
  
  

Group & Conc. (mg/L)		Temp.  (°C)	Rh (%)	O2 (%)	CO2  (ppm)	Air inlet flow rate (L/min)*	BZC (mg/L)
G1 & 0	Mean	22.51	55.87	20.41	619.18	20.00	-
	±SD	0.14	1.36	0.16	5.07	0.00	-
G2 & 0.01	Mean	22.55	56.15	20.45	618.82	20.00	0.011
	±SD	0.17	1.47	0.18	6.14	0.00	0.001
G3 & 0.03	Mean	22.52	55.66	20.43	619.51	20.00	0.030
	±SD	0.18	1.47	0.17	5.52	0.00	0.001
G4 & 0.1	Mean	22.52	55.92	20.43	618.81	20.00	0.102
	±SD	0.18	1.45	0.16	5.82	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;


•  


•  


•  


•  


•  

 

• 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 (Air Only)	-	20	360	-
	G2, G3  and G4  (Test Item)	5210	20	360	0.72
		7400			1.03
		11070			1.54
2	G1 (Air Only)	-	20	360	-
	G2, G3  and G4  (Test Item)	5240	20	360	0.73
		7350			1.02
		11140			1.55
3	G1 (Air Only)	-	20	360	-
	G2, G3  and G4  (Test Item)	5110	20	360	0.71
		7430			1.03
		11120			1.54
4	G1 (Air Only)	-	20	360	-
	G2, G3  and G4  (Test Item)	5260	20	360	0.73
		7480			1.04
		11220			1.56
5	G1 (Air Only)	-	20	360	-
	G2, G3  and G4  (Test Item)	5150	20	360	0.72
		7340			1.02
		11070			1.54
6	G1 (Air Only)	-	20	360	-
	G2, G3  and G4  (Test Item)	5190	20	360	0.72
		7390			1.03
		11150			1.55
7	G1 (Air Only)	-	20	360	-
	G2, G3  and G4  (Test Item)	5130	20	360	0.71
		7320			1.02
		11110			1.54

 

• 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)
8	G1 (Air Only)	-	20	360	-
	G2, G3  and G4  (Test Item)	5190	20	360	0.72
		7430			1.03
		11150			1.55
9	G1 (Air Only)	-	20	360	-
	G2, G3  and G4  (Test Item)	5060	20	360	0.70
		7340			1.02
		11020			1.53
10	G1 (Air Only)	-	20	360	-
	G2, G3  and G4  (Test Item)	5190	20	360	0.72
		7300			1.01
		11150			1.55
11	G1 (Air Only)	-	20	360	-
	G2, G3  and G4 (Test Item)	5110	20	360	0.71
		7380			1.03
		11090			1.54
12	G1 (Air Only)	-	20	360	-
	G2, G3  and G4  (Test Item)	5210	20	360	0.72
		7400			1.03
		11060			1.54
13	G1 (Air Only)	-	20	360	-
	G2, G3  and G4  (Test Item)	5140	20	360	0.71
		7240			1.01
		11040			1.53
14	G1 (Air Only)	-	20	360	-
	G2, G3  and G4  (Test Item)	5170	20	360	0.72
		7240			1.01
		11120			1.54

• SUMMARY OF GROSS PATHOLOGY FINDINGS

Route of administration	Inhalation route
Sex	Male
Treatment	Air only	Low Dose	Mid Dose	High Dose
Group	G1	G2	G3	G4
Dose (mg/L)	0	0.01	0.03	0.1
Number of animals	5	5	5	5
No. of dead rats during treatment	-	-	-	-
No. of moribund sacrificed rats	-	-	-	-
No. of terminally sacrificed rats	5	5	5	5
No. of animals with gross pathology	-	-	5	5
Caecum- contents, discoloration, reddish	-	-	5	5

-: No incidence.

 

Route of administration	Inhalation route
Sex	Female
Treatment	Air only	Low Dose	Mid Dose	High Dose
Group	G1	G2	G3	G4
Dose (mg/L)	0	0.01	0.03	0.1
Number of animals	5	5	5	5
No. of dead rats during treatment	-	-	-	-
No. of moribund sacrificed rats	-	-	-	-
No. of terminally sacrificed rats	5	5	5	5
No. of animals with gross pathology	-	-	5	5
Caecum- contents, discoloration, reddish	-	-	5	5

-: No incidence.

•  


•  


•  


•  

• SUMMARY OF HISTOPATHOLOGY FINDINGS

Refer Appendices 3, 4, 5 & 6

Route of administration		Inhalation route
Treatment		Air only		Low Dose		Mid Dose		High Dose
Dose (mg/L)		G1		G2		G3		G4
Group		0		0.01		0.03		0.1
Sex		M	F	M	F	M	F	M	F
Number of Animals		5	5	5	5	5	5	5	5
Lung (Left lobe)	Number examined	5	5	5	5	5	5	5	5
	Within normal limits	-	-	-	1	-	-	-	-
Pigment, Reddish Brown	Minimal	-	-	4	4		-	-	-
	Mild	-	-	1	-		5	-	2
	Moderate	-	-	-	-		-	5	3

 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, PV-Fast Scarlet 4RF was found to be 0.1 mg/L when exposed for 6 hours/day for 14 days by flow-past nose-only inhalation route to Sprague Dawley rats. The Maximum Tolerated Concentration (MTC) is more than the maximum concentration tested in the study i.e., >0.1 mg/L. Hence, for the subsequent repeated dose study, the concentrations of 0.01, 0.03 and 0.1 mg/L can be selected.

Executive summary:

 

The objective of this study was to determine the toxic potential of test item, PV-Fast Scarlet 4RF, when Sprague Dawley rats are exposed for 6 hours/day for 14 days by flow-past nose-only inhalation route. This study provides information on toxic effects, data for quantitative inhalation risk assessment, possible target organs, the estimated No Observed Adverse Effects Concentration (NOAEC), and maximum Tolerated Concentration (MTC), and information on the concentration levels to be selected for the main study.

 

A total of 40 (20 males + 20 females) healthy young Sprague Dawley rats were distributed to four groups viz., control (G1), low dose (G2), mid dose (G3) and high dose (G4). Each group comprised of 10 animals (5 males and 5 females). Animals allocated to Groups G2, G3 and G4 were exposed to test item PV-Fast Scarlet 4RF for 6 hours per day, for 14 days, at a nominal target concentration of 0.01(G2), 0.03(G3) and 0.1(G4) mg/L. Animals of the control group (G1) received air only inhalation for 6 hours/day for 14 days. 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) and further twice weekly during treatment period. Feed consumption was recorded weekly. Ophthalmoscopic examination was performed during acclimatization (pre-treatment) and during week 2 of the treatment period for control and high dose group animals (G1 and G4).

At the end of the exposure period, all animals were fasted overnight (water was provided ad libitum) and the next day the animals were subjected to necropsy. Blood, urine samples, and broncho-alveolar lavage fluid (BALF) were collected for analysis. Haematology, coagulation, clinical chemistry, urinalysis, and BALF fluid analysis were performed. All animals were subjected to necropsy and detailed gross pathology examination. The tissues mentioned in the study plan were collected, weighed, and preserved. Histopathological examination was carried out for left lung from control and high dose group animals (G1 and G4).

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 clinical signs of toxicity or mortality were noted in any of the dose groups.   

There were no treatment-related changes in mean body weight, percent change in body weight with respect to day 1. There was no treatment related variation noted in feed consumption and no ocular changes were observed during the ophthalmoscopy examination.

No adverse treatment related variations in haematology, clinical chemistry and urinalysis parameters were noted.

The test item being a fine powder, there is a chance that it has reached intestine through mouth during nose only exposure owing to continuous exposure and some inevitable movements of animal in the restrainer. This is a routine observation with such test items as per laboratory experience.

There were no treatment-related adverse changes noted in organ weights or their ratios. Grossly, reddish discoloration of the caecal contents was observed in animals of both sexes from group G3 and G4 and this was attributable to the physical nature of the test item.

Microscopically, test item-related changes were observed in lungs of all the test item administered animals and were characterized by, presence of multifocal, minimal to moderate, variably sized, pigmented (reddish brown) granular material throughout parenchyma of lung. This pigmented granular material was distributed both in alveolar and bronchiolar spaces. In the absence of accompanying inflammation or tissue destruction to the lung parenchyma, this change could be considered as non-adverse from dose level of 0.01mg/L.

During BALF analysis, few significant changes were noted: increase in absolute and relative eosinophils (G4) and decrease in LDH (G2, G3 and G4). The noted variations are considered to be secondary due to accumulation of test item particles in the lungs, and in the absence of any inflammation and/or microscopic changes of the lungs, the noted variations are considered not to be adverse.

There were no test item-related, microscopic findings in the study.