N-(5-chloro-2-methoxyphenyl)-2-[(2-methoxy-4-nitrophenyl)azo]-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:

19 September 2022 - 07 November 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 (28-Day (Subacute) Inhalation Toxicity Study

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

N-(5-chloro-2-methoxyphenyl)-2-[(2-methoxy-4-nitrophenyl)azo]-3-oxobutyramide

EC Number:

240-131-2

EC Name:

N-(5-chloro-2-methoxyphenyl)-2-[(2-methoxy-4-nitrophenyl)azo]-3-oxobutyramide

Cas Number:

15993-42-7

Molecular formula:

C18H17ClN4O6

IUPAC Name:

N-(5-chloro-2-methoxyphenyl)-2-[(2-methoxy-4-nitrophenyl)diazenyl]-3-oxobutanamide

Test material form:

solid: nanoform

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Laboratory bred animals
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 8 weeks
- Weight at study initiation: 197-199 g (male) and 159-161 g (female)
- Fasting period before study: no
- Housing: Maximum of three animals per cage were housed in a standard polycarbonate cage (size: L 430 x B 280 x H 210 mm) with stainless steel mesh top grill having facilities for holding pelleted feed and water bottle. Clean sterilized corn cob was provided as bedding material.
- Diet (e.g. ad libitum): Altromin Maintenance diet for rats and mice (manufactured by Altromin Spezialfutter GmbH & Co. KG) was provided ad libitum to the animals throughout the experimental period (except during restraining and exposure period).
- Water (e.g. ad libitum): Water was provided ad libitum throughout the acclimatization and experimental period. Deep bore-well water passed through a reverse osmosis unit was provided in plastic water bottles with stainless steel sipper tubes.
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.6°C to 22.8°C
- Humidity (%): 44% to 66%
- 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: To: 19 September 2022 to 07 November 2022

Administration / exposure

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

air

Mass median aerodynamic diameter (MMAD):

>= 1.83 - <= 1.93 µm

Geometric standard deviation (GSD):

2.67

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: flow-past, nose-only dynamic inhalation exposure system supplied by CH Technologies, USA. The exposure unit consisted of stackable exposure tiers with top and bottom sections or plates for introduction and exhaust of test item. Each tier has 12 exposure ports which were used for exposing up to 10 animals and sampling of test atmosphere. The volume of each inner plenum of inhalation chamber was 0.76 liters (11 cm diameter and 8 cm height) that consisted of total 12 port hole (one tier). Each tier was made of an inner plenum and an outer plenum that were connected to each other through rectangular trumpets (tubes) and connector cones.
- Method of holding animals in test chamber: animals were confined separately in the transparent polycarbonate restraining tubes which were positioned radially around the exposure chamber.
- Source and rate of air: Air compressor. The actual flow rate of chamber inlet was 20 L/min with 60 psi pressure and outlet air from chamber was 15 L/min maintained throughout the exposure period.
- Method of conditioning air: charcoal column, silica gel column and humidifier (if required).
- System of generating particulates/aerosols: Rotating Brush Generator (Palas RBG 1000 - supplied by Palas GmbH)
- Temperature, humidity, pressure in air chamber: 22±3°C, 30% to 70% humidity and 60 psi pressure.
- Air flow rate: 20 L/min
- Air change rate: 12 air changes per hour
- Method of particle size determination: Particle size distribution was determined gravimetrically once in a week by using a 7 stage Cascade Mercer Impactor (supplied by CH Technologies, USA).
- Treatment of exhaust air: Exhaust air was treated with 1% NaOH and passed through absorbent cotton before evacuated into the atmosphere.

TEST ATMOSPHERE
- Brief description of analytical method used: Actual aerosol concentration was determined by gravimetric method i.e. dividing the mass of test item collected on a filter paper by the volume of air passed through the filter paper and time. The 2.40 L/min critical orifice was used to draw the air from the inhalation chamber for one minute at animal breathing zone.
- Samples taken from breathing zone: yes, It was carried out at least four times during exposure, initially before putting the animals into the chambers to ensure the chamber concentration and approx. every two hours after equilibration period for each exposure day.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Actual aerosol concentration was determined by gravimetric method i.e. dividing the mass of test item collected on the filter paper by the volume of air passed through the filter paper and time. The 2.40 L/min critical orifice was used to draw the air from the inhalation chamber for one minute at animal breathing zone. It was carried out at least four times during exposure, initially before putting the animals into the chambers to ensure the chamber concentration and every two hours [i.e. 120 minutes (±15 minutes), 240 minutes (±15 minutes) and 360 minutes (±15 minutes)] after equilibration period for each exposure day.

Actual aerosol concentration of active ingredients during exposure was determined once for each exposure level during the first week and last week of exposure by a validated UV-visible spectrophotometer method (Study No. BIO-ANM 1838). The stability of the test item in dose formulations was established in this preliminary study. The test item formulations were stable at room temperature for 0, 6 and 24 hours at lowest dose (0.1102 mg/mL) and
highest dose (0.3904 mg/mL) in glass fiber filter paper.

The Test item deposited on the filter paper was taken in a separate 100 mL beaker, 10 mL of diluent (DMSO) was added and sonicated for 10 minutes, the solution filtered through 0.45 μm syringe filter and analysed.

Formulations were considered acceptable, as the mean results were within the range of 90 to 110% of the nominal concentration and the relative standard deviation (% RSD) was ≤10%.

Duration of treatment / exposure:

28 days

Frequency of treatment:

6 hours per day, 5 days per week.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/L air

Remarks:

G1, air only
G1R (air only recovery group)

Doses / concentrations 2

Dose / conc.:

0.45 mg/L air

Remarks:

G2, low dose, actual target concentration.

Doses / concentrations 3

Dose / conc.:

0.9 mg/L air

Remarks:

G3, mid dose, actual target concentration.

Doses / concentrations 4

Dose / conc.:

1.35 mg/L air

Remarks:

G4, high dose, actual target concentration.
G4R (high dose recovery group)

No. of animals per sex per dose:

5

Control animals:

yes

Details on study design:

- Dose selection rationale: doses were selected based on results obtained in a dose range finding study in which no toxic effects were found up to the highest dose tested (1.35 mg/L). Moreover, maximum achievable concentration was determined to be 1.35 mg/L.
- Fasting period before blood sampling for clinical biochemistry: yes, overnight (approximately at least 16 to 18 hours).
- Post-exposure recovery period in satellite groups: Following the 28 days exposure period, two recovery groups (0 and 1.35 mg/L) did not receive any treatment and were maintained for 14 days post treatment period and observed for reversibility or persistence of toxic effects.

Positive control:

no

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All the animals were observed for clinical signs and pre-terminal deaths at 1.5 h, 3 h, 4.5 h and 6 h during exposure period. Post-exposure clinical signs were observed at 30 to 40 minutes and 1 hour following exposure to the test chemical, and twice daily for mortality.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: animals will be subjected to detailed clinical examinations before initiation of the treatment (day of randomization) and at weekly intervals thereafter during the study.

BODY WEIGHT: Yes
- Time schedule for examinations: shortly before the first exposure (day 1), twice weekly thereafter for first two weeks. If there were no significant body weight effects in the first 2 weeks, body weights were recorded weekly once and recovery period body weight were recorded weekly once.

FOOD CONSUMPTION: yes
-Individual animal feed consumption was recorded weekly. Feed consumption (g/rat/day) was calculated using the amount of feed given and left over in each cage.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: once during acclimatization (Pre-treatment) and during week 3/4.
- Dose groups that were examined: control and high dose group.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on day 27 (within 24 hours after the last given dose) and day 43 of recovery groups respectively.
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes, fasted overnight (approximately at least 16 to 18 hours) (water provided ad libitum)
- How many animals: all rats of each sex/group
- Parameters checked: Haemoglobin concentration (HGB), Haematocrit (HCT), Erythrocyte count (RBC), Total leukocyte count (WBC), Mean corpuscular volume (MCV), Mean corpuscular hemoglobin (MCH), Mean corpuscular haemoglobin concentration (MCHC), Platelet count (PLT), Mean platelet volume (MPV), Reticulocyte count (Retic), Absolute reticulocyte count, Differential leucocytes count (DLC), Absolute differential leucocytes count (DLC). Prothrombin Time (PT) and Activated Partial Thromboplastin Time (APTT) were estimated by a coagulation analyzer.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on day 27 (within 24 hours after the last given dose) and day 43 of recovery groups respectively.
- Animals fasted: Yes, fasted overnight (approximately at least 16 to 18 hours) (water provided ad libitum)
- How many animals: all rats of each sex/group
- Parameters checked: Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Alkaline phosphatase (ALP), Total Protein, Albumin, Total bilirubin, Glucose, Total Cholesterol, Creatinine, Urea, Blood urea nitrogen (BUN), Triglycerides, Phosphorous, Calcium, Globulin. Sodium (mmol/L), Potassium (mmol/L) and Chloride (mmol/L) were estimated using a Electrolyte analyzer (Medica Corporation/Diamond Diagnostics).

URINALYSIS: Yes
- Time schedule for collection of urine: on day 27
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes (water provided ad libitum)
- How many animals: all rats of each sex/group
- Parameters checked: Blood, Bilirubin, Urobilinogen, Ketones, Protein, Glucose, Microalbumin, Leucocytes. In addition pH, nitrite and specific gravity were also analyzed. After analysis of the above parameters, the urine was subjected for centrifugation at 1500 rpm for 3 minutes. Then the urine was subjected for microscopic examination for urine sediments.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: week 3/4 at main group animals and week 6 for recovery group animals.
- Dose groups that were examined: low dose and high dose levels for main and recovery groups.
- Battery of functions tested: Home cage observations, handling observations, open field observations, sensory observations, neuromuscular observations, physiological observation (Rectal temperature), grip strength and locomotor activity.

BRONCHOALVEOLAR LAVAGE FLUID (BALF): Yes
- Time schedule for analysis: within 24 hours after the last dose given in main group animals. In the recovery group animals, BALF was collected on day 43.
- Dose groups that were examined: all groups.
- Number of animals: 5 females and 5 males for each group.
- Parameters checked: total protein, lactate dehydrogenase (LDH), total cells and differential cells counts.

LUNG BURDEN: No, no appropriate analytical method available.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes. Necropsy included an examination of external surfaces, external orifices, abdominal, thoracic and cranial cavities, organs, and tissues. See organs/tissues examined in table 1 below. Organ weights and organ weight ratios were also included.
HISTOPATHOLOGY: Histopathological examination was carried out on the preserved organs from all control and high dose animals including recovery animals (see table 1 below).
No gross pathological changes were observed in the study. Thus, no further microscopic examination of gross lesions required. Also, no test item related microscopic changes were observed in tissues of high dose animals (G4) and thus lower dose groups (G2 and G3) were not evaluated.

Other examinations:

Bone marrow smear (from one femur) was prepared for all the animals at the time of scheduled necropsy for the case of treatment related changes noticed in hematology parameters and/or microscopic changes in certain tissues/organs such as thymus, spleen and lymph node.

Statistics:

Data obtained 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, haematology, and clinical chemistry estimations, FOB, 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).

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical signs of toxicity were noted during the experiment.

Mortality:

no mortality observed

Description (incidence):

No mortality was noted during the experiment.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

No statistically significant treatment-related variations were observed in all the tested dose groups when compared with control group.
In recovery group, statistically significant increase in female body weight on day 22 & 29 (G4R) was noted. This variation is considered incidental due to lack of dose responsiveness.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

No toxicologically significant changes in food intake were observed. However, statistically significant increase in feed consumption was noted in G3 and G4 females (week 3).

The observed statistically significant changes in food intake were not accompanied by any changes in mean body weight. Therefore, the observed changes in food intake were considered to be incidental.

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, increase in MCH (G2) and decrease in HCT (G3) was noted. In females, decrease in MPV (G3) was noted.
All the variations noted are considered to be incidental in the absence of dose dependency.
In recovery males, decrease in percent eosinophils and absolute eosinophils (G4R) was noted. The noted variations were at the end of treatment period and hence considered to be incidental.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

In males of group G4 statistically significant increase in Chloride (G4) was noted. This change is considered incidental due to lack of dose responsiveness and/or could be due to random biological variation.

The bronchio-alveolar lavage fluid (BALF) collected during necropsy revealed the following statistically significant changes:
In main group males, increase in percent neutrophils (G4), absolute eosinophils (G3) and total protein (G3 and G4), decrease in percent lymphocytes (G4) and total protein (G2) was noted. In females, increase in total protein (G3 and G4), in percent monocyte and LDH (G2) was noted.
In recovery group, in males increase in percent neutrophils and absolute neutrophils (G4R), decrease in percent lymphocytes (G4R) was noted. In females decrease in total protein (G4R) was noted.
The noted variations are considered to be secondary due to the absence of any inflammation and/or microscopic changes of the lungs. Thus, these noted variations are considered not to be an adverse treatment-related effect.

Endocrine findings:

not examined

Urinalysis findings:

no effects observed

Description (incidence and severity):

No adverse treatment-related effects were observed in urinalysis parameters when compared to the control group.

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 females, recovery group increase in body temperature (G4R) was noted. In the absence of changes in all other neuromuscular parameters, the noted changes were considered as incidental.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

In main group males, increase in relative weight (to brain) of testis (G3) was noted.
In recovery group females, increase in relative weight (to brain) of liver (G4R) was noted.
These variations noted in organ weights are considered incidental as they were not associated with any macroscopic finding and also no microscopic changes were noted at the high dose main group animals.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no gross pathological changes in the study.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related, microscopic findings in the study.
Few microscopic findings observed in this study such as epithelial cyst(s) in thymus, luminal dilatation 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 (Elizabeth F. McInnes, 2012).

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Details on results:

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

Effect levels

Key result

Dose descriptor:

NOAEC

Effect level:

1.35 mg/L air

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No effects observed at the highest dose tested.

Target system / organ toxicity

Key result

Critical effects observed:

no

Any other information on results incl. tables

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
	Mean	197.29	210.57	226.09	239.13	258.22	290.43	295.95
G1, M & 0	±SD	9.90	11.14	11.66	13.17	14.78	18.92	20.21
	n	5	5	5	5	5	5	5
	Mean	197.77	207.63	223.46	242.25	255.41	281.58	287.35
G2, M & 0.45	±SD	10.21	12.57	13.07	11.81	15.26	27.65	26.88
	n	5	5	5	5	5	5	5
	Mean	199.25	213.90	231.69	246.24	260.23	292.73	299.64
G3, M & 0.90	±SD	12.40	14.97	14.21	14.57	16.06	16.29	16.69
	n	5	5	5	5	5	5	5
	Mean	199.52	210.33	228.00	243.10	258.09	291.81	299.49
G4, M & 1.35	±SD	11.97	12.93	11.74	13.43	17.97	24.38	25.29
	n	5	5	5	5	5	5	5
	Mean	159.91	165.81	175.51	181.39	185.25	192.65	195.06
G1, F & 0	±SD	10.78	12.26	12.10	12.77	13.40	12.83	12.16
	n	5	5	5	5	5	5	5
	Mean	160.49	163.82	177.89	183.49	187.01	199.26	203.26
G2, F & 0.45	±SD	9.43	9.05	9.64	9.33	10.08	11.45	11.20
	n	5	5	5	5	5	5	5
	Mean	160.81	164.26	178.46	186.93	189.94	201.58	205.51
G3, F & 0.90	±SD	7.43	7.28	7.99	5.97	6.36	7.07	6.16
	n	5	5	5	5	5	5	5
	Mean	159.44	166.41	181.01	185.20	189.86	200.93	203.87
G4, F & 1.35	±SD	12.43	9.28	11.17	10.25	10.82	10.25	10.59
	n	5	5	5	5	5	5	5

Group, Sex & Dose (mg/L)		Body Weight (g) on Days
		1	5	8	12	15	22	29	36	42
	Mean	199.93	212.10	227.48	241.94	260.34	289.46	306.15	326.68	348.56
G1R, M & 0	±SD	13.64	14.48	14.74	13.21	19.35	26.50	26.71	25.73	27.01
	n	5	5	5	5	5	5	5	5	5
G4R, M & 1.35	Mean	202.18	214.68	232.25	247.96	262.26	292.41	305.03	333.12	353.24
	±SD	11.24	10.85	15.33	19.61	22.75	26.59	28.22	30.72	33.97
	n	5	5	5	5	5	5	5	5	5
	Mean	156.67	162.13	173.44	178.42	182.18	192.95	198.94	210.08	217.02
G1R, F & 0	±SD	6.05	7.84	6.58	8.27	8.30	8.86	9.27	10.01	11.19
	n	5	5	5	5	5	5	5	5	5
G4R, F & 1.35	Mean	158.96	165.34	181.06	184.71	187.90	205.28*	211.73*	220.47	228.78
	±SD	5.23	8.67	8.23	7.11	7.63	4.16	4.17	4.09	4.65
	n	5	5	5	5	5	5	5	5	5

*: Statistically significant (p<0.05)

 

Table 3. 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
	Mean	20.42	25.31	23.77	24.31	-	-
G1, M & 0	±SD	1.54	3.87	2.34	1.48	-	-
	n	3	3	3	3	-	-
	Mean	19.56	23.12	23.42	22.60	-	-
G2, M & 0.45	±SD	2.04	0.62	1.93	1.18	-	-
	n	3	3	3	3	-	-
	Mean	20.18	24.39	24.83	24.43	-	-
G3, M & 0.90	±SD	0.52	0.78	0.96	1.10	-	-
	n	3	3	3	3	-	-
	Mean	21.18	24.11	24.85	24.36	-	-
G4, M & 1.35	±SD	0.59	0.15	0.82	0.73	-	-
	n	3	3	3	3	-	-
	Mean	15.35	16.03	15.55	15.17	-	-
G1, F & 0	±SD	1.05	0.90	0.50	0.55	-	-
	n	3	3	3	3	-	-
	Mean	15.55	17.28	17.82*	18.35	-	-
G2, F & 0.45	±SD	0.82	0.74	0.30	1.73	-	-
	n	3	3	3	3	-	-
	Mean	15.87	16.47	13.81	19.15	-	-
G3, F & 0.90	±SD	0.63	1.74	0.75	2.92	-	-
	n	3	3	3	3	-	-
	Mean	16.10	16.92	18.27*	18.68	-	-
G4, F & 1.35	±SD	0.99	0.47	1.28	1.21	-	-
	n	3	3	3	3	-	-
	Mean	20.29	23.31	22.05	23.26	24.30	25.36
G1R, M & 0	±SD	2.13	2.64	1.55	2.51	1.12	1.24
	n	3	3	3	3	3	3
	Mean	20.03	24.59	24.85	22.48	25.85	29.52
G4R, M & 1.35	±SD	0.77	2.35	1.65	0.77	1.46	3.23
	n	3	3	3	3	3	3
	Mean	13.52	15.41	15.17	15.57	17.13	17.54
G1R, F & 0	±SD	1.15	1.87	0.89	0.97	1.84	2.08
	n	3	3	3	3	3	3
	Mean	14.14	16.25	17.26	16.13	18.65	20.15
G4R, F & 1.35	±SD	1.03	0.72	1.18	0.37	1.48	1.14
	n	3	3	3	3	3	3
	Mean	20.29	23.31	22.05	23.26	24.30	25.36
G1R, M & 0	±SD	2.13	2.64	1.55	2.51	1.12	1.24
	n	3	3	3	3	3	3

*: Statistically significant (p<0.05)

 

Table 4. 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)
		(10^3 cells/µL)	(10^6 cells/µL)	(g/dL)	(%)	(fL)	(pg)	(g/dL)	(10^3 cells/µL)	(fL)
	Mean	11.18	8.80	16.94	51.70	58.82	19.26	32.76	913.60	7.44
G1, M & 0	±SD	2.65	0.39	0.41	1.20	1.82	0.75	0.47	215.17	0.49
	n	5	5	5	5	5	5	5	5	5
	Mean	11.26	8.70	16.88	50.20	57.84	19.48	33.62*	806.40	7.16
G2, M & 0.45	±SD	1.67	0.69	0.79	2.46	2.09	0.89	0.48	126.26	0.30
	n	5	5	5	5	5	5	5	5	5
	Mean	10.73	8.34	16.24	49.06*	58.88	19.48	33.08	860.00	6.90
G3, M & 0.90	±SD	2.82	0.18	0.34	0.89	0.93	0.22	0.52	139.53	0.23
	n	5	5	5	5	5	5	5	5	5
	Mean	9.75	8.75	16.42	50.26	57.46	18.78	32.68	725.60	7.32
G4, M & 1.35	±SD	0.91	0.29	0.51	1.02	2.24	1.08	0.55	330.68	1.05
	n	5	5	5	5	5	5	5	5	5
	Mean	9.80	8.76	16.26	50.18	57.38	18.58	32.38	673.60	7.96
G1R, M & 0	±SD	3.31	0.59	0.98	3.38	3.21	0.88	0.37	340.00	1.35
	n	5	5	5	5	5	5	5	5	5
G4R, M & 1.35	Mean	10.47	8.48	16.08	48.30	57.08	19.02	33.32	771.40	7.60
	±SD	1.78	0.56	0.19	1.66	2.11	1.27	1.06	139.11	0.31
	n	5	5	5	5	5	5	5	5	5

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)
	Mean	2.68	28.68	64.76	4.38	0.74	0.36	236.18
G1, M & 0	±SD	0.33	8.66	8.83	0.63	0.47	0.05	31.12
	n	5	5	5	5	5	5	5
	Mean	2.20	25.40	68.84	3.94	0.62	0.34	186.90
G2, M & 0.45	±SD	1.00	5.94	6.11	0.75	0.18	0.09	70.27
	n	5	5	5	5	5	5	5
	Mean	2.59	21.14	73.56	3.40	0.78	0.30	214.20
G3, M & 0.90	±SD	1.00	4.25	4.09	0.24	0.31	0.00	76.57
	n	5	5	5	5	5	5	5
	Mean	3.46	25.84	68.34	3.66	0.70	0.34	298.28
G4, M & 1.35	±SD	1.87	5.01	5.46	0.95	0.21	0.11	149.67
	n	5	5	5	5	5	5	5
	Mean	2.58	23.60	70.68	3.34	1.22	0.34	220.88
G1R, M & 0	±SD	1.21	2.33	2.83	0.56	0.48	0.05	86.17
	n	5	5	5	5	5	5	5
	Mean	3.03	23.02	72.22	2.96	0.54*	0.32	252.54
G4R, M & 1.35	±SD	1.11	6.56	7.19	1.08	0.21	0.15	78.55
	n	5	5	5	5	5	5	5

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)
	Mean	3.16	7.30	0.49	0.08	0.04	16.14	23.52
G1, M & 0	±SD	1.23	2.37	0.12	0.04	0.02	1.72	3.64
	n	5	5	5	5	5	5	5
	Mean	2.86	7.73	0.45	0.07	0.04	16.54	28.52
G2, M & 0.45	±SD	0.73	1.23	0.16	0.03	0.01	1.11	4.43
	n	5	5	5	5	5	5	5
	Mean	2.27	7.90	0.36	0.08	0.03	16.90	27.18
G3, M & 0.90	±SD	0.72	2.20	0.08	0.02	0.01	1.03	7.48
	n	5	5	5	5	5	5	5
	Mean	2.54	6.63	0.36	0.07	0.04	18.26	22.14
G4, M & 1.35	±SD	0.67	0.44	0.12	0.02	0.01	1.78	2.70
	n	5	5	5	5	5	5	5
	Mean	2.32	6.93	0.33	0.12	0.03	15.74	23.96
G1R, M & 0	±SD	0.88	2.32	0.13	0.04	0.02	0.73	2.23
	n	5	5	5	5	5	5	5
	Mean	2.32	7.66	0.30	0.06*	0.03	17.36	22.34
G4R, M & 1.35	±SD	0.38	2.09	0.10	0.02	0.02	2.01	3.98
	n	5	5	5	5	5	5	5

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)
		(10^3 cells/µL)	(10^6 cells/µL)	(g/dL)	(%)	(fL)	(pg)	(g/dL)	(10^3 cells/µL)	(fL)
	Mean	11.08	8.61	16.12	48.02	55.84	18.76	33.56	996.80	6.98
G1, F & 0	±SD	2.57	0.46	0.52	1.45	2.33	0.61	0.40	109.26	0.18
	n	5	5	5	5	5	5	5	5	5
	Mean	11.15	8.22	15.86	46.88	57.12	19.36	33.86	1010.40	6.98
G2, F & 0.45	±SD	3.25	0.66	1.15	3.07	2.45	0.99	1.00	129.41	0.16
	n	5	5	5	5	5	5	5	5	5
	Mean	10.97	8.60	16.26	48.54	56.46	18.92	33.52	946.20	6.64*
G3, F & 0.90	±SD	2.47	0.41	0.45	1.32	1.84	0.67	0.69	112.31	0.26
	n	5	5	5	5	5	5	5	5	5
	Mean	14.73	8.76	16.06	48.66	55.58	18.34	32.98	1009.60	6.76
G4, F & 1.35	±SD	2.18	0.45	0.70	1.97	2.18	0.74	0.27	175.75	0.18
	n	5	5	5	5	5	5	5	5	5
	Mean	10.34	8.74	16.20	48.28	55.22	18.54	33.58	820.20	7.74
G1R, F & 0	±SD	1.91	0.38	0.86	2.40	0.50	0.68	1.06	305.50	0.65
	n	5	5	5	5	5	5	5	5	5
	Mean	10.42	8.60	15.80	47.68	55.62	18.38	33.10	827.60	7.66
G4R, F & 1.35	±SD	2.54	0.60	0.60	1.22	2.85	0.82	0.52	262.09	0.15
	n	5	5	5	5	5	5	5	5	5

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)
	Mean	1.63	21.42	72.06	3.98	1.08	0.36	141.16
G1, F & 0	±SD	0.58	3.86	4.47	0.69	0.56	0.05	51.56
	n	5	5	5	5	5	5	5
	Mean	2.20	21.40	72.74	3.50	0.90	0.34	179.64
G2, F & 0.45	±SD	0.69	4.74	5.11	1.34	0.42	0.09	53.39
	n	5	5	5	5	5	5	5
	Mean	2.46	20.32	74.14	3.28	0.76	0.34	210.12
G3, F & 0.90	±SD	1.19	3.01	2.14	1.08	0.32	0.18	94.89
	n	5	5	5	5	5	5	5
	Mean	2.47	20.06	74.04	3.52	0.76	0.46	215.68
G4, F & 1.35	±SD	0.53	3.59	3.49	0.62	0.78	0.09	46.68
	n	5	5	5	5	5	5	5
	Mean	1.83	33.90	60.10	3.58	0.88	0.54	158.94
G1R, F & 0	±SD	0.65	23.21	24.58	0.70	0.61	0.28	52.74
	n	5	5	5	5	5	5	5
	Mean	2.78	21.78	71.86	3.64	1.30	0.38	231.64
G4R, F & 1.35	±SD	2.28	5.27	7.66	1.60	1.20	0.15	179.53
	n	5	5	5	5	5	5	5

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)
	Mean	2.35	8.01	0.44	0.12	0.04	17.50	25.44
G1, F & 0	±SD	0.55	2.05	0.12	0.05	0.01	3.73	5.22
	n	5	5	5	5	5	5	5
	Mean	2.39	8.12	0.38	0.10	0.04	17.38	29.88
G2, F & 0.45	±SD	0.92	2.47	0.14	0.04	0.02	0.61	6.29
	n	5	5	5	5	5	5	5
	Mean	2.19	8.16	0.37	0.08	0.04	17.74	23.66
G3, F & 0.90	±SD	0.44	1.96	0.15	0.04	0.03	1.22	2.64
	n	5	5	5	5	5	5	5
	Mean	3.02	10.84	0.51	0.12	0.07	21.16	23.72
G4, F & 1.35	±SD	0.93	1.14	0.09	0.13	0.02	4.79	3.85
	n	5	5	5	5	5	5	5
	Mean	3.68	6.03	0.37	0.09	0.06	17.08	24.46
G1R, F & 0	±SD	3.22	2.66	0.12	0.08	0.05	1.01	2.57
	n	5	5	5	5	5	5	5
	Mean	2.20	7.60	0.36	0.12	0.04	16.84	22.88
G4R, F & 1.35	±SD	0.38	2.41	0.11	0.08	0.02	0.71	4.55
	n	5	5	5	5	5	5	5

*: Statistically significant (p<0.05)

 

Table 5. Summary of BALF record

		Total Leucocyte Count	Neutrophils	Lymphocytes	Monocytes	Eosinophils	Basophils	Absolute Neutrophils
Group, Sex & Dose (mg/L)
		(WBC)	(Neut)	(Lymph)	(Mono)	(Eos)	(Baso)	(Neut)
		(10^3 cells/µL)	(%)	(%)	(%)	(%)	(%)	(10^3 cells/µL)
	Mean	0.56	14.30	76.34	1.20	0.62	3.18	0.09
G1, M & 0	±SD	0.41	3.61	4.87	0.58	0.95	1.43	0.08
	n	5	5	5	5	5	5	5
	Mean	0.45	22.52	68.06	2.66	1.04	2.74	0.10
G2, M & 0.45	±SD	0.28	4.79	8.73	2.26	0.43	0.59	0.07
	n	5	5	5	5	5	5	5
	Mean	0.75	27.24	65.56	2.32	1.28	3.44	0.21
G3, M & 0.90	±SD	0.57	3.99	4.22	1.11	0.41	1.54	0.16
	n	5	5	5	5	5	5	5
	Mean	0.45	39.34*	53.22*	1.68	1.66	3.18	0.19
G4, M & 1.35	±SD	0.13	14.41	15.94	1.23	1.28	1.78	0.12
	n	5	5	5	5	5	5	5
	Mean	0.25	14.04	72.42	2.56	0.34	3.28	0.04
G1R, M & 0	±SD	0.09	4.69	9.91	1.86	0.48	1.02	0.02
	n	5	5	5	5	5	5	5
	Mean	0.39	35.28*	56.22*	2.14	1.00	2.78	0.14*
G4R, M & 1.35	±SD	0.18	9.81	11.94	0.67	0.78	1.07	0.06
	n	5	5	5	5	5	5	5

		Absolute Lymphocytes	Absolute Monocytes	Absolute Eosinophils	Absolute Basophils	Lactate Dehydrogenase	Total Protein
Group, Sex & Dose (mg/L)
		(Lymph)	(Mono)	(Eos)	(Baso)	(LDH)	(TPR)
		(10^3 cells/µL)	(10^3 cells/µL)	(10^3 cells/µL)	(10^3 cells/µL)	(u/L)	(g/dL)
	Mean	0.42	0.01	0.00	0.02	51.20	326.47
G1, M & 0	±SD	0.31	0.01	0.00	0.02	28.00	16.32
	n	5	5	5	5	5	5
	Mean	0.30	0.01	0.00	0.01	60.00	300.83*
G2, M & 0.45	±SD	0.17	0.01	0.01	0.00	34.04	6.55
	n	5	5	5	5	5	5
	Mean	0.48	0.02	0.01*	0.03	78.80	423.00*
G3, M & 0.90	±SD	0.36	0.03	0.01	0.02	56.81	16.06
	n	5	5	5	5	5	5
	Mean	0.23	0.01	0.00	0.01	45.80	377.87*
G4, M & 1.35	±SD	0.04	0.01	0.01	0.01	21.32	11.10
	n	5	5	5	5	5	5
	Mean	0.18	0.01	0.00	0.01	50.80	176.07
G1R, M & 0	±SD	0.06	0.01	0.00	0.00	24.81	8.12
	n	5	5	5	5	5	5
	Mean	0.22	0.01	0.00	0.01	74.20	195.74
G4R, M & 1.35	±SD	0.12	0.01	0.01	0.01	28.26	37.67
	n	5	5	5	5	5	5

		Total Leucocyte Count	Neutrophils	Lymphocytes	Monocytes	Eosinophils	Basophils	Absolute Neutrophils
Group, Sex & Dose (mg/L)
		(WBC)	(Neut)	(Lymph)	(Mono)	(Eos)	(Baso)	(Neut)
		(10^3 cells/µL)	(%)	(%)	(%)	(%)	(%)	(10^3 cells/µL)
	Mean	0.44	21.26	58.92	0.92	1.14	4.26	0.10
G1, F & 0	±SD	0.10	7.02	10.32	0.35	0.92	1.23	0.06
	n	5	5	5	5	5	5	5
	Mean	0.44	30.56	57.56	2.32*	1.36	3.48	0.13
G2, F & 0.45	±SD	0.14	8.08	12.54	1.47	0.44	0.98	0.07
	n	5	5	5	5	5	5	5
	Mean	0.44	35.52	57.90	1.60	1.98	3.04	0.17
G3, F & 0.90	±SD	0.22	10.54	13.39	0.45	1.70	2.15	0.11
	n	5	5	5	5	5	5	5
	Mean	0.37	33.82	60.16	1.40	1.18	3.32	0.13
G4, F & 1.35	±SD	0.10	12.33	13.46	0.44	1.05	1.66	0.08
	n	5	5	5	5	5	5	5
	Mean	0.36	25.82	64.72	1.64	1.48	3.72	0.11
G1R, F & 0	±SD	0.22	15.16	14.99	0.80	1.68	1.53	0.14
	n	5	5	5	5	5	5	5
	Mean	0.28	27.38	62.92	3.20	0.58	3.12	0.09
G4R, F & 1.35	±SD	0.15	15.42	18.04	2.11	0.70	2.62	0.07
	n	5	5	5	5	5	5	5

		Absolute Lymphocytes	Absolute Monocytes	Absolute Eosinophils	Absolute Basophils	Lactate Dehydrogenase	Total Protein
Group, Sex & Dose (mg/L)
		(Lymph)	(Mono)	(Eos)	(Baso)	(LDH)	(TPR)
		(10^3 cells/µL)	(10^3 cells/µL)	(10^3 cells/µL)	(10^3 cells/µL)	(u/L)	(g/dL)
	Mean	0.26	0.00	0.00	0.02	34.80	233.92
G1, F & 0	±SD	0.07	0.00	0.01	0.01	13.59	9.52
	n	5	5	5	5	5	5
	Mean	0.24	0.01	0.01	0.01	58.80*	227.95
G2, F & 0.45	±SD	0.08	0.01	0.01	0.01	14.96	9.41
	n	5	5	5	5	5	5
	Mean	0.24	0.01	0.01	0.01	47.60	299.55*
G3, F & 0.90	±SD	0.09	0.01	0.01	0.01	6.19	4.15
	n	5	5	5	5	5	5
	Mean	0.22	0.00	0.00	0.01	41.80	390.17*
G4, F & 1.35	±SD	0.07	0.01	0.01	0.01	8.70	12.96
	n	5	5	5	5	5	5
	Mean	0.21	0.01	0.01	0.01	68.20	717.27
G1R, F & 0	±SD	0.09	0.01	0.01	0.01	34.80	41.96
	n	5	5	5	5	5	5
	Mean	0.16	0.01	0.00	0.01	69.20	248.44*
G4R, F & 1.35	±SD	0.08	0.01	0.00	0.02	45.90	8.05
	n	5	5	5	5	5	5

*: Statistically significant (p<0.05)

 

Table 6. Summary of clinical chemistry record

		Glucose	Urea	Creatinine	Total Cholesterol	Triglycerides	Total Protein	Albumin	Alanine aminotransferase	Aspartate aminotransferase
Group, Sex & Dose (mg/L)
		(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)
	Mean	86.60	23.92	0.54	47.60	53.00	7.38	3.35	58.60	114.20
G1, M & 0	±SD	3.78	3.96	0.01	13.61	13.10	0.29	0.12	7.37	7.43
	n	5	5	5	5	5	5	5	5	5
	Mean	89.60	22.08	0.55	57.60	64.60	7.42	3.38	62.20	114.00
G2, M & 0.45	±SD	14.74	1.63	0.02	8.91	10.88	0.35	0.17	14.13	21.04
	n	5	5	5	5	5	5	5	5	5
	Mean	98.40	22.50	0.53	57.80	46.60	7.22	3.27	60.40	114.40
G3, M & 0.90	±SD	13.33	3.46	0.03	5.54	19.20	0.11	0.11	7.23	9.91
	n	5	5	5	5	5	5	5	5	5
	Mean	103.40	22.66	0.55	51.20	41.60	7.30	3.40	58.60	117.40
G4, M & 1.35	±SD	18.72	1.70	0.02	12.21	4.83	0.32	0.10	3.21	7.33
	n	5	5	5	5	5	5	5	5	5
	Mean	113.20	23.04	0.60	56.20	40.40	7.22	3.17	54.00	102.60
G1R, M & 0	±SD	10.71	3.72	0.03	6.91	13.87	0.24	0.22	7.97	9.76
	n	5	5	5	5	5	5	5	5	5
	Mean	114.80	23.78	0.60	55.20	41.00	7.38	3.25	57.40	110.40
G4R, M & 1.35	±SD	14.41	2.01	0.03	8.11	13.98	0.33	0.13	7.23	6.73
	n	5	5	5	5	5	5	5	5	5

		Alkaline phosphatase	Total Bilirubin	Calcium	Phosphorous	Globulin	Blood Urea Nitrogen	Sodium	Potassium	Chloride
Group, Sex & Dose (mg/L)
		(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)
	Mean	197.60	0.06	10.26	6.45	4.03	11.18	142.30	3.36	106.44
G1, M & 0	±SD	19.45	0.03	0.34	0.53	0.19	1.85	1.11	0.04	1.17
	n	5	5	5	5	5	5	5	5	5
	Mean	199.40	0.08	10.14	6.03	4.04	10.32	141.18	3.56	106.16
G2, M & 0.45	±SD	73.97	0.02	0.29	0.69	0.22	0.77	1.47	0.14	1.78
	n	5	5	5	5	5	5	5	5	5
	Mean	198.40	0.08	10.26	6.35	3.95	10.52	143.24	3.51	107.94
G3, M & 0.90	±SD	26.99	0.02	0.22	0.43	0.10	1.62	2.12	0.23	2.27
	n	5	5	5	5	5	5	5	5	5
	Mean	221.40	0.07	10.20	6.55	3.90	10.59	144.12	3.65	109.88*
G4, M & 1.35	±SD	34.75	0.02	0.17	0.22	0.33	0.80	1.68	0.26	1.64
	n	5	5	5	5	5	5	5	5	5
	Mean	205.00	0.06	9.90	6.06	4.05	10.77	142.58	3.90	109.94
G1R, M & 0	±SD	41.97	0.01	0.29	0.43	0.21	1.74	1.28	0.35	2.20
	n	5	5	5	5	5	5	5	5	5
	Mean	237.00	0.06	9.96	6.01	4.13	11.11	142.02	3.80	107.80
G4R, M & 1.35	±SD	47.31	0.01	0.15	0.11	0.30	0.93	1.49	0.24	1.74
	n	5	5	5	5	5	5	5	5	5

		Glucose	Urea	Creatinine	Total Cholesterol	Triglycerides	Total Protein	Albumin	Alanine aminotransferase	Aspartate aminotransferase
Group, Sex & Dose (mg/L)
		(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)
	Mean	92.40	24.26	0.57	53.20	42.60	7.52	3.36	55.40	124.00
G1, F & 0	±SD	10.78	2.36	0.02	13.12	7.37	0.26	0.11	12.05	7.21
	n	5	5	5	5	5	5	5	5	5
	Mean	91.60	26.88	0.57	64.00	51.40	7.46	3.36	48.40	111.20
G2, F & 0.45	±SD	8.17	3.56	0.02	10.75	12.54	0.21	0.16	5.68	10.69
	n	5	5	5	5	5	5	5	5	5
	Mean	100.80	26.28	0.56	57.80	42.20	7.70	3.30	53.20	127.80
G3, F & 0.90	±SD	16.32	4.42	0.02	12.87	15.37	0.31	0.04	6.69	16.16
	n	5	5	5	5	5	5	5	5	5
	Mean	111.80	29.96	0.59	63.40	48.40	7.70	3.27	71.00	157.00
G4, F & 1.35	±SD	28.19	4.22	0.06	12.84	13.50	0.40	0.12	17.73	40.82
	n	5	5	5	5	5	5	5	5	5
	Mean	99.80	27.52	0.65	63.20	37.00	7.96	3.30	50.00	110.80
G1R, F & 0	±SD	10.52	5.51	0.03	10.35	9.30	0.21	0.15	6.52	24.14
	n	5	5	5	5	5	5	5	5	5
	Mean	110.00	29.84	0.64	61.80	38.40	8.08	3.32	56.60	120.40
G4R, F & 1.35	±SD	14.56	3.46	0.04	16.36	5.32	0.27	0.28	12.05	21.14
	n	5	5	5	5	5	5	5	5	5

		Alkaline phosphatase	Total Bilirubin	Calcium	Phosphorous	Globulin	Blood Urea Nitrogen	Sodium	Potassium	Chloride
Group, Sex & Dose (mg/L)
		(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)
	Mean	124.20	0.04	10.12	6.28	4.16	11.33	142.74	3.74	110.24
G1, F & 0	±SD	24.10	0.02	0.13	0.39	0.29	1.10	0.76	0.22	0.41
	n	5	5	5	5	5	5	5	5	5
	Mean	126.20	0.06	10.04	5.90	4.10	12.65	143.12	3.48	110.24
G2, F & 0.45	±SD	27.28	0.03	0.38	0.49	0.23	1.50	1.27	0.16	1.00
	n	5	5	5	5	5	5	5	5	5
	Mean	182.80	0.06	10.06	5.89	4.40	12.28	141.92	3.65	108.62
G3, F & 0.90	±SD	48.89	0.01	0.29	0.48	0.32	2.07	0.59	0.23	0.63
	n	5	5	5	5	5	5	5	5	5
	Mean	172.80	0.06	9.82	5.58	4.43	14.00	141.96	3.63	110.20
G4, F & 1.35	±SD	102.93	0.03	0.31	0.66	0.36	1.97	1.65	0.22	1.83
	n	5	5	5	5	5	5	5	5	5
	Mean	97.00	0.06	10.00	5.20	4.66	12.86	141.14	4.08	108.58
G1R, F & 0	±SD	44.27	0.02	0.25	0.38	0.23	2.57	1.18	0.34	1.75
	n	5	5	5	5	5	5	5	5	5
	Mean	126.40	0.07	10.32	5.32	4.76	13.94	140.10	3.86	107.52
G4R, F & 1.35	±SD	50.22	0.01	0.36	0.51	0.20	1.61	1.35	0.21	1.57
	n	5	5	5	5	5	5	5	5	5

*: Statistically significant (p<0.05)

 

Table 7. Summary of organ weight relative to brain weight (%) record

Group, Sex & Dose (mg/L)		Adrenals	Thymus	Spleen	Testes	Heart	Kidneys	Liver	Lungs
	Mean	2.6239	18.6359	52.3330	151.6008	55.2265	114.8712	453.5459	33.1393
G1, M & 0	±SD	0.2615	3.8696	6.2439	28.3528	7.1456	15.8632	21.4163	5.6510
	n	5	5	5	5	5	5	5	5
	Mean	3.1040	16.9810	43.8455	163.4445	53.7945	102.4403	412.2849	33.6013
G2, M & 0.45	±SD	0.5223	3.7623	10.6286	9.7424	4.9041	13.3324	66.8444	5.0837
	n	5	5	5	5	5	5	5	5
	Mean	3.2023	19.7048	53.8340	188.9486*	57.3237	111.4965	455.4316	38.4036
G3, M & 0.90	±SD	0.4411	4.9123	11.1870	14.2842	6.8871	5.2631	37.5671	7.5139
	n	5	5	5	5	5	5	5	5
	Mean	3.0887	17.3573	55.4992	165.9436	54.8321	108.0893	427.0642	32.9342
G4, M & 1.35	±SD	0.3887	3.7876	7.7364	5.2984	5.0048	11.5863	43.6424	5.7248
	n	5	5	5	5	5	5	5	5
	Mean	2.8546	21.1449	58.1065	173.3171	62.4969	115.2708	476.4784	33.3403
G1R, M & 0	±SD	0.1615	4.6944	15.3525	15.6432	5.9475	10.5048	42.4854	6.9822
	n	5	5	5	5	5	5	5	5
	Mean	3.0972	18.6033	57.9657	176.1758	62.6480	120.3310	523.0348	35.6010
G4R, M & 1.35	±SD	0.2058	2.1827	5.0866	8.1472	5.9316	10.0432	24.1082	4.0151
	n	5	5	5	5	5	5	5	5

Group, Sex & Dose (mg/L)		Adrenals	Thymus	Spleen	Ovaries	Uterus	Heart	Kidneys	Liver	Lungs
	Mean	3.8389	15.4284	33.2141	6.7355	24.0757	44.7360	80.1322	340.3334	25.8951
G1, F & 0	±SD	0.3948	4.0386	3.7998	1.1010	6.8533	4.1938	4.1657	36.0787	4.5852
	n	5	5	5	5	5	5	5	5	5
	Mean	3.2638	15.1472	40.7743	6.0692	25.5404	45.8893	76.4689	322.0287	29.0793
G2, F & 0.45	±SD	0.3230	3.0798	12.1654	0.4693	7.4461	6.9579	7.0577	25.1409	6.2521
	n	5	5	5	5	5	5	5	5	5
	Mean	3.8636	20.8785	45.5387	8.0718	27.7099	47.9261	83.7124	352.0770	28.6681
G3, F & 0.90	±SD	0.5924	5.6797	9.3563	1.9733	6.8682	4.7736	4.7026	22.0136	4.9923
	n	5	5	5	5	5	5	5	5	5
	Mean	3.9734	13.0026	37.3632	6.1368	34.8117	44.1751	78.4971	341.1353	31.6755
G4, F & 1.35	±SD	0.2949	2.0340	7.9358	0.9178	15.8147	5.1260	9.7032	36.0690	4.0716
	n	5	5	5	5	5	5	5	5	5
	Mean	3.6097	16.5442	37.1143	7.9390	24.6369	45.9990	73.7782	353.7968	30.5367
G1R, F & 0	±SD	0.4674	1.9561	7.9939	0.5656	3.3209	4.4562	6.7636	20.1929	6.3837
	n	5	5	5	5	5	5	5	5	5
	Mean	4.0385	17.6504	47.1822	8.1697	34.1243	49.8156	88.7540	410.5678*	34.6716
G4R, F & 1.35	±SD	0.5259	5.7481	12.2743	0.9867	8.8173	3.5639	18.0629	18.9730	4.5659
	n	5	5	5	5	5	5	5	5	5

*: Statistically significant (p<0.05)

 

Table 8. Summary of chamber exposure conditions

Group & Conc. (mg/L)	Temp. (°C)	Rh (%)	O2 (%)	CO2 (ppm)	Air inlet flow rate (L/min)*	BZC (mg/L)
Mean	22.49	55.66	20.38	618.10	20.00	-
G1/G1R & 0
±SD	0.13	1.39	0.15	4.87	0.00	-
Mean	22.47	55.57	20.38	617.92	20.00	0.46
G2 & 0.45
±SD	0.15	1.47	0.15	4.78	0.00	0.02
Mean	22.44	55.81	20.37	617.69	20.00	0.91
G3 & 0.90
±SD	0.16	1.37	0.16	5.36	0.00	0.02
Mean	22.46	55.47	20.38	618.53	20.00	1.34
G4/G4R & 1.35
±SD	0.16	1.58	0.16	5.04	0.00	0.02

*: 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

 

Table 9. Summary of histopathology findings of main group

Route of administration		Inhalation
Group Description		Air only		High Dose
Actual Target Dose (mg/L)		0		1.35
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	4
Ectopic tissue, adrenocortical	Minimal	-	-	-	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	Number examined	5	5	5	5
	Within normal limits	5	5	5	5
Eye lids	Number examined	5	5	5	5
	Within normal limits	3	3	4	5
Infiltrate, mixed cell	Minimal	1	-	-	-
Infiltrate, mononuclear cell	Minimal	1	2	1	-
Heart	Number examined	5	5	5	5
	Within normal limits	5	5	5	4
Infiltrate, mononuclear cell	Minimal	-	-	-	1
Hilar region 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	2	3	2	4
Cyst	Present	1	1	-	-
Basophilia, tubules	Minimal	1	1	2	-
Dilatation, pelvis	Mild	1	-	-	-
Dilatation, tubules	Minimal	-	-	1	-
Infiltrate, mononuclear cell, interstitium	Minimal	-	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	5	5	5
Lungs	Number examined	5	5	5	5
	Within normal limits	3	4	4	5
Infiltrate, mononuclear cell, alveoli	Minimal	2	-	1	-
Macrophages, increased, alveoli	Minimal	-	1	-	-
Mandibular lymph nodes	Number examined	5	5	5	5
	Within normal limits	5	5	5	5
Mesenteric lymph nodes	Number examined	5	5	5	5
	Within normal limits	5	5	5	5
Nasopharyngeal tissue	Number examined	5	5	5	5
	Within normal limits	5	5	5	5

Route of administration		Inhalation
Group Description		Air only		High Dose
Actual Target Dose (mg/L)		0		1.35
Group		G1		G4
Sex		M	F	M	F
Number of Animals		5	5	5	5
Olfactory bulb	Number examined	5	5	5	5
	Within normal limits	5	5	5	5
Ovaries	Number examined	X	5	X	5
	Within normal limits	X	5	X	5
Parathyroid	Number examined	5	5	5	5
	Within normal limits	5	5	5	5
Seminal vesicles	Number examined	5	X	5	X
	Within normal limits	5	X	5	X
Spinal cord	Number examined	5	5	5	5
	Within normal limits	5	5	5	5
Spleen	Number examined	5	5	5	5
	Within normal limits	5	5	5	5
Stomach	Number examined	5	5	5	5
	Within normal limits	5	5	5	5
Testes	Number examined	5	X	5	X
	Within normal limits	5	X	4	X
Degeneration/atrophy, tubules	Mild	-	X	1	X
Thymus	Number examined	5	5	5	5
	Within normal limits	3	5	4	3
Cyst, epithelial	Present	2	-	1	2
Thyroid	Number examined	5	5	5	5
	Within normal limits	4	4	4	3
Cyst, ultimobranchial	Present	1	1	1	1
Ectopic tissue, thymus	Present	-	-	-	1
Trachea	Number examined	5	5	5	5
	Within normal limits	5	5	5	5
Urinary bladder	Number examined	5	5	5	5
	Within normal limits	5	5	5	5
Uterus	Number examined	X	5	X	5
	Within normal limits	X	4	X	3
Dilatation, lumen	Minimal	X	1	X	1
	Mild	X	-	X	1

 

Table 10. Summary of histopathology findings of recovery group

Route of administration		Inhalation
Group Description		Air only Recovery		High Dose Recovery
Actual Target Dose (mg/L)		0		1.35
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	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	Number examined	5	5	5	5
	Within normal limits	5	5	5	5
Eye lids	Number examined	5	5	5	5
	Within normal limits	3	3	3	3
Infiltrate, mononuclear cell	Minimal	1	2	1	2
Infiltrate, mixed cell	Minimal	1	-	1	-
Heart	Number examined	5	5	5	5
	Within normal limits	5	5	5	5
Hilar region 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	2	2	4	2
Infiltrate, mononuclear cell, interstitium	Minimal	1	1	-	2
Basophilia, tubules	Minimal	2	1	1	1
Cyst	Present	1	1	-	-
Infiltrate, mononuclear cell, urothelium	Minimal	-	1	-	-
Larynx	Number examined	5	5	5	5
	Within normal limits	5	5	5	5
Liver	Number examined	5	5	5	5
	Within normal limits	5	5	5	5
Lungs	Number examined	5	5	5	5
	Within normal limits	4	4	5	5
Infiltrate, mononuclear cell, alveoli	Minimal	1	-	-	-
Macrophages, increased, alveoli	Minimal	-	1	-	-
Mandibular lymph nodes	Number examined	5	5	5	5
	Within normal limits	5	5	5	5
Mesenteric lymph nodes	Number examined	5	5	5	5
	Within normal limits	5	5	5	5
Nasopharyngeal tissue	Number examined	5	5	5	5
	Within normal limits	5	5	5	5

Route of administration		Inhalation
Group Description		Air only Recovery		High Dose Recovery
Actual Target Dose (mg/L)		0		1.35
Group		G1R		G4R
Sex		M	F	M	F
Number of Animals		5	5	5	5
Olfactory bulb	Number examined	5	5	5	5
	Within normal limits	5	5	5	5
Ovaries	Number examined	X	5	X	5
	Within normal limits	X	5	X	5
Parathyroid	Number examined	5	5	5	5
	Within normal limits	5	5	5	5
Seminal vesicles	Number examined	5	X	5	X
	Within normal limits	5	X	5	X
Spinal cord	Number examined	5	5	5	5
	Within normal limits	5	5	5	5
Spleen	Number examined	5	5	5	5
	Within normal limits	5	5	5	5
Stomach	Number examined	5	5	5	5
	Within normal limits	5	5	5	5
Testes	Number examined	5	X	5	X
	Within normal limits	5	X	5	X
Thymus	Number examined	5	5	5	5
	Within normal limits	4	4	4	4
Cyst, epithelial	Present	1	1	1	1
Thyroid	Number examined	5	5	5	5
	Within normal limits	4	3	5	3
Cyst, ultimobranchial	Present	1	2	-	2
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	3
Dilatation, lumen	Mild	X	-	X	2

 

 

 

 

 

Applicant's summary and conclusion

Conclusions:

In a repeated dose toxicity study with the test item, no treatment related effects were observed up to the highest dose tested of 1.35 mg/L after 28 days, 6 hours/day, 5 days/week of aerosolized test item inhalation exposure to rats. Thus, the NOEC is determined to be 1.35 mg/L.

Executive summary:

A 28-day repeated dose toxicity study by inhalation route in rats was conducted for the test substance following the OECD guideline 412, under GLP conditions. A total of 60 (30 males + 30 females) healthy young Sprague Dawley rats were distributed to four main groups and two recovery groups. Each group consisted of 10 animals (5 males and 5 females). Animals allocated to groups G1/G1R (air only), G2 (low dose), G3 (mid dose) and G4/G4R (high dose) were exposed to aerosolized test item for 6 hours per day, 5 days per week for 28 days, at an actual target concentration of 0 (air only, control), 0.45, 0.90 and 1.35 mg/L respectively. Following the 28 days exposure period, the animals in the recovery groups (G1R and G4R) were not given any treatment and maintained for 14 days post treatment period and observed for reversibility or persistence of toxic effects if any. Doses were selected based on results obtained in a dose range finding study in which no toxic effects were found up to the highest dose tested (1.35 mg/L). Moreover, the highest dose tested was selected based on maximum achievable concentration in the exposure system. The inhalation exposure of test item/air was achieved by a flow-past, nose-only dynamic inhalation exposure system.

All animals were observed for clinical signs, mortality and morbidity, body weight, feed consumption and ophthalmological and neurological/functional examinations. At the end of the exposure period, all animals were subjected to necropsy. Blood, urine samples, and broncho-alveolar lavage fluid (BALF) were collected for analysis. Haematology, clinical chemistry and urinalysis were performed. BALF from the right lobe of lung of all rats was analyzed for total protein, lactate dehydrogenase (LDH), total cell counts and differential cell counts. Histopathological examination was carried out on preserved organs from all control and high dose animals including recovery animals.

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 ranges. 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. There were no adverse effects observed during neurological/functional examinations. No adverse treatment related variations in haematology, clinical chemistry, BALF fluid analysis and urinalysis parameters were noted. There were no treatment-related adverse changes noted in organ weights or their ratios. No gross pathological changes were observed in the study. Thus, no further microscopic examination of gross lesions was required. Also, no test item related microscopic changes were observed in tissues of high dose animals (G4) and thus lower dose groups (G2 and G3) were not evaluated. Only few microscopic findings observed in this study such as epithelial cyst(s) in thymus, luminal dilatation 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 (Elizabeth F. McInnes, 2012).

Therefore, based on the above results, the NOAEC for the test item is considered to be 1.35 mg/L.