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

Endpoint summary

Description of key information

Key study: Repeated dose toxicity by oral route, following OECD Guideline 422. The NOAEL of the test substance after a repeated oral exposure of 31 days in males and from 40 to 58 days in females was determined to be 1000 mg/kg bw/day since no adverse effects were observed at the highest dose tested.

 

Key study. Repeated dose toxicity by inhalation, following OECD Guideline 412. 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.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 September 2022 – 08 December 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Hylasco Biotechnology India Pvt. Ltd, Charles River Technology Licensee, CPCSEA (Committee for the Purpose of Control and Supervision of Experiments on Animals). Registration No.: 1808/PO/RcBt/S/15/CPCSEA.
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 8 weeks (age at receipt)
- Weight at study initiation: 383-384 g (males); 265-266 g (females)
- Housing: Animals were housed in a standard polycarbonate cage (size: L 43 x B 28 x H 21 cm with stainless steel mesh top grill having facilities for holding pelleted food and drinking water in water bottle fitted with stainless steel sipper tube. Processed corncob granules were provided as bedding material
Acclimatization: 2 animals of same sex per cage.
Pre mating: 2 animals of the same sex and group per cage.
Mating: 2 animals (one male and one female) of the same group per cage.
Post mating: After confirming presence of sperm in the vaginal smear (Day 0 of pregnancy), the mated pairs were separated. Males were housed with their former cage mates while females were housed individually. Sterilized paper shreds were provided as a nesting material from gestation day 20 onwards.
- 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.
- Water (e.g. ad libitum): Water was provided ad libitum throughout the 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: 5 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.3-23.7ºC
- Humidity (%): 43-66 %
- Air changes (per hr): 12-15
- Photoperiod: 12 hrs dark / 12 hrs light

IN-LIFE DATES: From: To: 15 September 2022 to 08 December 2022

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

at 0.5% w/v

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The required quantity of test item was weighed in a beaker and grind well in a mortar using pestle with a small quantity of vehicle until a homogenous suspension was formed. Thereafter the entire quantity of test formulation was transferred into a measuring cylinder. A small quantity of vehicle was added to rinse the mortar and this was transferred into the measuring cylinder. The rinsing procedure of mortar and pestle was repeated many times to ensure the transfer of contents to the measuring cylinder. Finally, the volume was made up to the required quantity with vehicle to get the desired concentration for the different dose levels.

VEHICLE
- Justification for use and choice of vehicle (if other than water): The test item was insoluble in distilled water and formed homogeneous suspension in 0.5% w/v Carboxymethyl cellulose at the concentration of 100 mg/mL (the highest dose concentration selected for the study considering the dose volume of 10 mL/kg body weight). Hence, 0.5% w/v Carboxymethyl cellulose was used as vehicle for test item formulations as it is a routinely used and universally accepted vehicle of choice for the oral toxicity studies.
- Concentration in vehicle: 10, 30 and 100 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg bw
- Lot/batch no. (if required): SLCJ7163

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dose concentration verification was determined during the first week and last week of treatment by a validated UV-visible spectrophotometer method (Study No. BIO-ANM 1837). 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 48 hours at the concentrations of 5 mg/mL and 100 mg/mL in vehicle and the results were within the acceptance criteria.

Formulations were considered acceptable, as the mean results were within the range of 85 to 115% of the nominal concentration and the relative standard deviation (% RSD) was ≤10%.

Prepared test item formulations were administered to the animals within established stability conditions.

Duration of treatment / exposure:

Main group males: two weeks pre-mating, during mating and up to the day before sacrifice during the post-mating period (total of 31 days of treatment).
Main group females: two weeks pre-mating period, during mating, pregnancy (gestation) and up to lactation day 13 (ranging from a total period of 40 to 58 days).
Recovery group animals (both sexes): until the first scheduled sacrifice of dams (total of 50 days of administration) and kept without treatment for further 15-days of observation period.

Frequency of treatment:

Once a day

Dose / conc.:

0

Remarks:

G1/G1R - Vehicle control / vehicle control recovery

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

G2 - Low dose

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

G3 - Mid dose

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

G4/G4R - High dose / high dose recovery

No. of animals per sex per dose:

12 (main groups)
5 (Recovery groups)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
There was no information available from repeated dose toxicity of the test substance. The estimated LD50 of the test substance is > 2000 mg/kg body weight obtained from a study conducted as per OECD test guidelines 420 (Acute Oral Toxicity) in male and female rats.
As per the ECHA Registration Dossier, the estimated NOAEL of 2-[(4-methyl-2-nitrophenyl)azo]-3-oxo-N-phenylbutyramide, a read across substance was 1000 mg/kg body weight/day obtained from a study conducted as per OECD Guideline 422. There was no indication of systemic, reproductive and developmental toxicity effects in any of the tested doses i.e. 100, 300 and 1000 mg/kg body weight/day in both the sexes.
Based on the above-mentioned information, a dose range finding study (BIO-DTX 080) was conducted with the doses of 100, 300 and 1000 mg/kg bw/day. There was no indication of systemic, reproductive and developmental toxicity effects noted in any of the tested doses and thus the same doses were selected for the present main study.

- Fasting period before blood sampling for clinical biochemistry: yes, overnight.

Positive control:

None

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily for clinical signs of toxicity and twice daily for mortality and morbidity.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: on day 1 before treatment and weekly thereafter during treatment. These observations were made outside the home cage and preferably at the same time. Observations included, but not be limited to, changes in the skin, fur, eyes, mucous membranes, the occurrence of secretions and excretions and autonomic activity such as lacrimation, piloerection, pupil size, and unusual respiratory pattern.

BODY WEIGHT: Yes
- Time schedule for examinations:
Males: at receipt, on the first day of dosing, weekly thereafter and at termination.
Females: at receipt, on the first day of dosing, and weekly once thereafter during pre-mating and until confirmation of mating. Pregnant animals were weighed on gestation days (GD) 0, 7, 14, and 20 and on lactation days 1, 4, 7, 13 and 14 (terminal body weight). The non-pregnant animals were weighed on the day of mating and weekly once until termination.
Recovery animals: at receipt, on the first day of dosing, weekly once thereafter and at termination.

FOOD CONSUMPTION: yes
Feed consumption (cage wise) was measured during following occasions:
Main Group Males: once in a week during premating period coinciding with body weight recording.
Main Group Females:
- once in a week during premating period coinciding with body weight recording.
- during gestation days 0 to 7, 7 to 14 and 14 to 20, during lactation days 1 to 4, 4 to 7 and
7 to 13.
- once in a week for all mated, but not littered females.
- for all non-pregnant animals once in a week coinciding with body weight recording.
Recovery group animals (both sexes): once in a week throughout the experimental period coinciding with body weight recording.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: for all animals before initiation of treatment i.e., after grouping and randomization (day 1). At the end of dosing period for all males shortly prior to scheduled sacrifice, i.e., day 30, during lactation period for littered females i.e., on lactation day 13, one day prior to scheduled sacrifice, i.e., 24th to 26th day from the day of confirmation of mating for mated but not littered/non-pregnant females and during last week i.e., day 64 for the recovery group animals.
- Dose groups that were examined: all groups.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on the day of scheduled terminal sacrifice (for main group males on day 32; for main group females on lactation day 14 and for recovery group animals on day 66).
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (water provided ad libitum)
- How many animals: 5 males and 5 females randomly selected from each main group and from all recovery group animals.
- 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 an "OptiClot-4 coagulation analyzer".

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on the day of scheduled terminal sacrifice (for main group males on day 32; for main group females on lactation day 14 and for recovery group animals on day 66).
- Animals fasted: Yes (water provided ad libitum)
- How many animals: 5 males and 5 females randomly selected from each main group and from all recovery group animals.
- Parameters checked: Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Alkaline phosphatase (ALP), Cholinesterase, 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 Na/K/Cl analyzer (Medica Corporation).

PLASMA/SERUM HORMONES/LIPIDS: Yes,
- Time of blood sample collection: in adult main group males, at termination (after completion of 31 days of treatment); in adult main group females, at termination (on lactation day 14); in pups, at termination (postnatal day 13).
- Animals fasted: Yes (water provided ad libitum)
- How many animals: The assessment of serum T4 levels was performed in all adult males and 2 pups per litter (PND 13 pups). Further assessment of T4 levels in serum samples from the dams and PND 4 pups was not assessed as there were no treatment-related effects observed in the T4 levels of adult males and PND 13 pups.

URINALYSIS: Yes
- Time schedule for collection of urine: on the day of scheduled terminal sacrifice.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes (water provided ad libitum)
- How many animals: 5 randomly selected males of each main group and all recovery animals.
- 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: towards the end of the dosing period for males (shortly prior to scheduled sacrifice, i.e., on day 30) and during the lactation period for females (shortly prior to scheduled sacrifice, i.e., on lactation day 13). For recovery group animals towards the end of the recovery period (shortly prior to scheduled sacrifice, i.e.,
on day 64).
- Dose groups that were examined: 5 males and 5 females, randomly selected from each main group and all recovery group animals.
- Battery of functions tested: Home Cage Observations (convulsions, tremors and palpebral closure), Handling Observations, Open Field Observations, Sensory Observations, Neuromuscular Observations (Hind limb foot splay), Physiological Observation (Rectal temperature), Grip strength assessment and Motor activity assessment.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table 1 and 2)

HISTOPATHOLOGY: Yes (see table 1 and 2)

Other examinations:

Oestrous Cyclicity: Oestrus cyclicity was monitored for two weeks after five days of acclimatization to evaluate the normal oestrus cycle (4 to 5 days). Only females with normal oestrus cyclicity were selected for the treatment. Oestrus cyclicity was monitored daily from the beginning of the treatment period until evidence of mating.

Reproductive Performance Evaluation: mating and fertility index, cohabitation Record and Copulatory Interval (Pre-coital Interval), gestation Length, gestation index, parturation index and pregnancy index.

Delivery and Litter Observations: number of male/female pups born (live/dead/cannibalized) per litter, sex ratio (m/f) and live birth index.

Uteri Observations: number of implantation sites per dam/litter. Post implantation loss per litter was calculated based on the number of implantation sites and number of pups born. The postnatal loss per litter was calculated based on the number of pups born and number of pups survived until termination. The uterus of each female was observed for the presence of resorptions.

Pup Observations:
Individual pup from each litter was observed for external deformities or clinical signs once daily and for mortalities twice daily.
Pup weight: Individual live pup weight from each litter was recorded on
PND 1 (within 24 hours of parturition), 4, 7, and 13. The mean pup weight per litter (sex-wise) was reported.
Anogenital Distance: The anogenital distance of each live pup from each litter was measured on PND 4. The mean pup anogenital distance and its ratio was reported per litter (sex-wise).
Observation of male pups for nipples/areolae retention: The number of nipples/areolae in male pups was counted on PND 13 and presented litter wise.

Statistics:

After verification, the data was subjected to statistical analysis using SPSS software, version 27. All analysis and comparisons were evaluated at the 95% level of confidence (P<0.05). The statistical analysis was followed to the parameters as mentioned in the table 3 below.

Clinical signs:

no effects observed

Description (incidence and severity):

There were no clinical signs of toxicity in any of the animals of both sexes from all the tested dose groups throughout the experimental period. However, in group G4/G4R all animals were noted with yellow-coloured faeces from treatment day 2 and continued until termination. This coloration was due to coloured nature of the test item, but not an adverse test item related effect.
The detailed clinical examination conducted once in a week did not reveal any changes in any of the animals of both sexes from all the tested dose groups and vehicle control group.

Mortality:

no mortality observed

Description (incidence):

There were no mortality/morbidity observed at any dose group during the experimental period.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no changes noted in mean body weight and percent change in mean body weight gain with respect to day 1 in all the tested dose groups (main and recovery) of both sexes throughout the experimental period.

There were no changes noted in mean gestational body weight and percent change in mean gestational body weight gain in all the tested dose groups when compared with the vehicle control group.

There were no changes noted in mean lactation body weight and percent change in mean lactation body weight gain in all the tested dose groups when compared with the vehicle control group.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

There were no changes noted in mean feed consumption in all the tested dose groups (main and recovery) of both sexes when compared with the vehicle control group.

There were no changes noted in mean feed consumption measured during gestation period in all the tested dose groups when compared with the vehicle control group.

There were no test item-related changes in mean lactation feed consumption in all the tested dose groups when compared with the vehicle control group.
The noted statistically significant differences across the dose groups (decrease during lactation day 1 to 4 and 4 to 7 in group G2; decrease during lactation day 4 to 7 and 7 to 13 in group G3; decrease during lactation day 4 to 7 in group G4) when compared with vehicle control group are considered as incidental and unrelated to treatment. These changes are toxicologically irrelevant and not occurred in a dose dependant manner.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

There were no ocular changes observed in any of the animals from all the tested dose or vehicle control groups (main and recovery) of both sexes during ophthalmoscopic examination conducted shortly before scheduled sacrifice.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related changes noted in obtained mean haematological values in all the tested dose groups (main and recovery) of both sexes when compared with the vehicle control group.

The noted statistically significant increase in PT (seconds) in group G3 males and decrease in APTT (seconds) in group G4R females when compared with vehicle control groups are considered as incidental and unrelated to treatment. The obtained mean values are well within lab historical control range of same species and strain and also similar changes were not occurred in other sex at the same dose level.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related changes in mean clinical chemistry values in all the tested dose groups (main and recovery) of both sexes when compared with the vehicle control groups.

However, the following statistically significant differences were noted across the dose groups when compared with vehicle control group.
- increase in calcium level in groups G2 and G3 males.
- increase in phosphorous levels in group G4 males.
- increase in glucose levels in group G3 females.
- decrease in alkaline phosphatase levels in group G4 females.
- decrease in alkaline phosphatase levels in group G4R males.

These noted statistically significant changes are considered as incidental and unrelated to treatment as the obtained mean values are well within lab historical control range of same species and strain and also similar changes were not occurred in other sex at the same dose level.

Endocrine findings:

no effects observed

Description (incidence and severity):

There were no changes noted in mean serum T4 levels in any of the tested dose group males (main groups) when compared with the vehicle control group.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related changes noted in mean urinalysis values in all the tested dose groups (main group males and recovery group males and females) when compared with the vehicle control group.

The noted statistically significant increase in pH from group G4R females when compared with vehicle control group is considered as incidental and unrelated to treatment, as the obtained mean value is well within lab historical control range of same species and strain and also similar change was not occurred in other sex at the same dose level.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

The neurological/functional observations such as, home cage, handling, open-field, sensory, physiological observations did not reveal any test item related changes in any of the animals of both sexes from all the tested dose groups performed towards end of the dosing period for main groups (on day 30 for males and on lactation day 13 for dams) and performed towards end of recovery period for recovery groups (day 64).

There were no changes noted in mean fore/hind limb grip strengths, mean motor activity assessments and mean hind limb foot splay in all tested dose groups of both sexes when compared with vehicle control groups during conduct of neurological/functional examinations.

The noted statistically significant decrease in mean no. of movements from group G4 males when compared with vehicle control group is considered as incidental and unrelated to treatment. This noted observation is an alone occurrence of neurological assessments and also the obtained mean value is within lab historical control range of same species and strain.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related changes noted in mean absolute and relative organ weights in all the tested dose groups of both sexes (main and recovery) when compared with vehicle control group.

However, the following statistically significant differences in absolute and relative organ weights were noted across the dose groups when compared with vehicle control group.
- decrease in relative kidneys weight in group G4 males.
- decrease in relative liver weight in G3 and G4 males.
- increase in absolute brain weight in group G4R males.

These statistically significant changes are considered as incidental and unrelated to treatment, as the obtained mean values are well within lab historical control range of same species and strain and also there were no gross pathological changes noted in any of the animals from all the tested dose groups.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no gross pathological changes observed during conduct of necropsy in any of the parental animals.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related irregularities observed in oestrus cyclicity of females from any of the tested dose groups during pre-mating and mating treatment periods. However, one female from group G2 was noted with single irregular oestrus cycle (6-day cycle). This noted irregularity in duration of oestrus cyclicity in group G2 is considered as incidental and unrelated to treatment as the same female was noted with normal reproductive performance.

There were no test item-related changes in mean length of oestrus cycle per female during treatment period in all the tested dose groups when compared with the vehicle control group. The noted statistically significant increase in mean length of oestrus cycle in group G2 when compared with vehicle control group is considered as incidental and unrelated to treatment, as the obtained mean value is within lab historical control range of same species and strain and also there were no effects noted in any of the reproductive end points at this dose level.

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: No adverse effects were found at any dose tested.

Key result

Critical effects observed:

no

Table 4. Summary of clinical signs of toxicity, detailed clinical examination and mortality record

Group, Sex & Dose (mg/kg body weight/day)	Total No. of Animals	Clinical Signs of Toxicity^a: Observation (No. of Animals revealed)	Detailed Clinical Examination^b: Observation (No. of Animals revealed)	Mortality^c: No. of Mortalities (Total No. of Animals)
G1, M & 0	12	N (12)	NAD (12)	0 (12)
G2, M & 100	12	N (12)	NAD (12)	0 (12)
G3, M & 300	12	N (12)	NAD (12)	0 (12)
G4, M & 1000	12	N (12), Y (12)	NAD (12)	0 (12)

Group, Sex & Dose (mg/kg body weight/day)	Total No. of Animals	Clinical Signs of Toxicity^a: Observation (No. of Animals revealed)	Detailed Clinical Examination^b: Observation (No. of Animals revealed)	Mortality^c: No. of Mortalities (Total No. of Animals)
G1, F & 0	12	N (12)	NAD (12)	0 (12)
G2, F & 100	12	N (12)	NAD (12)	0 (12)
G3, F & 300	12	N (12)	NAD (12)	0 (12)
G4, F & 1000	12	N (12), Y (12)	NAD (12)	0 (12)

Group, Sex & Dose (mg/kg body weight/day)	Total No. of Animals	Clinical Signs of Toxicity^a: Observation (No. of Animals revealed)	Detailed Clinical Examination^b: Observation (No. of Animals revealed)	Mortality^c: No. of Mortalities (Total No. of Animals)
G1R, M & 0	5	N (5)	NAD (5)	0 (5)
G1R, F & 0	5	N (5)	NAD (5)	0 (5)

Group, Sex & Dose (mg/kg body weight/day)	Total No. of Animals	Clinical Signs of Toxicity^a: Observation (No. of Animals revealed)	Detailed Clinical Examination^b: Observation (No. of Animals revealed)	Mortality^c: No. of Mortalities (Total No. of Animals)
G4R, M & 1000	5	N (12), Y (12)	NAD (5)	0 (5)
G4R, F & 1000	5	N (12), Y (12)	NAD (5)	0 (5)

NAD: No Abnormality Detected; Y: Yellow coloured faeces.
a: observed once daily; b: observed once weekly; c: observed twice daily.

Note: All the animals were noted with yellow coloured faeces from treatment day 2 onwards.

 

Table 5. Summary of body weight (g) record

Group, Sex & Dose (mg/kg body weight/day)		Body Weight (g) on Day
		1	7	14	21	28
G1, M & 0	Mean	384.08	417.48	437.74	447.72	455.95
	±SD	23.38	32.16	36.54	37.32	41.65
	n	12	12	12	12	12
G2, M & 100	Mean	383.21	406.66	425.52	437.09	458.53
	±SD	21.85	36.78	45.68	47.77	41.85
	n	12	12	12	12	12
G3, M & 300	Mean	383.84	416.14	431.24	441.30	449.27
	±SD	21.28	26.26	26.71	30.77	31.80
	n	12	12	12	12	12
G4, M & 1000	Mean	383.53	408.50	421.64	434.04	444.44
	±SD	23.23	36.64	44.79	40.81	38.29
	n	12	12	12	12	12

Group, Sex & Dose (mg/kg body weight/day)		Body Weight (g) on Day
		1	7	14	21^#
G1, F & 0	Mean	265.48	273.08	277.18	297.46
	±SD	14.28	18.57	19.71	27.80
	n	12	12	12	3
G2, F & 100	Mean	266.41	274.84	280.12	295.80
	±SD	12.48	13.09	18.00	7.09
	n	12	12	12	2
G3, F & 300	Mean	265.88	273.13	274.86	282.09
	±SD	13.06	16.81	16.88	5.78
	n	12	12	12	3
G4, F & 1000	Mean	265.79	273.40	279.40	270.37
	±SD	13.61	15.46	16.89	18.94
	n	12	12	12	2

#: The data obtained from females in cohabitation only considered for mean calculations.

The data of day 21 body weight was not subjected to statistical analysis due to uneven number of variables.

 

Group, Sex & Dose (mg/kg body weight/day)		Body Weight (g) on Day
		1	7	14	21	28	35	42	49	56	63
G1R, M & 0	Mean	373.26	391.97	408.51	424.12	441.32	463.96	472.34	490.57	495.20	519.27
	±SD	18.27	32.44	37.69	32.32	30.60	27.00	22.20	18.58	25.89	17.65
	n	5	5	5	5	5	5	5	5	5	5
G4R, M & 1000	Mean	376.98	406.55	419.76	432.63	442.09	465.05	486.79	506.83	512.08	535.49
	±SD	14.87	18.04	28.29	32.29	36.04	36.27	36.96	35.73	35.94	45.56
	n	5	5	5	5	5	5	5	5	5	5

Group, Sex & Dose (mg/kg body weight/day)		Body Weight (g) on Day
		1	7	14	21	28	35	42	49	56	63
G1R, F & 0	Mean	262.49	267.22	269.32	275.76	284.04	288.97	298.01	304.78	317.44	322.01
	±SD	10.83	11.06	14.90	13.92	11.49	12.62	17.14	16.95	14.65	16.39
	n	5	5	5	5	5	5	5	5	5	5
G4R, F & 1000	Mean	261.74	270.89	277.16	279.41	279.22	284.29	291.74	296.80	299.69	305.70
	±SD	14.08	13.80	20.43	19.17	19.25	17.92	19.19	17.93	20.18	23.89
	n	5	5	5	5	5	5	5	5	5	5

 

 

Conclusions:

Based on the findings of this study, the NOAEL of the test substance after a repeated oral exposure of 31 days in males and from 40 to 58 days in females was determined to be 1000 mg/kg bw/day.

Executive summary:

A combined repeated dose toxicity study with the reproduction /developmental toxicity screening test of the test substance by oral administration in rats was conducted following the OECD Test Guideline 422. A total of 116 (58 males + 58 females) Sprague Dawley rats were selected for the study and distributed to four main (G1, G2, G3 and G4) and two recovery groups (G1R and G4R). Each main group consisted of 12 males and 12 females, and each recovery group consisted of 5 males and 5 females. The animals in G1/G1R group were administered with vehicle (0.5% w/v Carboxymethyl cellulose), and the animals in G2, G3 and G4/G4R groups were administered with test item at the dose levels of 100, 300 and 1000 mg/kg body weight. The test item was administered to males for a period of 31 days (two weeks pre-mating, during mating and up to the day before sacrifice during post-mating period) and to females for two weeks pre-mating, during mating, pregnancy (gestation) and up to lactation day 13. The vehicle and test item formulations were administered orally by gavage at the dose volume of 10 mL/kg body weight. The stability of test item formulations in vehicle was established before initiation of the treatment by a validated analytical method. Prepared test item formulations were administered to the animals within established stability conditions.

For systemic toxicity assessment, all the animals (main and recovery groups) of both sexes were observed once daily for clinical signs, twice daily for mortality and morbidity and once in a week for detailed clinical examination. Body weight of all animals (main and recovery groups) of both sexes were recorded once in a week until termination. Further, the body weight of all pregnant females was recorded on gestation day (GD) 0, 7, 14 and 20 during pregnancy and on lactation day (LD) 1, 4, 7, 13 and 14 during lactation. Feed consumption for all animals (main and recovery groups) of both sexes were recorded once in a week during pre-mating. Further, feed consumption of all pregnant females was recorded during GD 0 to 7, 7 to 14 and 14 to 20 and during LD 1 to 4, 4 to 7 and 7 to 13. Ophthalmological examination was carried out once before treatment and at the end of the dosing period for all males and females (main and recovery groups). Neurological/functional observations were performed for five randomly selected males and five randomly selected females from each main group towards end of the dosing period, for all animals from recovery groups towards end of the recovery period. The investigations of haematology and clinical chemistry for five randomly selected males and five randomly selected females from main group and all animals from recovery groups was conducted at termination. The investigations of urinalysis for five randomly selected males from each main group and for all recovery groups animals was conducted at termination. Serum thyroxine hormone (T4) levels were estimated for all main group males by ELISA method. The organs were collected and weighed on the day of termination for all animals (main and recovery groups) of both sexes and organ weight relative to terminal body weight was calculated. All the animals (main and recovery groups) of both sexes were observed for both external and internal gross pathological changes during conduct of necropsy. The histopathological examination was conducted on all the tissues collected from the vehicle control and high dose group animals (with special emphasis on stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure - stage aware histopathological evaluation).

For reproductive toxicity assessment, all the males (main groups) were evaluated for reproductive performances such as, mating and fertility indices. All the females (main groups) were evaluated for reproductive performances such as, mating, fertility, gestation and parturition indices. The females were also evaluated for pre-coital interval and gestation length. All females were evaluated for oestrus cyclicity during pre-mating period and the vaginal smear examination was continued until evidence of mating. At birth (delivery) parameters such as, number of live/dead pups born, litter size, sex ratio (m/f) and live birth index per litter were observed / calculated. The litter observations during lactation period such as number of survived or dead pups, sex ratio (m/f) and pup survival index per litter were observed / calculated. The total number of implantation sites was recorded for each litter during necropsy and the post-implantation and postnatal losses per litter was calculated.

For developmental toxicity assessment, all survived pups from each litter were observed once daily for external behavioural changes and twice daily for mortality until termination (PND 13). All pups from each litter were weighed individually on PND 1, 4, 7 and 13 and measured for anogenital distance on PND 4. All male pups were observed for retention of nipples on PND 13. All the pups were subjected for both external and internal gross pathological changes during conduct of necropsy. The collected serum from PND 13 pups was subjected to estimation of thyroxine hormone (T4) levels using ELISA method.

There were no clinical signs of toxicity and no mortality/morbidity noted in any of the animals from all the tested dose groups. However, in group G4/G4R all animals were noted with yellow-coloured faeces from treatment day 2 and this coloration was due to coloured nature of the test item. Detailed clinical examination did not reveal any changes in any of the tested dose group animals. No changes were noted in mean body weight, percent change in mean body weight gain and mean feed consumption of both sexes. The ophthalmological examination and neurological/functional examinations did not reveal any test item-related changes in any of the animals of both sexes. The obtained mean haematology, clinical chemistry and urinalysis values did not reveal any test item-related changes. The estimated serum T4 levels did not reveal any test item related changes. The absolute and relative organ weights of both sexes did not reveal any test item related changes. No gross pathological changes noted in any of the animals during necropsy.

There were no effects noted on reproductive performance of both sexes in all the tested dose groups. No test item-related irregularities observed in oestrus cyclicity of the females from all the tested dose groups during treatment period. No changes were noted in ‘at birth’ (delivery) and or ‘litter observations’ in all the tested dose groups. No effects were noted for live birth index and pup survival index in all tested dose groups. No test item-related post-implantation losses were noted in all the tested dose groups.

There were no test item-related developmental or external behavioural changes and no mortalities noted during postnatal period in any of the pups from all tested dose group litters. The mean pup weight, mean anogenital distance and its ratio in either sex of pups per litter were unaffected by the test item in all tested dose groups. There were no incidences of retention of nipples in male pups examined on PND 13 from all tested dose and control group litters. The estimated serum T4 levels of PND 13 (from all litters) pups did not reveal any changes in all tested dose group litters. There were no gross pathological changes noted in any of the pups during scheduled sacrifice from all the tested dose and control group litters.

Based on the above results, it can be concluded that the No Observed Adverse Effect Level (NOAEL) is 1000 mg/kg bw/day as no adverse effects were found at any dose level tested.

Reference 2

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 April 2022 – 19 June 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

yes

Remarks:

DRF study

GLP compliance:

no

Remarks:

DRF study

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: In-house bred animals.
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 11 weeks
- Weight at study initiation: 305.29-311.93 g (range of average values of each group of males at first day of dosing); 218.07-221.63 g (range of average values of each group of females at first day of dosing)
- Housing: Animals were housed in a standard polypropylene cage (size: L 430 x B 285 x H 150 mm) with a stainless-steel mesh top grill having facilities for holding pelleted food and drinking water in a water bottle fitted with a stainless steel sipper tube. Clean sterilized paddy husk was provided as bedding material.
Acclimatization: 2 animals of same sex per cage.
Pre mating: 2 animals of the same sex and group per cage.
Mating: 2 animals (one male and one female) of the same group per cage.
Post mating: After confirming presence of sperm in the vaginal smear (Day 0 of pregnancy), the mated pairs were separated. Males were housed with their former cage mates while females were housed individually. Sterilized paper shreds were provided as a nesting material from gestation day 20 onwards.
- 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.
- Water (e.g. ad libitum): Water was provided ad libitum throughout the 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: 5 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.4-24.0ºC
- Humidity (%): 46-65 %
- Air changes (per hr): 12-15
- Photoperiod: 12 hrs dark / 12 hrs light

IN-LIFE DATES: From: To: 15 April 2022 to 19 June 2022

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

at 0.5% w/v

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The required quantity of test item was weighed in a beaker or aluminum foil and grind well in a mortar using pestle with a small quantity of vehicle until a homogenous suspension was formed. Thereafter the entire quantity of test formulation was transferred into a measuring cylinder. A small quantity of vehicle was added to rinse the mortar and this was transferred into the measuring cylinder. The rinsing procedure of mortar and pestle was repeated many times to ensure the transfer of contents to the measuring cylinder. Finally, the volume was made up to the required quantity with vehicle to get the desired concentration for the different dose levels.

VEHICLE
- Justification for use and choice of vehicle (if other than water): The test item was insoluble in distilled water and formed homogeneous suspension in 0.5% w/v Carboxymethyl cellulose at the concentration of 100 mg/mL (the highest dose concentration selected for the study considering the dose volume of 10 mL/kg body weight). Hence, 0.5% w/v Carboxymethyl cellulose was used as vehicle for test item formulations as it is a routinely used and universally accepted vehicle of choice for the oral toxicity studies.
- Concentration in vehicle: 10, 30 and 100 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg bw
- Lot/batch no. (if required): SLCH1520

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

The stability of the test item in dose formulations was established in a preliminary study. The test item formulations were stable at room temperature for 48 hours at the concentrations of 5 mg/mL and 100 mg/mL in vehicle and the results were within the acceptance criteria (study nº BIO-ANM 1837).

Prepared test item formulations were administered to the animals within established stability conditions.

Duration of treatment / exposure:

Main group males: two weeks pre-mating, during mating and up to the day before sacrifice during the post-mating period (total of 34 days of treatment).
Main group females main group: two weeks pre-mating period, during mating, pregnancy (gestation) and up to lactation day 13.

Frequency of treatment:

Once a day

Dose / conc.:

0

Remarks:

G1 - Vehicle control

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

G2 - Low dose

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

G3 - Mid dose

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

G4 - High dose

No. of animals per sex per dose:

6

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
There was no information available from repeated dose toxicity of the test substance. The estimated LD50 of the test substance is > 2000 mg/kg body weight obtained from a study conducted as per OECD test guidelines 420 (Acute Oral Toxicity) in male and female rats.
As per the ECHA Registration Dossier, the estimated NOAEL of 2-[(4-methyl-2-nitrophenyl)azo]-3-oxo-N-phenylbutyramide, a read across substance was 1000 mg/kg body weight/day obtained from a study conducted as per OECD Guideline 422. There was no indication of systemic, reproductive and developmental toxicity effects in any of the tested doses i.e. 100, 300 and 1000 mg/kg body weight/day in both the sexes.
- Fasting period before blood sampling for clinical biochemistry: yes, overnight.

Positive control:

None

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily for clinical signs of toxicity and twice daily for mortality and morbidity.

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: The main group animals were weighed at receipt, on the first day of dosing, weekly thereafter and at termination. The main group females were weighed on gestation days 0, 7, 14 and 20 during pregnancy and on days 1, 4, 7 and 13 during lactation period. The non-pregnant animals were weighed weekly once until termination.

FOOD CONSUMPTION: yes
-feed consumption was measured for all animals once a week during premating period coinciding with body weight recording. Feed consumption was measured for all pregnant animals during GD 0 to 7, 7 to 14 and 14 to 20, during LD 1 to 4, 4 to 7 and 7 to 13. Feed consumption was measured for all non-pregnant animals once in a week.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: once before treatment for all animals (before randomization), at the end of the dosing period for all males (shortly prior to scheduled sacrifice, i.e., day 34) and females (shortly prior to scheduled sacrifice, i.e., on 26th day after confirmation of mating for mated but not littered females and on lactation day 13 for littered females).
- Dose groups that were examined: vehicle control and high dose main group.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on the day of scheduled terminal sacrifice (for males on day 35, for littered females on lactation day 14 and for mated but not littered females on 26th day from the day of confirmation of mating).
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (water provided ad libitum)
- How many animals: all animals
- 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 an "OptiClot-4 coagulation analyzer".

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on the day of scheduled terminal sacrifice (for males on day 35, for littered females on lactation day 14 and for mated but not littered females on 26th day from the day of confirmation of mating).
- Animals fasted: Yes (water provided ad libitum)
- How many animals: all animals
- Parameters checked: Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Alkaline phosphatase (ALP), Cholinesterase, 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 Na/K/Cl analyzer (Medica Corporation).

URINALYSIS: Yes
- Time schedule for collection of urine: on the day of scheduled terminal sacrifice.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes (water provided ad libitum)
- How many animals: all animals
- Parameters checked: Blood, Bilirubin, Urobilinogen, Ketones, Protein, Glucose, 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: all males (shortly prior to scheduled sacrifice, i.e., on day 34) and all females (shortly prior to scheduled sacrifice, i.e., on 26th day after confirmation of mating for mated but not littered females and on lactation day 13 for littered females).
- Dose groups that were examined: all main groups.
- Battery of functions tested: Home Cage Observations (convulsions, tremors and palpebral closure), Handling Observations, Open Field Observations, Sensory Observations, Neuromuscular Observations (Hind limb foot splay, Grip strength assessment and Motor activity assessment), Physiological Observation (Rectal temperature).

OTHER:

Oestrous Cyclicity: Oestrus cycles were monitored for two weeks during pre-mating treatment period to evaluate its normal oestrus cyclicity (4 to 5 days). Vaginal smears were also monitored daily from the beginning of treatment period until evidence of mating.

Reproductive Performance Evaluation: mating and fertility index, cohabitation Record and Copulatory Interval (Pre-coital Interval), gestation Length, gestation index, parturation index and pregnancy index.

Delivery and Litter Observations: number of male/female pups born (live/dead/cannibalized) per litter, sex ratio (m/f) and live birth index.

Uteri Observations: number of implantation sites per dam/litter. Post implantation loss per litter was calculated based on the number of implantation sites and number of pups born. The postnatal loss per litter was calculated based on the number of pups born and number of pups survived until termination. The uterus of each female was observed for the presence of resorptions.

Pup Observations and Pup Weight: Individual pup from each litter was observed externally and for mortalities twice daily. Individual live pup weight from each litter was recorded on PND 1 (within 24 hours of parturition), 4, 7 and 13.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table 1)

HISTOPATHOLOGY: No, there were no gross pathological changes noted in any of the animals from all the tested dose groups and no histopathology has been conducted.

Statistics:

After verification, the data was subjected to statistical analysis using SPSS software, version 27. All analysis and comparisons were evaluated at the 95% level of confidence (P<0.05). The statistical analysis was followed to the parameters as mentioned in the table 2 below.

Clinical signs:

no effects observed

Description (incidence and severity):

There were no clinical signs of toxicity recorded in any of the animals of both sexes from all the tested dose groups throughout the experimental period.
In group G4, all the animals of both the sexes were noted with slight yellow-coloured faeces from treatment day 2 onwards, which was due to coloured nature of the test item.

Mortality:

no mortality observed

Description (incidence):

There were no mortality/morbidity observed at any dose group during the experimental period.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

There were no changes noted in mean body weight and percent change in mean body weight gain with respect to day 1 in any of the tested dose groups of both sexes when compared with the vehicle control group throughout the experimental period.

There were no test item related changes noted in mean gestational body weight and percent change in mean gestational body weight gain in any of the tested dose groups when compared with the vehicle control group.
The noted statistically significant reduction in mean percent change in gestation body weight gain during GD 7 to 14 in group G4 is considered as incidental and unrelated to test item administration, as there were no clinical signs of toxicity noted throughout the experimental period and also no changes noted in mean feed consumption during this period.

There were no changes noted in mean lactation body weight and percent change in mean lactation body weight gain in any of the tested dose groups when compared with the vehicle control group.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There were no changes noted in mean feed consumption (including during gestation and lactation periods) in any of the tested dose groups of both sexes when compared with the vehicle control group.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

There were no ocular changes observed in any of the animals of both sexes from high dose and vehicle control groups during ophthalmological examination conducted shortly before scheduled sacrifice.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related changes noted in obtained mean haematological values in any of the tested dose groups of both sexes when compared with the vehicle control group.

However, statistically significant increase in mean reticulate count (% and absolute) in group G2 males, increase in mean basophils levels (%) in group G2 males, increase in mean APTT levels in group G2 males and increase in mean MCH levels in group G2 females were noted when compared with vehicle control group.
These statistically significant changes are considered as incidental and unrelated to treatment, as these changes did not occur in a dose dependant manner and also the obtained mean values are well within lab historical control range of same species and strain.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

There were no changes noted in obtained mean clinical chemistry values in any of the tested dose groups of both sexes when compared with the vehicle control group.

Endocrine findings:

not examined

Urinalysis findings:

no effects observed

Description (incidence and severity):

There were no changes noted in obtained mean urinalysis values in all the tested dose group males when compared with the vehicle control group.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

The neurological/functional observations such as, home cage, handling, open-field, sensory, physiological observations did not reveal any changes in any of the animals of both sexes from high dose and vehicle control groups performed towards end of the dosing period.
There were no changes noted in mean fore/hind limb grip strengths, mean motor activity assessments, and mean hind limb foot splay in all high dose group animals of both sexes when compared with vehicle control groups.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related changes in mean absolute and relative organ weights in any of the tested dose groups of both sexes when compared with vehicle control group.
The noted statistically significant decrease in mean kidney and prostate weights (absolute and relative) in group G4 males, increase in mean relative weight of uterus with cervix in group G3 females when compared with vehicle control group are considered as incidental and unrelated to treatment, as there were no gross pathological changes noted in any of these organs during necropsy. Also, all the obtained mean values of each organ are well within lab historical control range of same species and strain.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no gross pathological changes observed during conduct of necropsy in any of the parental animals.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related irregularities observed in oestrus cyclicity of females in any of the tested dose groups during pre-mating and mating treatment periods. The mean length of oestrus cycle per female during treatment period was unaffected by the test item administration from all tested dose groups and comparable with the vehicle control group.
However, one female from group G4 was noted with a single irregular oestrus cycle during mating period. These observed irregular cycle was considered as incidental and not due to test item exposure, as this female revealed normal cycles during pre-mating period and was also confirmed as pregnant.

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: No adverse effects were found at any dose tested.

Key result

Critical effects observed:

no

Table 3. Summary of clinical signs of toxicity and mortality record

Group, Sex & Dose (mg/kg body weight/day)	Total No. of Animals	Clinical Signs of Toxicity^a: Observation (No. of Animals revealed)	Mortality^c: No. of Mortalities (Total No. of Animals)
G1, M & 0	6	N (6)	0 (6)
G2, M & 100	6	N (6)	0 (6)
G3, M & 300	6	N (6)	0 (6)
G4, M & 1000	6	N^Y (6)	0 (6)

 

Group, Sex & Dose (mg/kg body weight/day)	Total No. of Animals	Clinical Signs of Toxicity^a: Observation (No. of Animals revealed)	Mortality^c: No. of Mortalities (Total No. of Animals)
G1, F & 0	6	N (6)	0 (6)
G2, F & 100	6	N (6)	0 (6)
G3, F & 300	6	N (6)	0 (6)
G4, F & 1000	6	N^Y (6)	0 (6)

a: observed once daily; c: observed twice daily.

^Y: There were no clinical signs of toxicity, however all animals from group G4 were noted with slight yellow coloured faeces which was due to coloured nature of the test item.

 

Table 4. Summary of body weight (g) record

 

Group, Sex & Dose (mg/kg body weight/day)		Body Weight (g) on Day
		1	7	14	21	28	34
G1, M & 0	Mean	305.29	322.67	337.80	355.29	379.50	394.85
	±SD	24.00	33.23	39.05	39.78	40.08	45.31
	n	6	6	6	6	6	6
G2, M & 100	Mean	311.93	324.33	338.86	364.55	390.48	408.41
	±SD	18.97	18.07	12.59	24.40	23.39	24.70
	n	6	6	6	6	6	6
G3, M & 300	Mean	310.62	325.88	342.80	358.68	372.79	383.83
	±SD	22.20	24.66	27.00	29.96	29.66	29.96
	n	6	6	6	6	6	6
G4, M & 1000	Mean	311.04	325.70	337.11	361.96	382.31	394.83
	±SD	13.11	17.68	24.99	32.05	30.95	33.02
	n	6	6	6	6	6	6

 

Group, Sex & Dose (mg/kg body weight/day)		Body Weight (g) on Day
		1	7	14	21
G1, F & 0	Mean	221.63	225.17	233.60	247.03
	±SD	8.96	11.70	11.34	9.85
	n	6	6	6	3
G2, F & 100	Mean	218.07	220.57	223.34	239.21
	±SD	5.79	7.87	11.61	9.57
	n	6	6	6	4
G3, F & 300	Mean	218.57	222.18	226.89	231.61
	±SD	6.08	8.96	8.58	13.61
	n	6	6	6	4
G4, F & 1000	Mean	218.33	219.37	227.35	235.80
	±SD	5.40	5.04	8.32	12.24
	n	6	6	6	3

The day 21 body weights for females were not subjected to statistical analysis due to uneven number of variables.

 

Table 5. Summary of gestation body weight (g)

 

 

Group, Sex & Dose (mg/kg body weight/day)		Body Weight (g) on Gestation Day (GD)
		0	7	14	20
G1, F & 0	Mean	239.44	258.58	287.49	345.56
	±SD	14.14	13.40	14.19	21.73
	n	5	5	5	5
G2, F & 100	Mean	234.23	251.57	271.03	330.07
	±SD	15.62	12.00	10.15	7.45
	n	6	6	6	6
G3, F & 300	Mean	236.02	250.77	276.96	338.53
	±SD	6.31	6.17	3.56	9.76
	n	5	5	5	5
G4, F & 1000	Mean	241.03	257.45	281.76	341.31
	±SD	11.47	10.36	16.86	23.77
	n	5	5	5	5

 

Table 6. Summary of percent change in body weight gain (%) with respect to day 1 record

Group, Sex & Dose (mg/kg body weight/day)		Percent Change in Body Weight (%) during Day
		1 to 7	1 to 14	1 to 21	1 to 28	1 to 34
G1, M & 0	Mean	5.54	10.46	16.23	24.30	29.29
	±SD	3.68	6.69	7.11	8.41	9.90
	n	6	6	6	6	6
G2, M & 100	Mean	4.03	8.78	16.86	25.21	30.94
	±SD	2.25	3.18	2.93	2.24	1.60
	n	6	6	6	6	6
G3, M & 300	Mean	4.89	10.31	15.40	19.97	23.55
	±SD	0.68	1.45	2.33	2.02	2.93
	n	6	6	6	6	6
G4, M & 1000	Mean	4.71	8.32	16.27	22.83	26.83
	±SD	3.25	5.16	7.37	6.93	7.17
	n	6	6	6	6	6

 

 

Group, Sex & Dose (mg/kg body weight/day)		Percent Change in Body Weight (%) during Day
		1 to 7	1 to 14	1 to 21
G1, F & 0	Mean	1.59	5.41	8.29
	±SD	2.76	2.93	1.30
	n	6	6	3
G2, F & 100	Mean	1.14	2.38	8.77
	±SD	1.64	3.54	4.39
	n	6	6	4
G3, F & 300	Mean	1.63	3.80	5.41
	±SD	2.19	2.51	4.68
	n	6	6	4
G4, F & 1000	Mean	0.48	4.12	8.70
	±SD	0.96	2.18	3.28
	n	6	6	3

Note: The data of day 1 to 21 body weight gain in females was not subjected to statistical analysis due to uneven number of variables.

Table 7. Summary of percent change in body weight gain (%) during gestation period

 

Group, Sex & Dose (mg/kg body weight/day)		Percent Change in Body Weight Gain (%) during Gestation Day (GD)
		0 to 7	7 to 14	14 to 20
G1, F & 0	Mean	8.05	11.25	20.18
	±SD	2.20	4.03	3.92
	n	5	5	5
G2, F & 100	Mean	7.53	7.83	21.84
	±SD	2.48	3.62	2.15
	n	6	6	6
G3, F & 300	Mean	6.28	10.49	22.21
	±SD	2.34	2.76	2.26
	n	5	5	5
G4, F & 1000	Mean	6.85	9.43*	21.15
	±SD	1.71	4.58	4.47
	n	5	5	5

*: Statistically significant (P<0.05) change than the vehicle control group.

 

Table 8. Summary of haematology record

 

Group, Sex & Dose (mg/kg body weight/day)		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	10.59	8.82	15.20	49.90	57.18	17.32	30.77	1066.33	8.85
	±SD	2.20	0.74	0.82	5.21	10.36	0.73	3.58	162.89	2.63
	n	6	6	6	6	6	6	6	6	6
G2, M & 100	Mean	12.17	8.28	14.97	48.12	58.62	18.15	31.10	828.83	8.97
	±SD	2.12	0.90	1.05	2.35	7.01	1.27	1.61	232.92	1.30
	n	6	6	6	6	6	6	6	6	6
G3, M & 300	Mean	9.05	9.05	15.98	48.85	53.95	17.68	32.75	1131.00	7.65
	±SD	2.83	0.18	0.60	1.45	1.12	0.59	0.57	203.38	0.32
	n	6	6	6	6	6	6	6	6	6
G4, M & 1000	Mean	9.33	9.13	15.70	49.37	54.07	17.20	31.85	1208.83	7.48
	±SD	2.95	0.48	0.64	2.40	1.12	0.37	0.36	54.25	0.17
	n	6	6	6	6	6	6	6	6	6

 

 

Group, Sex & Dose (mg/kg body weight/day)		Reticulocyte Count	Neutrophils	Lymphocytes	Monocytes	Eosinophils	Basophils
		(Retic)	(Neut)	(Lymph)	(Mono)	(Eos)	(Baso)
		(%)	(%)	(%)	(%)	(%)	(%)
G1, M & 0	Mean	2.26	24.93	68.95	3.50	1.37	0.23
	±SD	0.42	5.11	6.08	1.66	0.53	0.05
	n	6	6	6	6	6	6
G2, M & 100	Mean	7.08*	27.93	66.07	2.53	1.80	0.50*
	±SD	6.16	14.33	15.43	0.77	1.67	0.30
	n	6	6	6	6	6	6
G3, M & 300	Mean	1.70	26.05	69.03	2.82	0.93	0.22
	±SD	0.38	1.79	2.98	1.73	0.29	0.10
	n	6	6	6	6	6	6
G4, M & 1000	Mean	1.93	23.78	71.50	2.45	0.98	0.18
	±SD	0.46	3.68	2.88	0.41	0.19	0.08
	n	6	6	6	6	6	6

 

Group, Sex & Dose (mg/kg body weight/day)		Absolute Reticulocyte Count	Absolute Neutrophils	Absolute Lymphocytes	Absolute Monocytes	Absolute Eosinophils	Absolute Basophils	Prothrombin Time	Activated Prothrombin Time
		(Retic)	(Neut)	(Lymph)	(Mono)	(Eos)	(Baso)	(PT)	(APTT)
		(10^9 cells/L)	(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	200.60	2.61	7.33	0.37	0.15	0.03	17.97	18.47
	±SD	49.94	0.57	1.81	0.19	0.07	0.01	2.33	1.99
	n	6	6	6	6	6	6	6	6
G2, M & 100	Mean	542.58*	3.62	7.80	0.32	0.23	0.07	18.62	22.57*
	±SD	410.06	2.51	1.30	0.14	0.22	0.05	1.10	4.76
	n	6	6	6	6	6	6	6	6
G3, M & 300	Mean	154.20	2.36	6.24	0.26	0.09	0.02	19.30	20.40
	±SD	35.57	0.73	1.97	0.18	0.05	0.01	2.39	1.18
	n	6	6	6	6	6	6	6	6
G4, M & 1000	Mean	176.17	2.17	6.70	0.24	0.09	0.02	19.13	19.78
	±SD	41.95	0.56	2.30	0.11	0.04	0.02	1.10	1.09
	n	6	6	6	6	6	6	6	6

 

Group, Sex & Dose (mg/kg body weight/day)		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, F & 0	Mean	10.03	8.34	15.02	45.75	54.92	18.00	32.82	1157.83	7.53
	±SD	1.53	0.43	0.91	2.77	1.72	0.66	0.50	251.04	0.34
	n	6	6	6	6	6	6	6	6	6
G2, F & 100	Mean	12.17	7.66	14.85	45.85	60.27	19.47*	32.40	1132.33	7.80
	±SD	4.48	0.75	0.83	2.03	4.80	1.01	1.05	286.05	0.52
	n	6	6	6	6	6	6	6	6	6
G3, F & 300	Mean	8.02	8.21	15.17	45.85	56.20	18.50	33.02	1053.67	7.52
	±SD	2.72	0.76	0.97	1.81	4.61	0.85	1.18	201.85	0.44
	n	6	6	6	6	6	6	6	6	6
G4, F & 1000	Mean	11.51	8.40	15.40	46.67	55.67	18.35	32.98	1018.83	7.85
	±SD	4.08	0.53	0.73	2.48	2.77	1.01	0.32	277.50	0.93
	n	6	6	6	6	6	6	6	6	6

 

Group, Sex & Dose (mg/kg body weight/day)		Reticulocyte Count	Neutrophils	Lymphocytes	Monocytes	Eosinophils	Basophils
		(Retic)	(Neut)	(Lymph)	(Mono)	(Eos)	(Baso)
		(%)	(%)	(%)	(%)	(%)	(%)
G1, F & 0	Mean	2.41	31.45	63.18	2.95	1.45	0.23
	±SD	0.83	13.98	13.56	1.49	0.57	0.12
	n	6	6	6	6	6	6
G2, F & 100	Mean	4.81	33.20	61.42	3.12	1.13	0.23
	±SD	2.65	11.98	13.08	1.15	0.53	0.05
	n	6	6	6	6	6	6
G3, F & 300	Mean	2.92	26.33	67.98	2.25	2.35	0.27
	±SD	1.88	5.09	5.96	0.75	1.90	0.05
	n	6	6	6	6	6	6
G4, F & 1000	Mean	2.46	36.82	56.53	3.42	1.82	0.28
	±SD	0.68	7.75	9.41	1.98	0.95	0.08
	n	6	6	6	6	6	6

Group, Sex & Dose (mg/kg body weight/day)		Absolute Reticulocyte Count	Absolute Neutrophils	Absolute Lymphocytes	Absolute Monocytes	Absolute Eosinophils	Absolute Basophils	Prothrombin Time	Activated Prothrombin Time
		(Retic)	(Neut)	(Lymph)	(Mono)	(Eos)	(Baso)	(PT)	(APTT)
		(10^9 cells/L)	(10^3 cells/µL)	(10^3 cells/µL)	(10^3 cells/µL)	(10^3 cells/µL)	(10^3 cells/µL)	(Seconds)	(Seconds)
G1, F & 0	Mean	200.67	3.14	6.36	0.29	0.14	0.02	21.02	22.88
	±SD	69.88	1.55	1.87	0.14	0.05	0.01	2.20	4.35
	n	6	6	6	6	6	6	6	6
G2, F & 100	Mean	352.80	4.32	7.17	0.41	0.13	0.03	21.02	24.70
	±SD	154.17	3.00	2.18	0.29	0.06	0.01	3.75	4.89
	n	6	6	6	6	6	6	6	6
G3, F & 300	Mean	229.84	2.01	5.57	0.18	0.17	0.02	22.40	20.53
	±SD	119.66	0.40	2.31	0.07	0.11	0.01	2.02	2.33
	n	6	6	6	6	6	6	6	6
G4, F & 1000	Mean	206.37	4.43	6.26	0.31	0.20	0.03	22.32	22.23
	±SD	59.68	2.30	1.52	0.32	0.12	0.01	2.65	5.03
	n	6	6	6	6	6	6	6	6

*: Statistically significant (P<0.05) change than the vehicle control group.

Table 9. Summary of absolute organ weight (g) record

 

Group, Sex & Dose (mg/kg body weight/day)		Adrenals	Thymus	Spleen	Testes	Epididymides	Heart	Kidneys	Brain	Liver	Prostate	Seminal vesicles with coagulating glands
G1, M & 0	Mean	0.0535	0.4637	0.7456	3.4055	1.2239	1.3886	2.6745	1.9866	10.6389	1.2574	1.0129
	±SD	0.0077	0.1038	0.1659	0.2223	0.2484	0.1463	0.3503	0.1461	1.1788	0.1356	0.1424
	n	6	6	6	6	6	6	6	6	6	6	6
G2, M & 100	Mean	0.0531	0.3927	0.7238	3.3585	1.3526	1.4559	2.5636	2.0764	10.7855	1.3391	1.1541
	±SD	0.0069	0.1184	0.0651	0.1193	0.0588	0.1365	0.2839	0.0489	0.7569	0.0576	0.1047
	n	6	6	6	6	6	6	6	6	6	6	6
G3, M & 300	Mean	0.0476	0.3893	0.7210	3.3569	1.3377	1.3463	2.3584	1.9626	10.0424	1.1661	1.1036
	±SD	0.0044	0.0804	0.0619	0.2105	0.1007	0.0981	0.1124	0.0539	0.6729	0.0376	0.3899
	n	6	6	6	6	6	6	6	6	6	6	6
G4, M & 1000	Mean	0.0491	0.4140	0.7580	3.2237	1.2865	1.3239	2.2664*	2.0279	9.8080	1.1034*	1.1144
	±SD	0.0058	0.0557	0.1585	0.1919	0.0458	0.0826	0.1434	0.0991	0.6840	0.0480	0.2753
	n	6	6	6	6	6	6	6	6	6	6	6

 

Group, Sex & Dose (mg/kg body weight/day)		Adrenals	Thymus	Spleen	Ovaries	Uterus with Cervix	Heart	Kidneys	Brain	Liver
G1, F & 0	Mean	0.0723	0.3078	0.4983	0.1209	0.5553	0.9058	1.6783	1.8829	9.2964
	±SD	0.0195	0.0763	0.0786	0.0097	0.1280	0.3560	0.1976	0.0655	2.3159
	n	6	6	6	6	6	6	6	6	6
G2, F & 100	Mean	0.0707	0.2668	0.5616	0.1205	0.5045	0.9944	1.8263	1.7269	9.7643
	±SD	0.0172	0.0943	0.1022	0.0322	0.0671	0.0582	0.2799	0.0892	2.4392
	n	6	6	6	6	6	6	6	6	6
G3, F & 300	Mean	0.0684	0.2234	0.5423	0.1200	0.6906	0.9696	1.5334	1.8392	8.3310
	±SD	0.0112	0.0662	0.2112	0.0180	0.1472	0.0793	0.1901	0.1403	1.3353
	n	6	6	6	6	6	6	6	6	6
G4, F & 1000	Mean	0.0550	0.2274	0.6275	0.1047	0.5207	0.9550	1.5749	1.7492	9.2306
	±SD	0.0060	0.0597	0.2222	0.0215	0.1207	0.0822	0.1144	0.1237	1.6955
	n	6	6	6	6	6	6	6	6	6

*: Statistically significant (P<0.05) change than the vehicle control group.

 

Table 10. Summary of terminal body weight (g) and organ weight relative to terminal body weight (g) record

Group, Sex & Dose (mg/kg body weight/day)		Terminal Body Weight (g)	Adrenals	Thymus	Spleen	Testes	Epididymides	Heart	Kidneys	Brain	Liver	Prostate	Seminal vesicles with coagulating glands
G1, M & 0	Mean	366.40	0.0149	0.1263	0.2060	0.9381	0.3385	0.3809	0.7355	0.5458	2.9234	0.3470	0.2819
	±SD	40.80	0.0033	0.0238	0.0542	0.1071	0.0816	0.0401	0.1172	0.0524	0.3843	0.0536	0.0639
	n	6	6	6	6	6	6	6	6	6	6	6	6
G2, M & 100	Mean	395.93	0.0134	0.0999	0.1829	0.8516	0.3429	0.3700	0.6485	0.5267	2.7385	0.3398	0.2928
	±SD	24.82	0.0016	0.0309	0.0126	0.0696	0.0282	0.0517	0.0690	0.0437	0.3262	0.0318	0.0353
	n	6	6	6	6	6	6	6	6	6	6	6	6
G3, M & 300	Mean	371.10	0.0129	0.1048	0.1955	0.9099	0.3619	0.3644	0.6376	0.5325	2.7103	0.3161	0.2946
	±SD	31.10	0.0015	0.0180	0.0243	0.0944	0.0311	0.0335	0.0361	0.0547	0.0903	0.0293	0.0910
	n	6	6	6	6	6	6	6	6	6	6	6	6
G4, M & 1000	Mean	380.92	0.0130	0.1093	0.1994	0.8543	0.3402	0.3484	0.5976*	0.5356	2.5831	0.2909*	0.2948
	±SD	33.41	0.0021	0.0162	0.0379	0.1161	0.0370	0.0163	0.0476	0.0518	0.1725	0.0187	0.0778
	n	6	6	6	6	6	6	6	6	6	6	6	6

 

Group, Sex & Dose (mg/kg body weight/day)		Terminal Body Weight (g)	Adrenals	Thymus	Spleen	Ovaries	Uterus with Cervix	Heart	Kidneys	Brain	Liver
G1, F & 0	Mean	287.37	0.0252	0.1072	0.1732	0.0422	0.1928	0.3208	0.5832	0.6572	3.2303
	±SD	19.51	0.0070	0.0255	0.0225	0.0036	0.0418	0.1292	0.0419	0.0405	0.7746
	n	6	6	6	6	6	6	6	6	6	6
G2, F & 100	Mean	270.83	0.0261	0.0977	0.2078	0.0448	0.1865	0.3680	0.6768	0.6397	3.6156
	±SD	13.46	0.0060	0.0315	0.0386	0.0136	0.0238	0.0297	0.1168	0.0579	0.9315
	n	6	6	6	6	6	6	6	6	6	6
G3, F & 300	Mean	264.88	0.0258	0.0840	0.2039	0.0451	0.2625*	0.3659	0.5780	0.6949	3.1403
	±SD	11.88	0.0036	0.0228	0.0762	0.0047	0.0639	0.0216	0.0587	0.0535	0.4558
	n	6	6	6	6	6	6	6	6	6	6
G4, F & 1000	Mean	273.77	0.0201	0.0831	0.2297	0.0384	0.1904	0.3491	0.5755	0.6402	3.3744
	±SD	7.15	0.0020	0.0219	0.0833	0.0089	0.0454	0.0334	0.0432	0.0603	0.6334
	n	6	6	6	6	6	6	6	6	6	6

*: Statistically significant (P<0.05) change than the vehicle control group.

 

Conclusions:

Based on the findings of this study, the NOAEL of the test substance after a repeated oral exposure of 34 days in males and from 48 to 60 days in females was determined to be 1000 mg/kg bw/day.

Executive summary:

A combined repeated dose toxicity study with the reproduction /developmental toxicity screening test of the test substance by oral administration in rats was conducted following the OECD Test Guideline 422. A total of 48 (24 males + 24 females) Sprague Dawley rats were distributed to four groups. Each group (G1, G2, G3 and G4) consisted of 6 males and 6. The animals in G1 group were administered with vehicle (0.5% w/v Carboxymethyl cellulose), and the animals in G2, G3 and G4 groups were administered with test item at the dose levels of 100, 300 and 1000 mg/kg body weight. The test item was administered to males for a period of 34 days (two weeks pre-mating, during mating and up to the day before sacrifice during post-mating period) and to females for two weeks pre-mating, during mating, pregnancy (gestation) and up to lactation day 13. The vehicle and test item formulations were administered orally by gavage at the dose volume of 10 mL/kg body weight. The stability of test item formulations in vehicle was established before initiation of the treatment by a validated analytical method. Prepared test item formulations were administered to the animals within established stability conditions.

All animals were observed for clinical signs, mortality and morbidity, body weight, feed consumption, ophthalmological examinations, neurological/functional observations and clinical pathological (haematology, clinical chemistry and urinalysis) examinations. The gross pathology and organ weighing were performed on the day of termination for all animals. All females were observed for maternal body weight and feed consumption during gestation and lactation. Females were evaluated for oestrus cyclicity. Each dam was observed for mating performance, fertility performance, gestation length, litter size, number and percentage of live/dead pups, live birth index, sex ratio and observation of litter throughout the lactation period. The pups were observed once daily for clinical signs and external examinations, weighed individually on postnatal day (PND) 1, 4, 7 and 13 and observed for gross pathological observations at termination.

There were no test item related clinical signs of toxicity noted in any of the animals from all tested dose groups, except slight, yellow-coloured faeces from group G4 which was due to coloured nature of the test item. No mortality/morbidity noted in any of the animals from all tested dose groups. No test item related changes noted in mean body weight, percent change in mean body weight gain and mean feed consumption in all tested dose groups of both sexes. The ophthalmological examination and neurological/functional examinations did not reveal any changes in any of the animals from high dose group of both sexes. The obtained mean haematology, clinical chemistry and urinalysis values did not reveal any test item-related changes in all tested dose groups. The absolute and relative organ weights of both sexes did not reveal any changes from all tested dose groups.

No gross pathological changes were noted in any of the animals from all tested dose groups during necropsy.

There were no effects noted on reproductive performance of both sexes in all tested dose groups. No test item-related irregularities observed in oestrus cyclicity of the females from all the tested dose groups. No changes were noted in ‘at birth’ (delivery) and or ‘litter observations’ in all tested dose groups. No effects were noted for live birth index and pup survival index in all tested dose groups. No test item-related post-implantation losses were noted in all tested dose groups.

There were no developmental/external behavioural changes noted and no test item-related mortalities noted during postnatal period in any of pups from all tested dose group litters. The mean pup weight in either sex of pups per litter were unaffected by the test item in all tested dose groups. There were no gross pathological changes noted in any of the pups during scheduled sacrifice from all tested dose and control group litters.

It can be concluded that the No Observed Adverse Effect Level (NOAEL) is 1000 mg/kg bw/day as no adverse effects were found at any dose level tested.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The study has a Klimisch score = 1

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Reference 1

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

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

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

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.

Dose / conc.:

0 mg/L air

Remarks:

G1, air only
G1R (air only recovery group)

Dose / conc.:

0.45 mg/L air

Remarks:

G2, low dose, actual target concentration.

Dose / conc.:

0.9 mg/L air

Remarks:

G3, mid dose, actual target concentration.

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

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).

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.

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.

Key result

Critical effects observed:

no

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

 

 

 

 

 

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.

Reference 2

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 April 2022 - 08 June 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

yes

Remarks:

DRF study

GLP compliance:

no

Remarks:

DRF study.

Limit test:

no

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: 179-183 g (male) and 158-161 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: 13 April 2022 to 05 May 2022

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.

Duration of treatment / exposure:

14 days

Frequency of treatment:

6 hours per day, 7 days per week.

Dose / conc.:

0 mg/L air

Remarks:

G1, air only

Dose / conc.:

0.45 mg/L air

Remarks:

G2, low dose, actual target concentration.

Dose / conc.:

0.9 mg/L air

Remarks:

G3, mid dose, actual target concentration.

Dose / conc.:

1.35 mg/L air

Remarks:

G4, high dose, actual target concentration.

No. of animals per sex per dose:

5

Control animals:

yes

Details on study design:

- Dose selection rationale: doses were selected based on maximum achievable concentration (1.35 mg/L).
- Fasting period before blood sampling for clinical biochemistry: yes, overnight (approximately at least 16 to 18 hours).

Positive control:

no

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, thereafter once a day for clinical signs and twice daily for mortality.

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: shortly before the first exposure (day 1), twice weekly thereafter and at the time of euthanasia.

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 and maximum of three animals per cage were housed.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: once during acclimatization (Pre-treatment) and during week 2.
- Dose groups that were examined: control and high dose group.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on day 15 (within 24 hours after the last given dose)
- 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 15 (within 24 hours after the last given dose)
- 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 15
- 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.

BRONCHOALVEOLAR LAVAGE FLUID (BALF): Yes
- Time schedule for analysis: within 24 hours after the last dose given group animals.
- 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.

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 only for left lung from all control (G1), high dose group (G4) and also mid dose group (G3) due to coloration found in gross examination.
For other organs no histopathological examination was carried out as no gross pathological changes were observed.

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, 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).

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:

no effects observed

Description (incidence and severity):

No statistically significant treatment-related variations were observed in all the tested dose groups when compared with control group.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No statistically significant treatment-related variations were observed in all the tested dose groups when compared with control group.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

No ocular abnormalities were noted.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Some statistically significant variations were noted: In males, decrease in absolute lymphocytes (G2, G3 and G4) and basophils (G3 and G4). In females, decrease in platelets (G2) and increase in APTT (G2).
Variations noted in males are secondary due to exposure of test item and hence considered not to be an adverse effect. Variations noted in Platelets and APTT in G2 females are considered incidental due to lack of dose dependency.
Thus, no toxicologically significant treatment related changes in haematology parameters were noted.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

The following statistically significant variations were noted: In males, decrease in glucose (G4), creatinine (G2, G3 and G4), globulin (G2 and G3). In females increase in Chloride (G3), triglycerides and Phosphorous (G2).
Decrease in creatinine in males is considered incidental as similar change was not noted in females. All other variations noted are considered incidental due to lack of dose responsiveness and/or could be due to random biological variation.
Thus, no toxicologically significant changes were observed in clinical chemistry parameters when compared to the vehicle control group.

The bronchio-alveolar lavage fluid measured at termination revealed the following statistically significant changes: In males, increase in absolute eosinophils (G4) and decrease in Total protein (G2, G3 and G4). In females decrease in percent basophils (G3) and increase in total protein (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.

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):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

No statistically significant treatment-related variations were observed in all the tested dose groups when compared with control group.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were gross changes observed in lungs from G3 and G4 groups. This gross change was characterized by yellowish discoloration of lungs and was attributed to physical appearance of test item. However, there were no correlating microscopic findings in lungs of both G3 and G4 groups of animals and were found to be within normal histological limits.

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 presence of minimal, multi focal, mononuclear cells infiltration and minimal or multifocal/diffuse increased macrophages in parenchyma of lung, were considered incidental as they occurred randomly across the dose groups including concurrent control groups.

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.

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.

Key result

Critical effects observed:

no

Table 2. Summary of body weights (g) record

Group, Sex & Dose (mg/L)		Body Weight (g) on Days
		1	5	8	12	14
G1, M & 0	Mean	179.59	193.26	211.04	226.11	230.07
	±SD	8.74	8.01	9.09	10.35	10.93
	n	5	5	5	5	5
G2, M & 0.45	Mean	179.81	192.94	210.20	223.73	227.60
	±SD	8.86	9.99	9.65	7.84	8.01
	n	5	5	5	5	5
G3, M & 0.90	Mean	179.77	193.75	210.30	224.57	227.92
	±SD	8.61	9.04	8.83	8.31	8.74
	n	5	5	5	5	5
G4, M & 1.35	Mean	183.42	196.48	215.31	229.74	233.81
	±SD	8.64	7.76	8.34	7.26	7.10
	n	5	5	5	5	5
G1, F & 0	Mean	158.55	166.49	176.69	184.69	186.90
	±SD	7.89	7.72	6.64	6.63	6.91
	n	5	5	5	5	5
G2, F & 0.45	Mean	158.36	165.71	175.64	181.73	183.61
	±SD	6.94	7.57	6.76	7.29	6.86
	n	5	5	5	5	5
G3, F & 0.90	Mean	161.00	168.40	178.46	184.81	186.45
	±SD	10.41	10.08	10.12	9.10	9.04
	n	5	5	5	5	5
G4, F & 1.35	Mean	161.36	168.63	178.86	185.62	187.07
	±SD	11.83	12.08	13.24	12.96	12.71
	n	5	5	5	5	5

Table 3. Summary of haematology record

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	9.92	7.52	15.38	48.14	64.74	20.58	31.92	1106.00	7.46
	±SD	0.46	0.95	1.33	1.94	7.98	1.62	2.11	274.52	0.54
	n	5	5	5	5	5	5	5	5	5
G2, M & 0.45	Mean	7.15	8.64	16.58	49.98	57.82	19.16	33.12	911.80	7.30
	±SD	0.76	0.44	1.22	3.19	1.59	0.97	0.86	76.75	0.48
	n	5	5	5	5	5	5	5	5	5
G3, M & 0.90	Mean	8.95	6.75	13.96	45.78	70.48	20.74	29.88	900.20	8.14
	±SD	7.73	2.17	4.48	11.61	11.30	1.90	4.05	181.38	1.30
	n	5	5	5	5	5	5	5	5	5
G4, M & 1.35	Mean	6.19	7.31	15.70	48.66	69.44	22.12	32.34	1044.40	7.72
	±SD	1.28	1.60	1.20	1.65	16.92	3.70	2.66	174.99	0.54
	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)
G1, M & 0	Mean	8.49	21.26	71.94	3.98	0.34	0.34	587.60
	±SD	9.25	7.91	8.26	1.31	0.21	0.05	579.58
	n	5	5	5	5	5	5	5
G2, M & 0.45	Mean	3.55	27.62	65.54	4.30	0.76	0.38	306.94
	±SD	1.36	2.67	2.90	1.02	0.18	0.24	123.09
	n	5	5	5	5	5	5	5
G3, M & 0.90	Mean	4.68	36.44	57.18	3.88	0.62	0.26	279.64
	±SD	2.22	19.38	20.43	1.44	0.38	0.15	77.04
	n	5	5	5	5	5	5	5
G4, M & 1.35	Mean	8.80	29.66	63.84	4.08	1.04	0.32	554.60
	±SD	7.75	5.16	5.55	0.68	0.75	0.11	405.18
	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)
G1, M & 0	Mean	2.11	7.15	0.40	0.03	0.04	21.14	21.00
	±SD	0.75	0.92	0.14	0.02	0.01	1.55	1.44
	n	5	5	5	5	5	5	5
G2, M & 0.45	Mean	1.99	4.67*	0.31	0.05	0.03	19.12	21.90
	±SD	0.37	0.37	0.09	0.01	0.01	1.29	4.34
	n	5	5	5	5	5	5	5
G3, M & 0.90	Mean	4.42	3.91*	0.37	0.04	0.02*	20.84	23.24
	±SD	6.38	0.72	0.37	0.02	0.01	2.10	6.15
	n	5	5	5	5	5	5	5
G4, M & 1.35	Mean	1.88	3.90*	0.25	0.07	0.02*	19.28	22.98
	±SD	0.66	0.54	0.05	0.06	0.01	1.19	3.38
	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)
G1, F & 0	Mean	11.69	8.09	14.98	43.56	53.84	18.52	34.36	1056.20	7.08
	±SD	3.02	0.31	0.47	1.09	1.19	0.75	0.69	175.43	0.04
	n	5	5	5	5	5	5	5	5	5
G2, F & 0.45	Mean	12.31	8.14	15.18	44.10	54.18	18.66	34.42	724.60*	7.52
	±SD	5.13	0.45	0.88	2.48	1.62	0.67	0.19	98.43	0.48
	n	5	5	5	5	5	5	5	5	5
G3, F & 0.90	Mean	11.19	7.95	15.10	44.02	55.42	19.00	34.30	980.60	7.20
	±SD	3.25	0.38	0.41	2.38	2.36	0.97	1.21	122.01	0.31
	n	5	5	5	5	5	5	5	5	5
G4, F & 1.35	Mean	14.45	8.23	15.04	44.18	53.70	18.22	33.96	842.00	7.60
	±SD	4.40	0.77	1.61	3.81	1.12	0.57	0.82	188.03	0.32
	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)
G1, F & 0	Mean	2.88	27.04	66.64	3.16	1.50	0.38	233.28
	±SD	0.68	10.11	10.45	1.06	0.65	0.15	59.12
	n	5	5	5	5	5	5	5
G2, F & 0.45	Mean	2.75	21.14	71.56	4.18	1.14	0.42	222.04
	±SD	0.81	6.74	7.04	0.86	0.43	0.08	56.68
	n	5	5	5	5	5	5	5
G3, F & 0.90	Mean	3.45	17.60	74.80	3.02	2.48	0.42	272.28
	±SD	0.95	4.52	3.83	0.49	2.58	0.19	63.51
	n	5	5	5	5	5	5	5
G4, F & 1.35	Mean	3.31	20.66	73.06	3.32	0.96	0.42	273.86
	±SD	1.59	9.38	9.96	1.32	0.70	0.15	130.76
	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)
G1, F & 0	Mean	3.01	7.97	0.35	0.18	0.04	20.34	19.56
	±SD	1.08	3.17	0.05	0.09	0.01	1.64	3.41
	n	5	5	5	5	5	5	5
G2, F & 0.45	Mean	2.34	9.07	0.51	0.13	0.05	20.88	30.50*
	±SD	0.14	4.79	0.23	0.02	0.02	1.37	9.91
	n	5	5	5	5	5	5	5
G3, F & 0.90	Mean	2.01	8.37	0.34	0.23	0.05	20.60	25.04
	±SD	0.96	2.52	0.10	0.22	0.02	2.99	5.38
	n	5	5	5	5	5	5	5
G4, F & 1.35	Mean	3.27	10.27	0.48	0.12	0.06	22.40	20.02
	±SD	2.60	1.95	0.21	0.07	0.02	0.65	1.60
	n	5	5	5	5	5	5	5

*: Statistically significant (p<0.05)

 

Table 4. Summary of BALF record

Group, Sex & Dose (mg/L)		Total Leucocyte Count	Neutrophils	Lymphocytes	Monocytes	Eosinophils	Basophils	Absolute Neutrophils
		(WBC)	(Neut)	(Lymph)	(Mono)	(Eos)	(Baso)	(Neut)
		(10^3 cells/µL)	(%)	(%)	(%)	(%)	(%)	(10^3 cells/µL)
G1, M & 0	Mean	0.88	31.20	56.26	5.40	2.40	4.76	0.37
	±SD	1.04	12.94	19.06	9.32	2.30	2.64	0.57
	n	5	5	5	5	5	5	5
G2, M & 0.45	Mean	0.29	32.28	53.48	1.64	11.44	4.12	0.09
	±SD	0.21	5.82	8.79	1.24	11.80	2.61	0.06
	n	5	5	5	5	5	5	5
G3, M & 0.90	Mean	0.27	39.70	48.12	2.76	8.22	3.96	0.11
	±SD	0.15	5.40	6.46	2.28	6.24	1.13	0.05
	n	5	5	5	5	5	5	5
G4, M & 1.35	Mean	0.74	43.90	48.46	2.60	3.72	3.22	0.33
	±SD	0.40	7.08	7.17	0.96	1.75	0.83	0.19
	n	5	5	5	5	5	5	5

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)	(g/dL)
G1, M & 0	Mean	0.35	0.12	0.01	0.06	117.60	0.54
	±SD	0.24	0.26	0.00	0.09	58.77	0.01
	n	5	5	5	5	5	5
G2, M & 0.45	Mean	0.16	0.01	0.02	0.02	73.20	0.38*
	±SD	0.13	0.01	0.01	0.01	30.98	0.01
	n	5	5	5	5	5	5
G3, M & 0.90	Mean	0.14	0.01	0.02	0.01	76.60	0.48*
	±SD	0.09	0.01	0.00	0.01	36.12	0.02
	n	5	5	5	5	5	5
G4, M & 1.35	Mean	0.35	0.02	0.03*	0.03	104.20	0.25*
	±SD	0.20	0.01	0.01	0.02	25.21	0.00
	n	5	5	5	5	5	5

Group, Sex & Dose (mg/L)		Total Leucocyte Count	Neutrophils	Lymphocytes	Monocytes	Eosinophils	Basophils	Absolute Neutrophils
		(WBC)	(Neut)	(Lymph)	(Mono)	(Eos)	(Baso)	(Neut)
		(10^3 cells/µL)	(%)	(%)	(%)	(%)	(%)	(10^3 cells/µL)
G1, F & 0	Mean	0.38	34.40	19.42	0.60	24.28	3.50	0.13
	±SD	0.12	17.19	8.71	0.54	10.86	1.74	0.07
	n	5	5	5	5	5	5	5
G2, F & 0.45	Mean	0.31	24.86	22.50	0.50	25.10	2.62	0.08
	±SD	0.09	5.41	5.96	0.31	8.97	1.20	0.03
	n	5	5	5	5	5	5	5
G3, F & 0.90	Mean	0.34	28.62	25.84	0.94	23.10	1.18*	0.09
	±SD	0.07	8.48	10.64	1.03	12.63	0.89	0.02
	n	5	5	5	5	5	5	5
G4, F & 1.35	Mean	0.38	32.62	24.24	1.18	18.92	2.28	0.13
	±SD	0.26	8.02	11.42	1.28	6.30	0.52	0.08
	n	5	5	5	5	5	5	5

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)	(g/dL)
G1, F & 0	Mean	0.07	0.00	0.09	0.01	56.20	0.16
	±SD	0.04	0.00	0.04	0.00	22.75	0.01
	n	5	5	5	5	5	5
G2, F & 0.45	Mean	0.07	0.00	0.08	0.01	64.40	0.34*
	±SD	0.02	0.00	0.04	0.00	17.53	0.02
	n	5	5	5	5	5	5
G3, F & 0.90	Mean	0.09	0.00	0.08	0.01	62.40	0.34*
	±SD	0.05	0.00	0.05	0.01	28.09	0.03
	n	5	5	5	5	5	5
G4, F & 1.35	Mean	0.10	0.01	0.07	0.01	68.40	0.45*
	±SD	0.09	0.01	0.06	0.00	27.75	0.01
	n	5	5	5	5	5	5

*: Statistically significant (p<0.05)

 

Table 5. Summary of clinical chemistry record

Group, Sex & Dose (mg/L)		Glucose	Urea	Creatinine	Total Cholesterol	Triglycerides	Total Protein	Albumin	Aspartate aminotransferase	Alanine aminotransferase
		(GLU)		(CRE)	(CHO)	(TRI)	(TPR)	(ALB)	(AST)	(ALT)
		(mg/dL)	(mg/dL)	(mg/dL)	(mg/dL)	(mg/dL)	(g/dL)	(g/dL)	(U/L)	(U/L)
G1, M & 0	Mean	103.80	34.90	0.55	46.20	54.00	7.08	3.21	135.00	73.00
	±SD	1.92	4.29	0.01	9.50	15.39	0.13	0.11	27.45	19.79
	n	5	5	5	5	5	5	5	5	5
G2, M & 0.45	Mean	102.00	30.10	0.49*	45.80	56.80	6.50*	3.32	117.80	48.60
	±SD	9.92	3.82	0.01	11.99	14.60	0.32	0.14	8.14	9.74
	n	5	5	5	5	5	5	5	5	5
G3, M & 0.90	Mean	96.00	42.28	0.50*	43.60	46.00	6.40*	3.11	191.00	74.60
	±SD	8.15	19.28	0.03	9.45	12.75	0.21	0.08	152.48	46.36
	n	5	5	5	5	5	5	5	5	5
G4, M & 1.35	Mean	91.00*	34.76	0.49*	42.80	58.20	6.66	3.16	143.40	58.80
	±SD	5.10	8.53	0.04	6.98	6.76	0.47	0.15	67.23	22.88
	n	5	5	5	5	5	5	5	5	5

Group, Sex & Dose (mg/L)		Alkaline phosphatase	Total Bilirubin	Phosphorous	Calcium	Globulin	Blood Urea Nitrogen	Sodium	Potassium	Chloride
		(ALP)	(BIT)	(PHO)	(CAL)	(GLO)	(BUN)	(Na)	(K)	(CLO)
		(U/L)	(mg/dL)	(mg/dL)	(mg/dL)	(g/dL)	(mg/dL)	(mmol/L)	(mmol/L)	(mmol/L)
G1, M & 0	Mean	267.00	0.10	6.33	10.04	3.87	16.31	142.38	4.08	107.40
	±SD	68.82	0.02	0.59	0.51	0.21	2.01	1.81	0.45	1.79
	n	5	5	5	5	5	5	5	5	5
G2, M & 0.45	Mean	237.00	0.08	6.60	10.06	3.18*	14.07	141.54	3.96	106.32
	±SD	46.63	0.01	0.48	0.22	0.24	1.78	1.40	0.18	3.14
	n	5	5	5	5	5	5	5	5	5
G3, M & 0.90	Mean	278.60	0.33	6.82	9.78	3.29*	19.76	141.44	3.94	106.32
	±SD	68.80	0.48	0.30	0.28	0.19	9.01	1.05	0.33	1.76
	n	5	5	5	5	5	5	5	5	5
G4, M & 1.35	Mean	229.20	0.20	6.88	9.94	3.50	16.24	140.98	4.19	106.92
	±SD	34.22	0.23	0.28	0.36	0.47	3.98	0.89	0.26	1.16
	n	5	5	5	5	5	5	5	5	5

Group, Sex & Dose (mg/L)		Glucose	Urea	Creatinine	Total Cholesterol	Triglycerides	Total Protein	Albumin	Aspartate aminotransferase	Alanine aminotransferase
		(GLU)		(CRE)	(CHO)	(TRI)	(TPR)	(ALB)	(AST)	(ALT)
		(mg/dL)	(mg/dL)	(mg/dL)	(mg/dL)	(mg/dL)	(g/dL)	(g/dL)	(U/L)	(U/L)
G1, F & 0	Mean	93.80	41.24	0.55	41.40	39.20	7.00	3.10	166.40	68.00
	±SD	13.33	4.01	0.02	8.26	5.02	0.31	0.21	50.40	20.46
	n	5	5	5	5	5	5	5	5	5
G2, F & 0.45	Mean	93.40	33.54	0.55	50.80	69.40*	6.98	3.27	122.80	48.00
	±SD	9.84	7.03	0.03	9.47	16.24	0.18	0.14	22.37	6.96
	n	5	5	5	5	5	5	5	5	5
G3, F & 0.90	Mean	100.20	33.70	0.56	59.00*	51.40	6.94	3.17	151.40	48.60
	±SD	8.93	7.18	0.04	6.96	11.76	0.36	0.18	36.73	9.07
	n	5	5	5	5	5	5	5	5	5
G4, F & 1.35	Mean	96.80	38.84	0.53	49.20	45.60	6.82	2.86	131.80	53.20
	±SD	16.15	2.98	0.03	15.93	8.59	0.34	0.32	19.87	12.79
	n	5	5	5	5	5	5	5	5	5

Group, Sex & Dose (mg/L)		Alkaline phosphatase	Total Bilirubin	Phosphorous	Calcium	Globulin	Blood Urea Nitrogen	Sodium	Potassium	Chloride
		(ALP)	(BIT)	(PHO)	(CAL)	(GLO)	(BUN)	(Na)	(K)	(CLO)
		(U/L)	(mg/dL)	(mg/dL)	(mg/dL)	(g/dL)	(mg/dL)	(mmol/L)	(mmol/L)	(mmol/L)
G1, F & 0	Mean	150.80	0.09	4.78	9.72	3.90	19.27	141.60	3.67	109.02
	±SD	30.80	0.02	0.74	0.38	0.44	1.87	1.22	0.36	0.88
	n	5	5	5	5	5	5	5	5	5
G2, F & 0.45	Mean	140.40	0.09	5.85*	10.14	3.71	15.67	141.96	4.07	109.20
	±SD	69.38	0.02	0.52	0.18	0.06	3.28	1.46	0.50	1.29
	n	5	5	5	5	5	5	5	5	5
G3, F & 0.90	Mean	177.20	0.11	5.53	9.80	3.77	15.75	141.04	3.77	108.86
	±SD	37.18	0.07	0.45	0.23	0.45	3.36	1.04	0.24	1.91
	n	5	5	5	5	5	5	5	5	5
G4, F & 1.35	Mean	204.80	0.09	5.63	9.56	3.96	18.15	141.00	3.70	108.84
	±SD	90.60	0.01	0.74	0.33	0.57	1.39	1.87	0.19	0.42
	n	5	5	5	5	5	5	5	5	5

*: Statistically significant (p<0.05)

 

Table 6. Summary of chamber (exposure) conditions

Group & Conc. (mg/L)		Temp.  (°C)	Rh (%)	O2 (%)	CO2  (ppm)	Air inlet flow rate (L/min)*	BZC (mg/L)
G1 & 0	Mean	22.47	55.83	20.37	620.04	20.00	-
	±SD	0.18	1.37	0.15	5.35	0.00	-
G2 & 0.45	Mean	22.48	56.02	20.40	620.05	20.00	0.47
	±SD	0.18	1.47	0.16	4.72	0.00	0.01
G3 & 0.90	Mean	22.51	55.82	20.43	620.21	20.00	0.93
	±SD	0.19	1.54	0.18	4.88	0.00	0.01
G4 & 1.35	Mean	22.47	55.97	20.42	620.01	20.00	1.35
	±SD	0.20	1.53	0.19	5.72	0.00	0.02

* Values were constant throughout the exposure; hence standard deviation is zero; BZC: Breathing Zone Concentration

 

Table 7. 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)	8360	20	360	1.16
		17780			2.47
		26210			3.64
2	G1 (Air Only)	-	20	360	-
	G2, G3  and G4  (Test Item)	8430	20	360	1.17
		17530			2.43
		25950			3.60
3	G1 (Air Only)	-	20	360	-
	G2, G3  and G4  (Test Item)	8410	20	360	1.17
		17930			2.49
		26830			3.73
4	G1 (Air Only)	-	20	360	-
	G2, G3  and G4  (Test Item)	8290	20	360	1.15
		19190			2.67
		24980			3.47
5	G1 (Air Only)	-	20	360	-
	G2, G3  and G4  (Test Item)	8380	20	360	1.16
		18090			2.51
		26190			3.64
6	G1 (Air Only)	-	20	360	-
		8680	20	360	1.21
		18410			2.56
		26060			3.62
7	G1 (Air Only)	-	20	360	-
	G2, G3  and G4  (Test Item)	8430	20	360	1.17
		18110			2.52
		26710			3.71

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)	8580	20	360	1.19
		18450			2.56
		26590			3.69
9	G1 (Air Only)	-	20	360	-
	G2, G3  and G4  (Test Item)	8410	20	360	1.17
		18020			2.50
		26510			3.68
10	G1 (Air Only)	-	20	360	-
	G2, G3  and G4  (Test Item)	8550	20	360	1.19
		18350			2.55
		25690			3.57
11	G1 (Air Only)	-	20	360	-
	G2, G3  and G4 (Test Item)	8370	20	360	1.16
		17630			2.45
		26330			3.66
12	G1 (Air Only)	-	20	360	-
	G2, G3  and G4  (Test Item)	8680	20	360	1.21
		18420			2.56
		26130			3.63
13	G1 (Air Only)	-	20	360	-
	G2, G3  and G4  (Test Item)	8540	20	360	1.19
		17870			2.48
		26570			3.69
14	G1 (Air Only)	-	20	360	-
	G2, G3  and G4  (Test Item)	8520	20	360	1.18
		18590			2.58
		25700			3.57

Table 8. Summary of gross pathology findings

Route of administration	Inhalation route
Sex	Male				Female
Treatment	Air only	Acetanil Yellow 7GT 1115C			Air only	Acetanil Yellow 7GT 1115C
Group	G1	G2	G3	G4	G1	G2	G3	G4
Nominal Target Dose  (mg/L )	0	0.45	0.90	1.35	0	0.45	0.90	1.35
Number of animals	5	5	5	5	5	5	5	5
No. of dead rats during treatment	-	-	-	-	-	-	-	-
No. of moribund sacrificed rats	-	-	-	-	-	-	-	-
No. of terminally sacrificed rats	5	5	5	5	5	5	5	5
No. of animals with gross pathology	-	-	5	5	-	-	5	5
Lungs- discoloration, yellow	-	-	5	5	-	-	5	5

Table 9. Summary of histopathology findings

Route of administration		Inhalation route
Treatment		Air only		Acetanil Yellow 7GT 1115C
Nominal Target Dose (mg/L )		0		0.90		1.35
Group		G1		G3		G4
Sex		M	F	M	F	M	F
Number of Animals		5	5	5	5	5	5
Lung (Left lobe)	Number examined	5	5	5	5	5	5
	Within normal limits	4	3	4	4	4	3
Infiltrate, mononuclear cells, alveoli	Minimal	1	-	1	1	-	1
Macrophage, increased, alveoli	Minimal	1	2	-	-	1	1

 

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 14 days, 6 hours/day of aerosolized test item inhalation exposure to rats. Thus, the NOEC is determined to be 1.35 mg/L.

Executive summary:

A 14 day repeated dose toxicity study by inhalation route in rats was conducted for test substance following the OECD guideline 412. A total of 40 (20 males + 20 females) healthy young Sprague Dawley rats were distributed to four groups. Each group consisted of 10 animals (5 males and 5 females). Animals allocated to groups G2 (low dose), G3 (mid dose) and G4 (high dose) were exposed to aerosolized test item for 6 hours per day, for 14 days, at an actual target concentration of 0.45, 0.90 and 1.35 mg/L respectively. Highest dose tested was selected based on maximum achievable concentration in the exposure system. 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.

All animals were observed for clinical signs, mortality and morbidity, body weight, feed consumption and ophthalmological 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, coagulation, clinical chemistry, urinalysis, and BALF fluid analysis were performed. Histopathological examination was carried out for left lung from all control (G1), high dose group (G4) and also mid dose group (G3) due to coloration found in gross examination.

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. 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 treatment related gross pathological lesions noted. There were gross changes observed in lungs from G3 and G4 groups. This gross change was characterized by yellowish discoloration of lungs and was attributed to physical appearance of test item. However, there were no correlating microscopic findings in lungs of both G3 and G4 groups of animals and were found to be within normal histological limits.

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

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

1.35 mg/L

Study duration:

subacute

Species:

rat

Quality of whole database:

The study has a Klimisch score = 1

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the available data, the substance is not classified for specific target organ toxicity by repeated exposure (STOT-RE) according to CLP Regulation (EC) no 1272/2008.