A dried sludge product resulting from the treatment process of domestic wastewater. The exact processes followed for the production of the dried sludge are: Preliminary + secondary treatment of the wastewater stream, thickening, dehydration and drying of the excess sludge.

Acute Toxicity: inhalation

Administrative data

Endpoint:

acute toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Study Initiation and Completion Date: From 09 NOV 2021 to 15 MAR 2022
In-life phase: From 03 JAN 2022 to 10 JAN 2022

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 403 (Acute Inhalation Toxicity)

Version / remarks:

adopted 7 September, 2009

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test type:

traditional method

Limit test:

yes

Test material

Test material information

Constituent 1

Reference substance name:

A dried sludge product resulting from the treatment process of domestic wastewater. The exact processes followed for the production of the dried sludge are: Preliminary & secondary treatment of the wastewater stream, thickening, dehydration and drying of the excess sludge.

EC Number:

943-834-9

Molecular formula:

Not applicable for UVCB substances

IUPAC Name:

A dried sludge product resulting from the treatment process of domestic wastewater. The exact processes followed for the production of the dried sludge are: Preliminary & secondary treatment of the wastewater stream, thickening, dehydration and drying of the excess sludge.

Specific details on test material used for the study:

Batch Manufactured by
(Name and Address) : EYDAP SA, Psyttalia wastewater treatment plant
Batch Supplied by (Name and Address) : SustChem Technical Consulting S.A., 3rd Septemvriou 144, Athens, 112 51
Test Item : Dried Sludge from Domestic wastewater
CAS No. : Not Applicable
Chemical Name (IUPAC) : Not Applicable
Physical Appearance : Dark Brown to black, porous, powder to
granular solid with small amounts of additional organic waste such as pine needles, Wood shards, etc
Purity as per Certificate of Analysis: - (UVCB substance)
Batch No. : 190221
Manufactured date as per CoA : 19/02/2021
Expiry date : 01.09.2022
pH : 6.96±0.15
Relative Density : 1.13±0.07
Recommended Storage Condition: Deep Frozen (-10 to -25°C)
Note: 1. Date of receipt of test item at test facility: 23 July 2021
2. Test item code by test facility: S098-01

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

Source : Hylasco Biotechnology (India) Pvt. Ltd.Telangana -500078
No. of group : One treatment group (G1)
No. of rats per group: Six (3 males and 3 females - For limit test)
Six (3 males and 3 females - For sighting test)
Age at receipt : 7 weeks age on the date of receipt 03.12.2021
Age at exposure : 11 weeks
Body weight range at grouping : Males : 284.5 to 287.9 g
Females : 277.6 to 280.5 g
The weights did not exceed ± 20 percent of the
mean weight in each sex on the day of grouping.

Body weight range at start of treatment: Males : 285.9 to 289.4 g
Females : 278.5 to 281.1 g

Identification : Rat accession number, cage card and turmeric
colour body marking. The temporary body marking during acclimatization period was done with crystal violet.

Acclimatization : After physical examination for good health and suitability for the experiment the animals were acclimatized for six days before treatment. The Females were nulliparous and non- pregnant. Animals were observed once daily during acclimatization period for any abnormalities. After grouping (prior to testing) i.e., on day 6 of acclimatization the animals were acclimatized to the test apparatus (restrainers) for at least one hour, to lessen the stress caused by introduction to the new environment.

Administration / exposure

Route of administration:

other: dust aerosol

Type of inhalation exposure:

nose only

Vehicle:

clean air

Remarks:

The Air compressor provides compressed air. The compressed air is dehumidified, filtered and reduced by filter reducer assembly to the required pressure (2 kg/cm2) at the use point.

Mass median aerodynamic diameter (MMAD):

>= 1 - <= 4 µm

Geometric standard deviation (GSD):

>= 1.5 - <= 3

Details on inhalation exposure:

Exposure chamber:
(Schematic drawing of inhalation exposure chamber is included as Figure 1)
The equipment is designed and operated in slight positive pressure so that the genuine test atmosphere is dynamically delivered to each port and exhaled air from exposed animals is immediately exhausted. The volume of the inhalation chamber is 0.7 liters. (Manufactured by SMIT Fabricators).
The nose only, flow-past exposure chamber consists of two chambers, the aerosol inlet chamber (Diameter 10 cm × Height 8 cm) and the aerosol exhaust chamber (Diameter 20 cm × Height 10 cm). The chambers are cylindrical in shape and placed one next to the other. The chamber has 12 ports into a central plenum arranged in a row and these ports are open at the outer cylindrical chamber for fixing the animal restrainers and provisions for sampling aerosol, recording of oxygen content, carbon dioxide content, chamber temperature and humidity. The Air compressor provides compressed air. The compressed air is dehumidified, filtered and reduced by filter reducer assembly to the required pressure (2 kg/cm2) at the use point.
Animals were restrained in glass exposure restrainers (size approximately Length 23 cm, Diameter 7 cm). These restrainers make an airtight seal with the plenum tube through a rubber gasket. Within the plenum is a central tube into which the aerosol enters at each port. Air flow carries the aerosol to the nose of the animals restrained in position at the open ends of the tubes.

Aerosol Generation System:
Based on physico-chemical properties of the test item, solid dust aerosol generation system was used for sighting study and limit test study.

Solid dust / aerosol Generation System:
The instrument has two modules, a) Operating panel and b) Dust feeder module. The operating panel is connected to the power source and to the dust feeder module. The dust feeder module is connected to the inhalation exposure unit.

Preparation of test item dust for Sighting study/ limit test:
Marshall mars mill and Sieve was used for powdering of test item before loading into the dust/aerosol generating system.

Seven stage cascade impactor:
Seven stage cascade impactor was used to determine particle size distribution, Mass Median Aerodynamic Diameter (MMAD) and Geometric standard deviation (GSD).

Unit of measurement: Aerosol particle size in µm.

UNIPHOS – (700) Inhalation Chamber Monitor:
The inhalation chamber monitor was used to measure oxygen and carbon dioxide content, temperature and humidity in the inhalation chamber during exposure.

Analytical verification of test atmosphere concentrations:

yes

Remarks:

Actual test item concentrations were analyzed (from the sample collected at animals breathing zone) for active ingredient concentration. The concentration of test item in air inhalation samples was determined by using an in-house developed and validated

Duration of exposure:

4 h

No. of animals per sex per dose:

3

Control animals:

no

Details on study design:

Conditions:
Animals were housed under standard laboratory conditions, air conditioned with adequate fresh air supply (13.7 to 14.0 air changes / hour). Environment: with temperature 21 to 23°C, relative humidity 65 to 67%, with 12 hours light and 12 hours dark cycle.
The maximum and minimum temperature and relative humidity in the experimental room were recorded once daily. The relative humidity in the experimental room was calculated from dry and wet bulb temperature recordings.
Housing:
Rats were housed individually in standard polysulfone cages (Size: approximately L 425 x B 266 x H 185 mm), with stainless steel top grill having facilities for pelletted food and drinking water. Additionally, polycarbonate rat huts were placed inside the cage as an enrichment object and was changed along with the cage at least once a week.
Bedding: Steam sterilized corn cob was used and changed along with the cage
once a week.
Diet: ad libitum
Rat & mice pellet feed, manufactured by Krishna Valley Agrotech LLP, MIDC Kupwad block, Sangli, Maharashtra, was provided to animals. Diet analysis report is enclosed as Annexure 1.
Water: ad libitum
The animals were provided deep bore-well water passed through activated charcoal filter and exposed to UV rays in ‘Aquaguard’ on-line water filtercum-purifier manufactured by Eureka Forbes Ltd., Mumbai 400 001, India, was provided to animals in polycarbonate bottles with stainless steel sipper tubes.

Technical pre-test
The test item was made into fine powder using marshall mars mill and sieve.A technical pre-test was conducted without animals to check the feasibility of generating adequate test atmosphere (Test item concentration, particle size distribution, O2, CO2, temperature and relative humidity) after chamber equilibrium. The chamber air actual concentration was estimated gravimetrically.

Sighting study:
The test item was made into fine powder using marshall mars mill and sieve. During technical pre-test, the fine powder was tested at 50 mm/hr piston speed and at 8 LPM airflow rate with using dust generator and generated good dust aerosol. Based on technical pre-test results, the sighting study was conducted as per OECD 403 Traditional Protocol using 3 male and 3 female rats and the rats were exposed to the test item dust aerosol.

The exposed rats were observed for 7 days for clinical signs and necropsied.
No abnormality was detected in surviving rats at necropsy.
Based on the sighting study results, the following piston speed was selected for the limit test.
G1 : Powder test item with 50 mm/h piston speed and at 8 LPM air flow rate

Limit test
The exposure conditions maintained during the sighting study were adopted for
the limit test.

Grouping
Three male and three female rats were assigned to the study group by body weight stratification during acclimatization. Animals with extreme body weights were not used in the study and were excluded from the treatment.

Animal restraint during exposure:
The rats were housed individually in the special rat restrainer (as detailed under exposure chamber) which provided nose exposure only. Food and water were withheld during the exposure period.
Treatment and duration of exposure / observation:
All the rats as per the group allotted was exposed to the dust for four hours on the day of exposure. Thereafter, all rats were subjected to a 14 days post-treatment observation period.

Conditions monitored during exposure:
The chamber temperature and humidity were recorded at hourly intervals, i.e. four times during the exposure period using the inhalation chamber monitor. The instrument uses a semiconductor sensor has been used for measuring the temperature inside the chamber and capacitive sensor is used for the measurement of relative humidity.

Atomizer pressure: The atomizer pressure of 2 kg/cm2 was maintained.
Airflow rate: The airflow rate was monitored continuously and the airflow of 8LPM was maintained.

Aerosol Particle Size Analysis:
Particle size of the aerosol generated in the chamber air was determined two times i.e., during the first and fourth hour of the exposure period using any one port. The air samples from the inner chamber (breathing zone level by the rats)was passed at sampling rate of 4 LPM and sampling duration of 1 minutethrough the seven stage cascade impactor. The MMAD and Geometric standard deviation (GSD) were calculated.
The value for Mass Median Aerodynamic Diameter (MMAD) 1-4 µm and
geometric standard deviation (GSD) 1.5-3.0 for the test item was within the
range for G1 group, as recommended by OECD guidance document no. 39 on
acute inhalation testing.

Oxygen (O2) and Carbon dioxide (CO2) content in the chamber air:
The inhalation chamber monitor was used to measure oxygen and carbon dioxide content in the inhalation chamber during exposure. These Parameters were determined three times (first, fourth hour and another during the third hour of exposure) during exposure period using any one port. The instrument uses a sealed electrochemical sensor for detecting oxygen and nondispersive infra-red sensor (NDIR) is used for the detection of carbon dioxide gas.

Determination of Breathing Zone Concentration (BZC) of test item concentration in chamber air (Sighting study / Limit test):
Sample collection:
The test item concentration at the animals’ breathing zone of the inhalation chamber was determined using gravimetric filter analysis method and active ingredient (a.i) analysis method also. Aerosol samples were drawn from one port at hourly intervals of the 4hour exposure period. The chamber air was passed through the gravimetric single filter paper (Whatman glass microfiber filters, No. 47 mm) by suction applied using vacuum pump.

Air Sample Analysis :
Actual test item concentrations were analyzed (from the sample collected at animals breathing zone) for active ingredient concentration. The concentration of test item in air inhalation samples was determined byusing an in-house developed and validated analytical method.

Clinical Signs and pre-terminal deaths:
The rats were observed for pre-terminal deaths four times during day 1 (at hourly intervals during the exposure) and twice after release of rats from the restrainers for clinical signs of toxicity / pre-terminal deaths and once daily during days 2-15. All observed clinical signs were recorded.

Observation included changes in skin and fur, eyes and mucous membranes, and also respiratory, circulatory, autonomic and central nervous systems and somatomotor activity and behaviour pattern. Attention was directed to observations of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma.

Body weights:
Body weights were recorded once during the acclimatization period and on day 1 (pre-exposure), 2, 4 (3 days post exposure), 8 (7 days post exposure) and 15 (14 days post exposure).

Necropsy:
At the end of the observation period, all rats were euthanised using isoflurane anesthesia and subjected to detailed necropsy by an experienced prosector. Particular attention was given to any changes in the respiratory tract.

Results and discussion

Effect levels

Sex:

male/female

Dose descriptor:

LC50

Effect level:

> 5.1 mg/L air

Based on:

test mat.

Exp. duration:

4 h

Mortality:

Τhere was no mortality at 5.1 mg/L of chamber air in both male and female Wistar rats. All Rats were normal from immediately after the exposure period.

Body weight:

All rats gained bodyweight throughout the experiment period. There were no
mortalities observed during the experimental period. No abnormality detected
at necropsy.

Any other information on results incl. tables

The results of the acute inhalation toxicity study results are presented in Tables 1, 2 and 3.

Aerosol Particle Size Distribution

The overall mean MMAD of 2.41 micrometers and GSD of 1.73 was observed for G1 group. 

The value for Mass Median Aerodynamic Diameter (MMAD) 1-4 µm and geometric standard deviation (GSD) 1.5 to 3.0 for the test item were within the range for G1 group as recommended by OECD guidance document no. 39 on acute inhalation testing.

Chamber Equilibrium

The exposure start was soon after the exposure chamber attained the equilibrium state within 0.26 minutes.

Chamber equilibrium (t₉₅) = 3(Chamber volume / Chamber air flow)

Chamber equilibrium (t₉₅)   = 3(0.7/8) = 0.26 minutes

Study Duration

The rats were observed for a minimum period of 14 days following the dose administration.

Clinical Signs and pre-terminal deaths

Refer Table 3 and Table 2

Αll Rats were normal from immediately after the exposure period.

Body Weights

Refer Table 2

All rats gained bodyweight throughout the experiment period. There were no mortalities observed during the experimental period. No abnormality detected at necropsy.

Necropsy

Refer Table 3

No abnormality was detected at necropsy in any of the rats.

Table 1. Particulars of sample collection, test conditions, aerosol generation and test item concentration obtained in the chamber during limit test study

Group No.	No. of rats exposed	Exposure@ Date and Time	Piston speed (mm/h)	Air flow rate, L/min	Sampling duration (min)	Sampling rate, L/min	Nominal concentration, mg of test item / L of chamber air*	Gravimetric Concentration, mg of test item / L of chamber air Mean ± SD	Analyzed Concentration, mg of test item / L of  chamber air Mean ± SD
G1	3M + 3F	03 January 2022 Start:  08.45 AM End :  12.45  PM	50	8	2	5	45.21	5.31 ± 0.04	5.1± 0.056

@: Exposure start time is soon after the chamber equilibrium time of about 0.26 minutes

M : Male     F : Female

 *: Nominal concentration is the mass of generated test item in the inhalation chamber divided by the total volume of air passed through the chamber system.

    

Nominal concentration (mg/L) For G1 group	=	The mass of generated test item in the inhalation chamber	=	(86810)mg	=	45.21 mg/L
		The total volume of air passed through the chamber system		(4×60×8)L

 

Table 2. Body weight, body weight change and pre-terminal deaths                                                                   

                                                                                                                                                                                                                                                                                      Refer Table 3

Group and Dose (mg of test item/L of chamber air)	Rat No.	Sex	Body weight (g)							No. dead / No. tested	Pre-terminal deaths (%)
			Initial	2^nd day	4^th day	8^th day	Weight change (day 8 – Initial)	15^th day	Weight change (day 15 – Initial)
G1   ‘5.1’	Rad2331	M	285.9	287.4	292.7	304.2	18.3	338.3	52.4	0/6 (0M/0F)	‘0’
	Rad2332	M	289.4	290.9	296.2	307.9	18.5	331.4	42.0
	Rad2333	M	286.8	288.2	293.4	302.8	16.0	339.8	53.0
	Rad2334	F	280.2	281.5	284.7	292.6	12.4	315.1	34.9
	Rad2335	F	278.5	279.8	284.1	297.0	18.5	317.6	39.1
	Rad2336	F	281.1	282.3	285.3	294.2	13.1	321.2	40.1

                 M: Male          F: Female

Table 3. Clinical Signs, pre-terminal deaths and necropsy findings of individual rats

 

Group: G1                                                                                                                                                                              Dose (mg/L of chamber air): 5.1

Rat No.

Sex

Days of observations

Necropsy Findings

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

@

#

Rad2331

M

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

NAD

Rad2332

M

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

NAD

Rad2333

M

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

NAD

Rad2334

F

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

NAD

Rad2335

F

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

NAD

Rad2336

F

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

N

NAD

M: Male                F: Female                N: Normal           NAD: No Abnormality Detected 

 

@: No mortality observed during the exposure period of four hours and the clinical signs recorded were at the time of release of animals from the restrainers. 

#: Observation of clinical signs approximately 1 hour after end of exposure      

Applicant's summary and conclusion

Interpretation of results:

Category 5 based on GHS criteria

Remarks:

The test item is classified as category 5 as per Globally Harmonized System of Classification and Labelling of Chemicals (GHS) Ninth Revised Edition, United Nations (2021). ST/SG/AC.10/30/Rev.9, as there was no mortality at 5.1 mg/L of chamber air in both male and female Wistar rats.

Conclusions:

The acute inhalation (4 hours) Median Lethal Concentration (LC50) value of “Dried Sludge from Domestic Waste Water” is more than 5.1 mg/L of chamber air in male and female Wistar rats.

Executive summary:

An acute inhalation toxicity study with “Dried Sludge from Domestic Waste Water” was conducted in Wistar rats with one treatment group (G1).
The inhalation toxicity study of “Dried Sludge from Domestic Waste Water” was determined in 3 male and 3 female Wistar rats by exposure to test item as a dust aerosol generated by dust generator with an air flow rate of 8LPM and a piston speed of 50 mm/h with 2.0 kg/cm2 of atomizer pressure. The rats were housed in special rat restrainers and continuously exposed to the test item aerosol (nose only) for 4 hours in an inhalation exposure chamber (dynamic state) for the G1 group. The post treatment observation period was 14 days. The dust aerosol was sampled from the inhalation chamber to determine particle size distribution, Mass Median Aerodynamic Diameter (MMAD) and Geometric standard deviation (GSD). Particle size of the aerosol generated in the chamber air was determined two times i.e., during the first and fourth hour of the exposure period using any one port. The MMAD and GSD was calculated using seven stage cascade impactor.
The overall mean MMAD of 2.41 micrometers and GSD of 1.73 was observed for G1 group.
The analytically determined average concentration of “Dried Sludge from Domestic Waste Water” was 5.1 mg/L of chamber air.
All animals were normal from immediately after the exposure period.
All rats gained bodyweight throughout the experiment period. There were no mortalities observed during the experimental period. No abnormality detected at necropsy.
The acute inhalation (4 hours) Median Lethal Concentration (LC50) value of“Dried Sludge from Domestic Waste Water” is more than 5.1 mg/L of chamber air in male and female Wistar rats.
Based on the results of the present study, the test item “Dried Sludge from Domestic Waste Water” is classified as follows:
• The test item is classified as category 5 as per Globally Harmonized System of Classification and Labelling of Chemicals (GHS) Ninth Revised Edition, United Nations (2021). ST/SG/AC.10/30/Rev.9, as there was no mortality at 5.1 mg/L of chamber air in both male and female Wistar rats.