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.

Endpoint summary

Description of key information

Dried sludge from domestic wastewater is of no intrinsic hazard following acute dermal, inhalation and oral exposure.

• The acute dermal LD50 was greater than 2000 mg/kg body weight


• The acute inhalation LC50 was determined to be >5.1 mg/L air.


• The acute oral LD50 was 5000 mg/kg body weight

 

Dermal

The acute dermal toxicity of Dried Sludge from Domestic Waste water was tested in accordance with OECD Guideline 402 (Acute Dermal Toxicity: Fixed Dose Procedure). The item was tested a group of 5 female Wistar rats. The body weight of all the animals were measured on the last day of acclimatization for the dose range finding study. Based on the individual body weight, the test item at the dose of 200, 1000 and 2000 mg/kg body weight was weighed in aluminum foil and made into paste by adding 0.1, 0.3 and 0.5 mL of Milli-Q water respectively and was completely transferred on to the cotton gauze (size: Females: 8 x 5 cm of 6 ply) and applied directly (semi-occlusive) to the clipped skin of the rat to cover about 10% of body surface of the rat and secured in position by adhesive tape wound around the torso. The test item contact period with the skin was for 24 hours. After the 24 hours contact period, the dressing was removed, and the applied area was washed with deionized water and wiped dry using clean towels.

 

The animals were observed for clinical signs and pre-terminal deaths (mortality) once during first 30 minutes after application, and at hourly intervals for 6 hours after application on the day of treatment (day 1) and once daily during Days 2 to 15. In addition, the site of applied area was observed for skin reactions at 24, 48 and 72 hours after removal of test chemical using the Draize criteria. There were no clinical signs and pre-terminal deaths (mortality) observed during the study and there were no skin reactions at the site of application at 24, 48 and 72 hours after test patch removal. Individual body weights of animals were recorded on test days 1 (Pre-application), 8 (7 days post application), and 15 (14 days post application). All rats gained body weight throughout the observation period.

At the end of the observation period, all rats were euthanised and exsanguinated under isoflurane anesthesia and subjected to detailed necropsy. No abnormalities were detected during the necropsy.

 

Based on the present study results, the acute dermal LD50 of Dried Sludge from Domestic Waste water is greater than 2000 mg/kg body weight in female Wistar rats. The test item, Dried Sludge from Domestic Wastewater is classified as “Category 5” (the dermal LD50 range of 2000 to 5000 mg/kg) as per Globally Harmonized System of Classification and Labelling of Chemicals 2021, as there was no mortality observed at 2000 mg/kg body weight.

 

Inhalation

The acute inhalation toxicity of Dried Sludge from Domestic Waste water was tested in accordance with OECD Guideline 403 (Acute Inhalation Toxicity). The item was tested in Wistar rats with one treatment group (G1). 3 male and 3 female Wistar rats were exposed to test item. 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. The rats were housed in special rat restrainers which provided nose exposure only. All rats were exposed to the dust for 4 hours on the day of exposure. Thereafter, all rats were subjected to a 14 days post-treatment observation period.

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 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. 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). All rats gained bodyweight throughout the experiment period. At the end of the observation period, all rats were euthanised using isoflurane anesthesia and subjected to detailed necropsy. There were no mortalities observed during the experimental period and there were 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 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.

 

 

Oral

The Dried Sludge from domestic Wastewater was tested according to OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method). No toxicity information is available the for the test item. Hence the test was initiated with starting dose of 300 mg/kg body weight. The test item was administered orally to a group of experimental animals at one of the defined dose (i.e. 300 mg/kg body weight) as a first step (G1-FTS). All the three female rats were survived. The test was continued with same dose of 300 mg/kg body weight with three additional female rats at next treatment step (G1-STS). All rats were normal and no pre-terminal deaths. The test item was administered to experimental animals at higher dose (2000 mg/kg body weight) as a first step (G2-FTS) and all the rats survived. The test was continued at the same dose of 2000 mg/kg bodyweight (G2-STS) and all rats were survived. Dosing was stopped. The subsequent dosing was done at approximately 48-72 hours after the previous step. The prepared test item dose formulations were administered at the dose volume of 10 mL/kg bodyweight to attain the dose of 300 mg/kg body weight (G1–FTS and G1-STS) and 2000 mg/kg body weight (G2–FTS and G2-STS) as a single oral gavage to overnight fasted rats (16 to 18 hours).

At each step, the animals were observed five times on test day 1 (day of administration). There was no mortality or pre-terminal deaths. The body weights were recorded on test day 1 (pre-administration), day 8 (7 days post administration) and day 15 (14 days post administration) and at death. All survived rats gained weight during experimental period. The rats surviving to the end of the observation period were euthanized by using isoflurane anaesthesia and subjected to detailed necropsy. No abnormality was detected at necropsy.

Based on the results of the present study, the acute oral LD50 of Dried Sludge from Domestic wastewater is 5000 mg/kg as per the LD50 cut-off value.

 

Key value for chemical safety assessment

Acute toxicity: via oral route

Link to relevant study records

Reference

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 06 June 2022 to 20 July 2022

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test type:

acute toxic class method

Species:

rat

Strain:

Wistar

Sex:

female

Details on test animals or test system and environmental conditions:

Conditions:
Rats were housed under standard laboratory conditions, air conditioned with adequate fresh air supply (14.2 air changes/hour). Environment: with temperature 20 to 25°C, relative humidity 64 to 68%, 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 pelleted food and drinking water in polycarbonate bottle. Additionally, polycarbonate rat huts were placed inside the cage as an enrichment object and were changed along with the cage once a week.

Bedding: Steam sterilized corn cob was used and changed once a week along with the cage.

Diet: ad libitum
Rat & mice pellet feed, manufactured by Krishna Valley Agrotech LLP, MIDC Kupwad block, Sangli, Maharashtra, India, was provided to animals. Diet analysis report is enclosed as Annexure - 2.

Water: ad libitum
Deep bore-well water passed through activated charcoal filter and exposed to UV rays in Aquaguard on-line water filter-cum-purifier manufactured by Eureka Forbes Ltd, Mumbai 400 001, India was provided to animals in polycarbonate bottles with stainless steel sipper tubes. Water analysis report is enclosed as Annexure - 3.

Route of administration:

oral: gavage

Vehicle:

other: Milli-Q-water

Details on oral exposure:

The prepared test item dose formulations were administered at the dose volume of 10 mL/kg bodyweight to attain the dose of 300 mg/kg body weight (G1–FTS and G1-STS) and 2000 mg/kg body weight (G2–FTS and G2-STS) as a single oral gavage to overnight fasted rats (16 to 18 hours). Each animal was administered orally by gavage using disposable plastic syringe attached with metal feeding cannula. Food was offered about 3 to 4 hours after dosing. Water was not withheld.

Doses:

No toxicity information was available the for the test item. Hence the test was initiated with starting dose of 300 mg/kg body weight.All the three rats were survived at starting dose of 300 mg/kg body weight (G1-FTS). The test was continued with same dose of 300 mg/kg body weight with three additional female rats at next treatment step (G1-STS) as per Annex-2C. The test was continued with higher dose of 2000 mg/kg body weight with three additional female rats at next treatment step
(G2-FTS) All rats were survived. The test was continued with same dose of 2000 mg/kg body weight with three additional female rats at next treatment step (G2-STS) as per Scheme and all rats were survived. Dosing was stopped. The subsequent dosing was done at approximately 48-72 hours after the previous step.

No. of animals per sex per dose:

3

Key result

Sex:

female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Mortality:

There were no clinical signs and pre-terminal deaths.

Body weight:

other body weight observations

Remarks:

The body weights of all the rats increased throughout the observation period.

Other findings:

No abnormality was detected at necropsy.

• Table 1. Body weight, body weight change

Group and Dose (mg/kg body weight)	Rat No.	Sex	Body weight (g)					No. dead/ No. tested	Pre-terminal deaths (%)
			Initial (Day 1)	8^th day	Weight change (day 8 – Initial)	15^th day	Weight change (day 15 – Initial)
G1 (FTS) 300	Rad4245	F	189.3	209.7	20.4	225.0	35.7	0/3	0
	Rad4246	F	190.7	211.7	21.0	231.6	40.9
	Rad4247	F	198.3	216.0	17.7	229.9	31.6
G1 (STS) 300	Rad4248	F	187.6	204.0	16.4	219.1	31.5	0/3	0
	Rad4249	F	186.3	210.6	24.3	224.6	38.3
	Rad4250	F	196.7	217.4	20.7	235.7	39.0
G2 (FTS) 2000	Rad4251	F	187.3	211.0	23.7	229.6	42.3	0/3	0
	Rad4252	F	189.5	216.7	27.2	237.5	48.0
	Rad4253	F	208.6	219.0	10.4	238.0	29.4
G2 (STS) 2000	Rad4254	F	206.0	223.1	17.1	250.4	44.4	0/3	0
	Rad4255	F	208.9	228.6	19.7	253.5	44.6
	Rad4256	F	209.0	223.9	14.9	251.6	42.6

         F: Female;                        FTS: First Treatment Step                   STS: Second Treatment Step                 NA: Not Applicable

 

Appendix 2. Individual clinical signs, dose administration and necropsy findings

        

Group and Dose (mg/kg body weight)

Date and time of administration

Rat No.

Sex

Body weight (Day 1)

Total volume administered (mL)

Day of observation

Necropsy Findings

Day 1

Day 2 to 15

30 min

1hour

2 hours

3 hours

4 hours

G1 (FTS) 300

11 June 2022 and  11:57 AM to 11:59 AM

Rad4245

F

189.3

1.9

N

N

N

N

N

N

NAD

Rad4246

F

190.7

1.9

N

N

N

N

N

N

NAD

Rad4247

F

198.3

2.0

N

N

N

N

N

N

NAD

F: Female         FTS: First treatment step                     N: Normal                        min: minutes                mg: milligrams                   

kg: kilograms    mL: millilitre      NAD: No Abnormality Detected          

 

APPENDIX 2 contd. Individual clinical signs, dose administration and necropsy findings                   

Group and Dose (mg/kg body weight)

Date and time of administration

Rat No.

Sex

Body weight (Day 1)

Total volume administered (mL)

Day of observation

Necropsy Findings

Day 1

Day 2 to 15

30 min

1hour

2 hours

3 hours

4 hours

G1 (STS) 300

14 June 2022 and 11:14 AM to 11:17 AM

Rad4248

F

187.6

1.9

N

N

N

N

N

N

NAD

Rad4249

F

186.3

1.9

N

N

N

N

N

N

NAD

Rad4250

F

196.7

2.0

N

N

N

N

N

N

NAD

     F: Female         STS: Second treatment step                 N: Normal                        min: minutes                mg: milligrams       

*: Observation at the end of the treatment day                kg: kilograms                mL: millilitre       NAD: No Abnormality Detected

 

APPENDIX 2 contd. Individual clinical signs, dose administration and necropsy findings

Group and Dose (mg/kg body weight)

Date and time of administration

Rat No.

Sex

Body weight (Day 1)

Total volume administered (mL)

Day of observation

Necropsy Findings

Day 1

Day 2 to 15

30 min

1hour

2 hours

3 hours

4 hours

G2 (FTS) 2000

17 June 2022 and 11:53 AM to 11:55 AM

Rad4251

F

187.3

1.9

N

N

N

N

N

N

NAD

Rad4252

F

189.5

1.9

N

N

N

N

N

N

NAD

Rad4253

F

208.6

2.1

N

N

N

N

N

N

NAD

F: Female         STS: Second treatment step                 N: Normal                        min: minutes                mg: milligrams       

*: Observation at the end of the treatment day                kg: kilograms                mL: millilitre       

NAD: No Abnormality Detected

 

APPENDIX 2 contd. Individual clinical signs, dose administration and necropsy findings

Group and Dose (mg/kg body weight)

Date and time of administration

Rat No.

Sex

Body weight (Day 1)

Total volume administered (mL)

Day of observation

Necropsy Findings

Day 1

Day 2 to 15

30 min

1hour

2 hours

3 hours

4 hours

G2 (STS) 2000

21 June 2022 and 12:15 PM to 12:18 PM

Rad4254

F

206.0

2.1

N

N

N

N

N

N

NAD

Rad4255

F

208.9

2.1

N

N

N

N

N

N

NAD

Rad4256

F

209.0

2.1

N

N

N

N

N

N

NAD

F: Female         STS: Second treatment step                 N: Normal                        min: minutes                mg: milligrams       

*: Observation at the end of the treatment day                kg: kilograms                mL: millilitre       

NAD: No Abnormality Detected

 

 

           

Interpretation of results:

Category 5 based on GHS criteria

Remarks:

The test item, Dried Sludge from Domestic wastewater is classified as follows: • The test item is classified as “Category 5 or unclassified” as per Globally Harmonized Classification system of Annex 2d of the Guideline, OECD 423. as there was no mortality observed at 2000 mg/kg body weight (Annexure 1). • The test item is classified as “Category 5” (2000

Conclusions:

Based on the results of the present study, the acute oral LD50 of Dried Sludge
from Domestic wastewater is 5000 mg/kg as per the LD50 cut-off value.

Executive summary:

No toxicity information was available for the test item Dried Sludge from Domestic wastewater. Hence the study was initiated with 300 mg/kg bodyweight. The dose formulation was prepared by using milli-Q-water and administered as a single oral gavage to overnight fasted (16 to 18 hours) three female rats (G1-FTS) at the dose of 300 mg/kg body weight. All rats were normal and no pre-terminal deaths. The treatment was continued by dosing three additional female rats at the same dose of 300 mg/kg body weight (G1-STS) and all rats were normal and no pre-terminal deaths. As per the scheme - The treatment was continued by dosing three additional female rats at the higher dose of 2000 mg/kg body weight (G2-FTS) and all rats were normal and no pre-terminal deaths. Again, treatment was done by dosing three additional female rats at the same dose of 2000 mg/kg body weight (G2-STS) and all rats were normal and no pre-terminal deaths. The dosing was stopped as per Annex 2c.

The prepared dose formulation was administered at the dose volume of               10 mL/kg bodyweight.

The rats were observed for mortality and clinical signs for 14 days post treatment. There was no mortality or pre-terminal deaths. Body weights were recorded prior to dosing on day 1 and again on days 8 and 15. Necropsy was performed for all the survived and dead rats. All survived rats gained weight during experimental period. There were no gross pathological changes at necropsy.

The dose formulation analysis was performed for the G1-FTS (300 mg/kg body weight) & G2-FTS (2000 mg/kg body weight). The results were considered acceptable, as the percent agreements of the analyzed concentrations are within the range of 70% to 120% of the claimed concentrations. Homogeneity of the dose formulation was considered acceptable as % RSD from six replicates at each dose level is ≤ 20.0%, hence back up samples discarded.

Based on the results of the present study, the test item, Dried Sludge from Domestic wastewater in Wistar rats is classified as follows:      

• The LD₅₀ cut off value is 5000 mg/kg body weight or unclassified.

The test item, Dried Sludge from Domestic wastewater is classified as follows:

• The test item is classified as “Category 5 or unclassified” as per Globally Harmonized Classification system of Annex 2c of the Guideline, OECD 423.


• The test item is classified as “Category 5” (2000<ATE≤5000 mg/kg) as per Globally Harmonized System of Classification and Labelling of Chemicals (GHS) Ninth Revised Edition, United Nations (2021).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LD50

Value:

5 000 mg/kg bw

Acute toxicity: via inhalation route

Link to relevant study records

Reference

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)

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

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

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.

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.

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.

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      

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LC50

Value:

> 5.1 mg/L air

Quality of whole database:

The study is a GLP compliant and has a Klimisch score 1.

Acute toxicity: via dermal route

Link to relevant study records

Reference

Endpoint:

acute toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From Nov. 18, 2021 to March 9, 2022

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 402 (Acute Dermal Toxicity: Fixed Dose Procedure)

GLP compliance:

yes (incl. QA statement)

Test type:

fixed dose procedure

Species:

rat

Strain:

Wistar

Sex:

female

Details on test animals or test system and environmental conditions:

Conditions:
Rats were housed under standard laboratory conditions, in an air conditioned with adequate fresh air supply (13.7 to 13.9 air changes/hour).

Environment:
Temperature: 21˚C - 24°C
Relative humidity: 65% - 67%
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:
Animals were housed individually (to avoid fighting injury in standard polysulfone cages (Size: approximately L 425 x B 266 x H 185 mm), with stainless steel top grill having facilities for pelleted food and drinking water from a polycarbonate bottle. Additionally, polycarbonate rat huts were placed inside the cage as enrichment objects and were changed along with the cage once a week.
Bedding: Steam sterilized corn cob was used and changed once a week along with the cage.

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
Deep bore-well water passed through activated charcoal filter and exposed to UV rays in Aquaguard on-line water filter-cum-purifier manufactured by Eureka Forbes Ltd, Mumbai 400 001, India, was provided to animals in polycarbonate bottles with stainless steel sipper tubes. Water analysis report is enclosed as Annexure 2.

Type of coverage:

semiocclusive

Vehicle:

water

Remarks:

Milli-Q water

Details on dermal exposure:

Preparation of test site for application:
Approximately 24 hours before treatment, the hair on the dorsolateral thoracic surface of the skin was clipped (approximately 10 x 8 cm) with an electric clipper (Aesculap - Germany). Care was taken to avoid abrading the skin, which could alter its permeability.

Test item application:
Based on the individual body weight, the test item at the dose of 200, 1000 and 2000 mg/kg body weight was weighed on an aluminium foil and made into a paste by adding sufficient volume (about 0.1, 0.3 and 0.5 mL respectively) of Milli-Q water and completely transferred on to the cotton gauze (size: Females: 8 x 5 cm of 6 ply) and applied directly (semi-occlusive) to the clipped skin of the rat to cover about 10% of body surface of the rat and secured in position by adhesive tape wound around the torso. The test item contact period with the skin was for 24 hours.
After the 24 hours contact period, the dressing was removed and the applied area was washed with deionized water and wiped dry using clean towel.

Duration of exposure:

24 hours

No. of animals per sex per dose:

2

Key result

Sex:

female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Mortality:

There were no clinical signs and pre-terminal deaths (mortality) observed during the study.

Body weight:

other body weight observations

Remarks:

All rats gained body weight throughout the observation period.

Other findings:

No abnormalities were detected during the necropsy.

Table 1. Individual body weight, body weight changes and pre-terminal deaths

Group and Dose (mg/kg body weight)	Rat No.	S e x	Body weight (g)					Pre-terminal deaths
			Initial (Day 1 - at treatment)	8^th   day	Weight change (day 8 – Initial)	15^th day	Weight change (day 15 – Initial)
G1 and 200 DRF	Rad2087	F	228.1	246.3	18.2	265.4	37.3	0
G2 and 1000 DRF	Rad2088	F	228.5	247.1	18.6	268.1	39.6	0
G3 and 2000 DRF	Rad2089	F	231.3	253.4	22.1	271.4	40.1	0
G3 and 2000 Main study	Rad2090	F	234.4	253.8	19.4	272.8	38.4	0
	Rad2091	F	233.8	250.6	16.8	271.6	37.8	0

 DRF: Dose Range Finding    F: Female

 

Table 2.Individual body weight, body weight changes and pre-terminal deaths

Dose range finding study

Group & Dose (mg/kg body weight)

Date and time of application

Rat Number

S e x

Initial Bwt (g)

Quantity (mg) applied

Observations and skin reaction

Days

1

2

3

4

5

30 min

1 h

2 h

3 h

4 h

5 h

6 h

*

Er @

Ed @

*

Er @

Ed @

*

Er @

Ed @

G1 and 200 DRF

24 November 2021 and 10:45AM

Rad2087

F

228.1

46.0

N

N

N

N

N

N

N

N

N

0

0

N

0

0

N

0

0

 

Group & Dose (mg/kg body weight)

Rat Number

S e x

Observation

Necropsy findings

Days

6

7

8

9

10

11

12

13

14

15

G1 and 200 DRF

Rad2087

F

N

N

N

N

N

N

N

N

N

N

NAD

F: Female              N: Normal           h: hour    min: minutes        NAD: No abnormality detected        Er: Erythema                       Ed: Edema  

Score 0: No Erythema / Edema  *: Clinical signs; @: Skin scoring as per Draize method (approximately 24, 48 and 72 hours) after test patch removal

 

Example:  body weight * dose /1000 = 45.62mg = round off 46 mg        

Table 2 contd. Individual test item application, clinical signs, skin reaction and necropsy findings

 

 

Dose range finding study

 

Group & Dose (mg/kg body weight)

Date and time of application

Rat Number

S e x

Initial Bwt (g)

Quantity (mg) applied

Observations and skin reaction

Days

1

2

3

4

5

30 min

1 h

2 h

3 h

4 h

5 h

6 h

*

Er @

Ed @

*

Er @

Ed @

*

Er @

Ed @

G2 and 1000 DRF

26 November 2021 and 10:46AM

Rad2088

F

228.5

229

N

N

N

N

N

N

N

N

N

0

0

N

0

0

N

0

0

 

Group & Dose (mg/kg body weight)

Rat Number

S e x

Observation

Necropsy findings

Days

6

7

8

9

10

11

12

13

14

15

G2 and 1000 DRF

Rad2088

F

N

N

N

N

N

N

N

N

N

N

NAD

F: Female              N: Normal           h: hour    min: minutes        NAD: No abnormality detected        Er: Erythema                       Ed: Edema  

Score 0: No Erythema / Edema  *: Clinical signs; @: Skin scoring as per Draize method (approximately 24, 48 and 72 hours) after test patch removal

 

Example:  body weight * dose /1000 = 228.5mg = round off 229 mg     

 

Table 2 contd. Individual test item application, clinical signs, skin reaction and necropsy findings

 

Dose range finding study

 

Group & Dose (mg/kg body weight)

Date and time of application

Rat Number

S e x

Initial Bwt (g)

Quantity (mg) applied

Observations and skin reaction

Days

1

2

3

4

5

30 min

1 h

2 h

3 h

4 h

5 h

6 h

*

Er @

Ed @

*

Er @

Ed @

*

Er @

Ed @

G3 and 2000 DRF

29 November 2021 and 10:47AM

Rad2089

F

231.3

463

N

N

N

N

N

N

N

N

N

0

0

N

0

0

N

0

0

 

Group & Dose (mg/kg body weight)

Rat Number

S e x

Observation

Necropsy findings

Days

6

7

8

9

10

11

12

13

14

15

G3 and 2000 DRF

Rad2089

F

N

N

N

N

N

N

N

N

N

N

NAD

F: Female              N: Normal           h: hour    min: minutes        NAD: No abnormality detected        Er: Erythema                       Ed: Edema  

Score 0: No Erythema / Edema  *: Clinical signs; @: Skin scoring as per Draize method (approximately 24, 48 and 72 hours) after test patch removal

 

Example:  body weight * dose /1000 = 462.6 = round off 463 mg      

     

Table 2 contd. Individual test item application, clinical signs, skin reaction and necropsy findings

Main study

 

Group & Dose (mg/kg body weight)

Date and time of application

Rat Number

S e x

Initial Bwt (g)

Quantity (mg) applied

Observations and skin reaction

Days

1

2

3

4

5

30 min

1 h

2 h

3 h

4 h

5 h

6 h

*

Er @

Ed @

*

Er @

Ed @

*

Er @

Ed @

G3 and 2000 (Main)

01 December 2021 and 10:50AM to 10:53AM

Rad2090

F

234.4

469

N

N

N

N

N

N

N

N

N

0

0

N

0

0

N

0

0

Rad2091

F

233.8

468

N

N

N

N

N

N

N

N

N

0

0

N

0

0

N

0

0

 

Group & Dose (mg/kg body weight)

Rat Number

S e x

Observations

Necropsy findings

Days

6

7

8

9

10

11

12

13

14

15

G3 and 2000 (Main)

Rad2090

F

N

N

N

N

N

N

N

N

N

N

NAD

Rad2091

F

N

N

N

N

N

N

N

N

N

N

NAD

F: Female              N: Normal           h: hour    min: minutes                        NAD: No abnormality detected        Er: Erythema       Ed: Edema  

Score 0: No Erythema / Edema           

*: Clinical signs; @: Skin scoring as per Draize method (approximately 24, 48 and 72 hours) after test patch removal

Example:  body weight * dose /1000 = 468.8 mg = round off 469 mg    

Interpretation of results:

Category 5 based on GHS criteria

Remarks:

The test item is classified as “Category 5 or Unclassified” (the dermal LD50 range of 2000 to 5000 mg/kg) 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 observed at 2000 mg/kg body weight

Conclusions:

Based on the present study results, the acute dermal LD50 of
Dried Sludge From Domestic Wastewater is greater than 2000 mg/kg body weight in female Wistar rats.

Executive summary:

The acute dermal toxicity of Dried Sludge from Domestic Wastewater was tested in 5 female wistar rats, in a stepwise study design, with an initial  3 animals tested in a dose range finding study and two additional animals used for the main study.

Based on the individual body weight, the test item at the dose of 200, 1000 and 2000 mg/kg body weight was weighed in aluminum foil and made into paste by adding 0.1, 0.3 and 0.5 mL of Milli-Q water respectively and mixed by using glass rod. and was completely transferred on to the cotton gauze (size: Females: 8 x 5 cm of 6 ply) and applied directly (semi-occlusive) to the clipped skin of the rat to cover about 10% of body surface of the rat.  It was secured in position by adhesive tape wound around the torso. The test item contact period with the skin was for 24 hours.

After the 24 hours contact period, the dressing was removed and the applied area was washed with deionized water and wiped dry using clean towels. 

All rats were observed for clinical signs of toxicity and mortality for 14 days post application. In addition, the treatment site was observed at 24, 48 and 72 hours after removal of test item using the Draize criteria. There were no clinical signs of toxicity and mortality. There was no skin reaction observed at test item applied area. Body weights were measured on days 1, 8 and 15 and all rats gained weight during experimental period. At the end of observation period, the animals were euthanized and subjected to necropsy. There were no abnormalities detected during the necropsy.

Based on the present study results, the acute dermal LD₅₀ of Dried Sludge from Domestic Wastewater is greater than 2000 mg/kg body weight in female Wistar rats.

The test item, Dried Sludge from Domestic Wastewater is classified as follows:

• The test item is classified as “Category 5” (the dermal LD₅₀ range of 2000 to 5000 mg/kg) 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 observed at 2000 mg/kg body weight.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LD50

Value:

> 2 000 mg/kg bw

Quality of whole database:

The study is a GLP compliant and has a Klimisch score 1.

Justification for classification or non-classification

Based on the available data, the substance is not classified.