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: oral

Administrative data

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)

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 423 (Acute Oral toxicity - Acute Toxic Class Method)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test type:

acute toxic class method

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.

Test animals

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.

Administration / exposure

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

Results and discussion

Effect levels

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.

Any other information on results incl. tables

• 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

 

 

           

Applicant's summary and conclusion

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).