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: dermal

Administrative data

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)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Materials and methods

Test guideline

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

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, 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.

Administration / exposure

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

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 (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.

Any other information on results incl. tables

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    

Applicant's summary and conclusion

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.