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.

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 13 July 2022 to 20 July 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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 429 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

Adopted 22th July 2010

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

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.

In vivo test system

Test animals

Species:

mouse

Strain:

Balb/c

Sex:

female

Study design: in vivo (LLNA)

Vehicle:

other: - Vehicle control groups: i) 4:1 Acetone-Olive Oil (AOO) ii) sesame oil iii) water

Concentration:

0.2g of test material per ml of solvent at (37 ± 1) °C for (72 ± 2) h

No. of animals per dose:

5

Details on study design:

Method
The LLNA identifies contact allergens as a function of proliferative responses induced in draining lymph nodes following topical exposure of mice to test chemicals. The mechanistic basis for selection of this endpoint is that the acquisition of skin sensitization is dependent upon, and correlates quantitatively with, T lymphocyte proliferation induced in regional lymph nodes draining the site of encounter with a contact allergen. Using the standard LLNA for the purposes of hazard identification, chemicals are classified as contact allergens if they elicit, at one or more test concentrations, a three-fold or greater increase in draining lymph node cell (LNC) proliferation compared with concurrent vehicle controls (a stimulation index [SI] of 3 or more).

Test Procedures
The experimental design of the assay is as follows:
Exaggerated extraction was performed in clean, chemically inert, closed containers with minimum dead space using 0.2g of test material per ml of solvent at (37 ± 1) °C for (72 ± 2) h; Double-distilled water was chosen as the polar solvent, whereas refined sesame oil as the non-polar solvent.
Τhe treatment groups, which consist of at least five mice, were the following:
- Vehicle control groups: i) 4:1 Acetone-Olive Oil (AOO) ii) sesame oil iii) water
- Positive control group (25% w/w α-Hexyl cinnamaldehyde in 4:1 AOO)
- Test material non-polar extract (aqueous)
- Test material polar extract (sesame oil)
A dose of 25μL/day of test material / positive control solution/ solvents was applied to the dorsal side of both ears of designated mice for three consecutive days.
Observation period: Daily (from Day 0 to Day 6)
Observations: Mortality, morbidity: at least once daily. Clinical signs, irritation: individual observation at least once daily for 6 days; body weight: on Day 1 and Day 6.
The protocol for determining the level of lymph node cell proliferation is described in ASOP and is in accordance with OECD 429 guideline.

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Results and discussion

Positive control results:

Concurrent positive control’s SI index was 3.60. The value was in accordance with the laboratory historical data of positive control.

In vivo (LLNA)

Results

Key result

Parameter:

SI

Value:

< 3

Test group / Remarks:

Test group’s SI (non-polar extract) 0.95
Test Group’s SI (polar extract) 1.20

Remarks on result:

no indication of skin sensitisation based on QSAR/QSPR prediction

Any other information on results incl. tables

Appendix 1

	NEGATIVE CONTROL GROUP		POSITIVE CONTROL GROUP
Mouse ID #	Individual dpm
	raw data	w/o blank	raw data	w/o blank
1	7259.71	4646.415	15704.8	13091.505
2	6253.59	3640.295	16701.4	14088.105
3	5821.47	3208.175	13178	10564.705
4	7632.12	5018.825	17301.2	14687.905
5	4286.66	1673.365
	Average dpm
	3637.415		13108.055
	Standard Deviation
	1319.860055		1818.912418
	Stimulation Index (SI)
	N/A		3.603673213
	VEHICLE CONTROL GROUP		TEST GROUP
	SESAME OIL		OILY EXTRACT
Mouse ID #	Individual dpm
	raw data	w/o blank	raw data	w/o blank
1	13185.7	10572.405	10408.6	7795.305
2	12738.9	10125.605	10502.1	7888.805
3	12995.1	10381.805	15754.2	13140.905
4	12885.9	10272.605	12000.9	9387.605
5	11705.4	9092.105
	Average dpm
	10088.905		9553.155
	Standard Deviation
	580.5741727		2500.630215
	Stimulation Index (SI)
	N/A		0.946897111
	The product is not considered a sensitizer.
	VEHICLE CONTROL GROUP		TEST GROUP
	WATER		AQUEOUS EXTRACT
Mouse ID #	Individual dpm
	raw data	w/o blank	raw data	w/o blank
1	6204.12	3590.825	4440.21	1826.915
2	5402.73	2789.435	5435	2821.705
3	5333.77	2720.475	6226.17	3612.875
4	3221.94	608.645	5731.25	3117.955
5			5869.84	3256.545
	Average dpm
	2427.345		2927.199
	Standard Deviation
	1275.197993		677.6359791
	Stimulation Index (SI)
	N/A		1.205926228
	The product is not considered a sensitizer.

 

Appendix 2

 

		Weight
Group	Mouse	Day 1			Day 6
1 (Negative Control)	1	20.68			21.6
	2	21.35			22.27
	3	20.15	AVERAGE	20.756	20.86	AVERAGE	22.012
	4	20.9	SD	0.387226	22.36	SD	0.721426
	5	20.7	% VARIATION	1.87%	22.97	% VARIATION	3.28%
2 (Positive Control)	1	21.94			22.26
	2	20.1			21.22
	3	22.41	AVERAGE	21.81	22.99	AVERAGE	22.236
	4	22.05	SD	0.883878	21.72	SD	0.698014
	5	22.55	% VARIATION	4.05%	22.99	% VARIATION	3.14%
3 (Aqueous Excipient)	1	22.09			20.11
	2	20.68			22.1
	3	21.39	AVERAGE	21.338	22	AVERAGE	21.672
	4	21.3	SD	0.450395	22.56	SD	0.839748
	5	21.23	% VARIATION	2.11%	21.59	% VARIATION	3.87%
4 (Oily Excipient)	1	20.92			21.34
	2	22.18			20.45
	3	23.11	AVERAGE	21.698	23.82	AVERAGE	21.664
	4	21.06	SD	0.832692	21.53	SD	1.13836
	5	21.22	% VARIATION	3.84%	21.18	% VARIATION	5.25%
5 (Aqueous Extract)	1	21.73			21.8
	2	23.13			24.67
	3	20.84	AVERAGE	22.442	20.44	AVERAGE	22.658
	4	25	SD	1.480451	24.69	SD	1.718713
	5	21.51	% VARIATION	6.60%	21.69	% VARIATION	7.59%
6 (Oily Extract)	1	23			23.54
	2	22			22.1
	3	23.67	AVERAGE	23.286	24.26	AVERAGE	23.778
	4	23.69	SD	0.729509	23.75	SD	1.024
	5	24.07	% VARIATION	3.13%	25.24	% VARIATION	4.31%
AVERAGE		21.88833			22.33667
SD		1.185943			1.303954
% VARIATION		5.42%			5.84%

 

T-test analysis: Mice weights on Day 6 vs Day 1

Group	Day 6 vs Day 1
	Mean of differences	95% CI	P value
All	0.4483	0,1227 to 0,7740	0.0087	↑
1 (Negative Control)	1.256	0,4722 to 2,040	0.0113	↑
2 (Positive Control)	0.426	-0,2217 to 1,074	0.1419	-
3 (Aqueous Excipient)	0.334	-1,363 to 2,031	0.6137	-
4 (Oily Excipient)	-0.034	-1,258 to 1,190	0.9423	-
5 (Aqueous Extract)	0.216	-0,7521 to 1,184	0.5692	-
6 (Oily Extract)	0.492	-0,06740 to 1,051	0.0711	-

 

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

“Dried sludge from domestic wastewater” should not be considered as a skin sensitizer according to the results of Murine Local Lymph Node Assay.

Executive summary:

The purpose of this study was to evaluate the skin sensitization potential of the product “Dried sludge from domestic wastewater” intended to be used as fuel. For this purpose, Local Lymph Node Assay (LLNA) was performed. LLNA is a validated and accepted method used for the identification of skin sensitizing chemicals. It is the initial requirement for sensitization testing within the new REACH (Registration, Evaluation, Authorization and Restriction of Chemical substances) regulations in the European Union. Due to its high dissolubility, exaggerated extraction with a polar and and a non polar solvent was performed. Extracts were applied to the dorsum of mice ears and lymph node proliferation was assessed by measuring the level of radioactivity. Both extracts of the product exhibited Stimulation Index < 3 and the product should not be considered as a skin sensitizer according to the results of Murine Local Lymph Node Assay.