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

The sensitisation potential of the UVCB -Dried Sludge from domestic wastewater was tested in vitro and in vivo:

• OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 luciferase KeratinoSens™


• OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)  

The results of the in vitro skin sensitisation test were inconclusive due to the solubility properties of the substance and therefore the second in vitro skin sensitisation test (OECD TG 442E - h-CLAT method) was waived as inconclusive results were also expected for the same reason. The test item did not show any sensitisation potential in the in vivo test conducted.

In Vitro Skin Sensitisation Test:

An in vitro skin sensitisation test according to OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 luciferase KeratinoSens™ test method was performed to assess the sensitisation potential of Dried Sludge from domestic wastewater.

The test item was diluted in DMSO. Maximal final tested concentrations of the test item were 12.5 µg/mL in the 1st experiment, 400 µg/mL in the 2nd experiment and 300 µg/mL in the 3rd experiment. In each experiment there were used KeratinoSens Assay Ready Cells, which do not need to be cultivated before experiments. The KeratinoSensTM cells were seeded into two 96-well plates. Three independent experiments were performed. All experiments fulfilled acceptance criteria and they were used for evaluation. In all experiments, 12 test item concentrations were tested. The concentration range of the test item was (final tested concentrations): 0.006 – 12.5 µg/mL, 0.2 – 400 µg/mL and 0.2 – 300 µg/mL. All experiments included corresponding positive controls (PC = Cinnamic aldehyde), negative controls (NC = untreated wells with cells and 1% DMSO) and blank of the negative controls (BL = untreated wells without cells with 1% DMSO).

Under the experimental design described above, the luciferase induction at the highest non-cytotoxic concentration is < 1.5 threshold in all acceptable experiments, EC1.5 is < 200 µg/mL and there is no clear-dose response. However, the test item is not fully soluble and the maximal tested concentrations of the test item were tested as a stable dispersions or soluble parts of solution. The result of the testing of the test item, Dried Sludge from domestic wastewater, was inconclusive predicted by the ARE Nrf2 Luciferase KeratinoSens™ Test Method.

Therefore, the effect of the test item Dried Sludge from domestic wastewater predicted by the ARE Nrf2 Luciferase Test Method to sensitisation potential was inconclusive.

In Vivo Skin Sensitisation Test:

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.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

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

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)

Species:

mouse

Strain:

Balb/c

Sex:

female

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)

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.

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

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	-

 

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

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

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information:

There is no information available for respiratory sensitisation. Therefore, there is a data gap in this respect. However, the data gap cannot be fulfilled with experimental data, since there is no internationally accepted animal model for respiratory sensitisation. 

The LLNA is a fully validated method for the identification of skin sensitizers, but it has been found that most, if not all, chemical respiratory sensitizers elicit a positive response on this assay (Arts et al. 2008; Dearman et al. 2013). Since the assay is based on the ability of the allergen to stimulate the activation and the proliferation of T lymphocytes in the draining lymph node, it is not surprising that both skin and respiratory chemical sensitizers result positive in the LLNA as both skin and respiratory chemical sensitizers are able to trigger T cell response. Skin and respiratory sensitizers only differ from the quality of the T cell response by inducing a TH1 or TH2 response, respectively. Therefore, LLNA is not able to discriminate skin from respiratory sensitizers, but this assay could be useful to determinate whether a chemical is able to induce sensitization. A chemical failing to induce a positive response in the LLNA could be safely regarded as lacking ability to induce both skin and
respiratory sensitization (Basketter et al. 2017). Based on the negative results of skin sesnitization, dried sludge from domestic water is not expected to have any respiratory sesnisitazing potential.

In case human data for respiratory sensitisation emerges, this will be taken into account.