Sodium 2,4,5-trichlorobenzenesulphonate

Endpoint summary

Description of key information

The results of three GLP guideline studies to examine in vitro the key events of the adverse outcome pathway (AOP) for skin sensitisation are as follows:

- In chemico Direct Peptide Reactivity Assay (DPRA): the results were found to be inconclusive because the test item co-eluted with the Cysteine peptide. Even with this interference, the results suggest that the substance probably does not significantly react with the peptides used in the study and thus, by this method, would not have been regarded as a skin sensitiser.

- In vitro KeratinoSens™ Assay: the test item did not induce the luciferase activity in the transgenic KeratinoSens™ cell line in either of the two independent experimental runs. Therefore, the test item can be considered as a non-sensitizer according to theARE-Nrf2 Luciferase Test Method

- In vitro Human Cell Line Activation Test (h-CLAT): The test substance did not upregulate the expression of the CD54 or CD86 cell surface markers in at least two independent experimental runs and thus can be regarded as not a skin sensitiser in the h-CLAT assay.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

13-Feb-2018 thru 1 March 2018

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Analytical interference resulted in inconclusive results

Qualifier:

according to guideline

Guideline:

OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))

Version / remarks:

adopted: February 04, 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Justification for non-LLNA method:

Current REACH protocol requires In vitro / In chemico assessment before in vivo.

Specific details on test material used for the study:

Name: STB-FR (Benzenesulfonic acid, 2,4,5-Trichloro-, Sodium salt)
Batch No.: 1710-06
Chemical name: Sodium Trichlorobenzene Sulfonate
CAS No: 53423-65-7
Composition: 90.5% STB; 9.5% other
Molecular Weight: 283.494 g/mol
Purity: 90.5%
Physical State: solid powder
Colour: white
Stability: stable
Storage Conditions: at room temperature
Expiry Date: October 2019

Details on the study design:

The test substance was dissolved in distilled water : acetonitrile 1:1 (v/v), based on the results of pre-experiments. The molecular weight and purity of the mixture were derived from all components (excluding water). Based on the derived molecular weight of 283.494 g/mol a 100 mM stock solution was prepared. The test item preparations were tested in parallel by incubating the samples with the peptides containing either cysteine or lysine for 24 ± 2 h at 25 ± 2.5 °C. Subsequently, samples were analysed by HPLC analysis. All test item solutions were freshly prepared immediately prior to use. For the 100 mM stock solution of the test item no turbidity or precipitation was observed when diluted with the cysteine peptide solution. After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples were inspected for precipitation, turbidity or phase separation. No precipitation, turbidity or phase separation was observed for any of the samples. For the 100 mM stock solution of the test item no turbidity or precipitation was observed when diluted with the lysine peptide solution. After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples were inspected for precipitation, turbidity or phase separation. No precipitation, turbidity or phase separation was observed for the samples of the test item. Phase separation was observed for the samples of the co-elution of the positive control and the positive control. Samples were not centrifuged prior to the HPLC analysis.

Cinnamic aldehyde ((2E)-3-phenylprop-2-enal) was solved in acetonitrile and was used as positive control. A stock concentration of 100 mM was prepared and was included in every assay run for both peptides. Co-elution controls were set up in parallel to sample preparation but without the respective peptide solution. The controls were used to verify whether a test chemical absorbs at 220 nm and co-elutes with the cysteine or lysine peptide. The co-elution controls were prepared for every test item preparation and the positive control and were included in every assay run for both peptides. Reference controls (RCs) were set up in parallel to sample preparation in order to verify the validity of the test run.

19.65 mg cysteine peptide with an amino acid sequence of Ac-RFAACAA were pre-weighed in a vial and dissolved in a defined volume (38.10 mL) of a phosphate buffer with pH 7.5 to reach a concentration of 0.667 mM. 19.84 mg lysine peptide with an amino acid sequence of Ac-RFAAKAA were pre-weighed in a vial and dissolved in a defined volume of ammonium acetate buffer with pH 10.2 (37.68 mL) to reach a concentration of 0.667 mM. All peptides used for this study were stored at -80 °C and protected from light. Peptides were thawed only immediately prior to use.

Dose groups included 100 mM stock solutions of the test item and positive control and a solvent control.

A threshold of 6.38% average peptide depletion is used to support the discrimination between skin sensitisers and non-sensitisers. In case of co-elution of the test item with a peptide peak, the peak cannot be integrated correctly and the calculation of the PPD is not possible. If severe co-elution occurs with both peptides then the analysis is reported as "inconclusive". In cases where the co-elution occurs only with the lysine peptide prediction, a threshold of 13.89% can be used to support the discrimination between skin sensitisers and non-sensitisers.

Positive control results:

The acceptance criteria for the depletion range of the positive control were fulfilled.

Key result

Run / experiment:

other: Average

Parameter:

other: Mean Cysteine Peptide Depletion (%)

Value:

17.11

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

not determinable because of methodological limitations

Remarks:

Test item co-eluted with cysteine peptide

Key result

Run / experiment:

other: Average

Parameter:

other: Mean Lysine Peptide Depletion (%)

Value:

0

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Other effects / acceptance of results:

A mayor peak in the co-elution control of the test item was observed at the retention time of the cysteine peptide. Therefore, co-elution of test item and peptide occurred and the given peak areas do not properly reflect the amount of cysteine peptide present in the test item samples. Therefore, these values can not be used for evaluation.

Depletion of the Cysteine Peptide

Sample	Peptide Depletion (%)	Mean Peptide Depletion (%)	SD of Peptide Depletion (%)	CV of Peptide Depletion (%)
Positive Control	77.40	77.79	0.34	0.44
	77.95
	78.02
Sample	16.54	17.11	0.51	2.95
	17.48
	17.32

Depletion of the Lysine Peptide

Sample	Peptide Depletion (%)	Mean Peptide Depletion (%)	SD of Peptide Depletion (%)	CV of Peptide Depletion (%)
Positive Control	66.56	66.10	0.50	0.75
	66.16
	65.57
Sample	0.00	0.00	0.00	----
	0.00
	0.00

Interpretation of results:

other: Study is inconclusive due to co-elution of test item with Cysteine peptide.

Conclusions:

Study is inconclusive due to co-elution of test item with Cysteine peptide.

Executive summary:

In this GLP guideline study conducted according to OECD 442c using the in chemico Direct Peptide Reactivity Assay (DPRA), the results were found to be inconclusive because the test item co-eluted with the Cysteine peptide. Even with this interference, the results suggest that the substance probably does not significantly react with the peptides used in this study and thus, by this method, would not have been regarded as a skin sensitiser.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 April 2018 - 09 May 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD 442E (In VItro Skin Sensitisation h-CLAT)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: DB-ALM Protocol n°158 (Skin Sensitisation h-CLAT)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of dendritic cells

Justification for non-LLNA method:

Current REACH protocol requires In vitro / In chemico assessment before in vivo.

Specific details on test material used for the study:

Name: STB-FR
Batch No.: 1710-06
Chemical name: Benzenesulfonic acid, 2,4,5-trichloro-, sodium salt
CAS No: 53423-65-7
Composition: 90.5% STB; 9.5% other
Molecular Weight: 283.494 g/mol
Purity: 90.5%
Physical State: solid powder
Colour: white
Log KOW: not specified by the sponsor
Stability: stable
Storage Conditions: at room temperature
Expiry Date: October 2019

Details on the study design:

The in vitro human cell line activation test (h-CLAT) enables detection of the sensitising potential of a test item by addressing the third molecular key event of the adverse outcome pathway (AOP), namely dendritic cell (DC) activation, by quantifying the expression of the cell surface markers CD54 and CD86 in the human monocytic cell line THP-1. The expression of the cell surface markers compared to the respective solvent controls is used to support discrimination between skin sensitiser and non-sensitisers. Prior to the main study the cell batch was checked for its reactivity towards known positive and negative controls and was found to be acceptable for further testing. In this study, the test substance was dissolved in DMSO. For the dose finding assay stock solutions with concentrations ranging from 500.00 mg/mL to 3.91 mg/mL were prepared by a serial dilution of 1:2. Cells were incubated with the test item for 24 h at 37°C. After exposure cells were stained with propidium iodide and cell viability was measured by FACS analysis.

The test meets acceptance criteria if:
• the cell viability of the solvent controls is >90%,
• the cell viability of at least four tested doses of the test item in each run is >50%,
• the RFI values of the positive control (DNCB) is ≥150% for CD86 and ≥200% for CD54 at a cell viability of >50%,
• the RFI values of the solvent control is not ≥150% for CD86 and not ≥200% for CD54,
• the MFI ratio of CD86 and CD54 to isotype IgG1 control for the medium and DMSO control, is >105%.

Sensitising potential of the test item was predicted from the mean percentage expression of CD86 and CD54. Any test chemical tested by the h-CLAT was considered positive if the RFI of CD86 was equal to or greater than 150% at any tested dose at a cell viability ≥ 50% in at least two independent runs or if the RFI of CD54 was equal to or greater than 200% at any tested dose at a cell viability ≥ 50% in at least two independent runs or if the RFIs of both the CD86 and CD54 were equal to or are greater than 150% and 200% respectively at any tested dose at a cell viability ≥ 50% in at least two independent runs. In case of not concordant results a third run would be conducted to make the final prediction. Otherwise the results were considered as inconclusive.
A negative test result of a test item was only accepted if the cell viability at a concentration of 1.2 x CV75 is <90%. In contrast, a positive test outcome was accepted irrespective of cell viabilities >90% at a concentration of 1.2 x CV75. If no CV75 could be derived negative test results can be accepted when the test item is tested at the highest soluble concentration (5000 µg/mL for 0.9% NaCl solution; 1000 µg/mL for DMSO or a different organic solvent) even if the cell viability is >90%.

Positive control results:

The acceptance criteria for the positive control were fulfilled.

Key result

Run / experiment:

other: 1

Parameter:

other: CD86 Relative Flourescence Intensity (RFI, %)

Value:

81

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Run / experiment:

other: 2

Parameter:

other: CD86 Relative Flourescence Intensity (RFI, %)

Value:

95

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Run / experiment:

other: 1

Parameter:

other: CD54 Relative Flourescence Intensity (RFI, %)

Value:

103

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Run / experiment:

other: 2

Parameter:

other: CD54 Relative Flourescence Intensity (RFI, %)

Value:

114

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Other effects / acceptance of results:

No cytotoxic effects were observed for the cells treated with the test item. Relative cell viability at the highest test item concentration was reduced to 92.4% (CD86), 91.8% (CD54) and 91.5% (isotype IgG1 control) in the first experiment and to 87.6% (CD86), 87.0% (CD54) and 85.4% (isotype IgG1 control) in the second experiment. The expression of the cell surface marker CD86 was not upregulated above the threshold of 150% in any of the experiments. The expression of cell surface marker CD54 was not upregulated above the threshold of 200% in any of the experiments. Therefore, the test item is not considered to be a skin sensitiser. The controls confirmed the validity of the study for all experiments.

CD54 and CD86 Expression Experiment 1

Sample	Conc. [μg/mL]	Cell Viability [%]			Mean Fluorescence Intensity			corrected Mean Fluorescence Intensity		Relative Flourescence Intensity (RFI)		Ratio Isotype IgG1 to [%]
		CD86	CD54	Isotype IgG1	CD86	CD54	Isotype IgG1	CD86	CD54	CD86	CD54	CD86	CD54
Medium Control	-	94.8	94.2	94.2	4504	1496	854	3650	642	89	86	527	175
Solvent Control	0.20%	93.4	93.6	93.6	4829	1457	712	4117	745	100	100	678	205
DNCB	4.00	82.0	82.8	82.3	11458	2405	702	10756	1703	261	229	1632	343
STB-FR	1000	92.4	91.8	91.5	4088	1599	1022	3066	577	74	77	400	156
	833.33	92.3	92.5	92.4	3846	1601	945	2901	656	70	88	407	169
	694.44	89.0	88.7	89.3	4176	1692	924	3252	768	79	103	452	183
	578.70	90.8	91.3	91.9	4196	1700	958	3238	742	79	100	438	177
	482.25	91.1	91.5	90.4	4073	1569	924	3149	645	76	87	441	170
	401.88	91.9	91.9	92.0	4052	1567	904	3148	663	76	89	448	173
	334.90	92.6	93.5	93.3	3854	1535	923	2931	612	71	82	418	166
	279.08	92.6	93.4	92.7	4255	1596	924	3331	672	81	90	460	173

CD54 and CD86 Expression Experiment 2

Sample	Conc. [μg/mL]	Cell Viability [%]			Mean Fluorescence Intensity			corrected Mean Fluorescence Intensity		Relative Flourescence Intensity (RFI)		Ratio Isotype IgG1 to [%]
		CD86	CD54	Isotype IgG1	CD86	CD54	Isotype IgG1	CD86	CD54	CD86	CD54	CD86	CD54
Medium Control	-	95.6	94.8	94.8	3501	1395	752	2749	643	90	110	466	186
Solvent Control	0.20%	94.0	94.4	95.0	3793	1314	731	3062	583	100	100	519	180
DNCB	4.0	83.1	83.1	82.1	9665	2532	1064	8601	1468	281	252	908	238
STB-FR	1000.00	87.6	87.0	85.4	3545	1433	924	2621	509	86	87	384	155
	833.33	90.2	88.8	89.0	3429	1570	993	2436	577	80	99	345	158
	694.44	91.1	91.0	91.1	3726	1476	892	2834	584	93	100	418	165
	578.70	91.2	92.5	92.6	3756	1432	853	2903	579	95	99	440	168
	482.25	94.3	92.4	93.3	3695	1574	907	2788	667	91	114	407	174
	401.88	93.7	93.5	93.4	3707	1441	849	2858	592	93	102	437	170
	334.90	93.4	93.7	93.3	3723	1405	835	2888	570	94	98	446	168
	279.08	94.0	94.5	93.5	3430	1344	839	2591	505	85	87	409	160

Interpretation of results:

GHS criteria not met

Conclusions:

The test substance was non-sensitising in the in vitro human cell line activation test (h-CLAT) assay.

Executive summary:

In this GLP guideline study conducted according to OECD 442E "In vitro Skin Sensitisation assays addressing the Key Event on activation of dendritic cells on the Adverse Outcome Pathway for Skin Sensitisation" human cell line activation test (h-CLAT) to assess a key event associated with skin sensitisation, namely dendritic cell activation, the test substance did not upregulate the expression of the cell surface marker in at least two independent experimental runs and thus can be regarded as not a skin sensitiser in the h-CLAT assay.

Reference 3

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

13 February 2018 - 12 April 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Justification for non-LLNA method:

Current REACH protocol requires In vitro / In chemico assessment before in vivo.

Specific details on test material used for the study:

Name: STB-FR
Batch No.: 1710-06
Chemical name Sodium Trichlorobenzene Sulfonate
CAS No 53423-65-7
Composition: 90.5% STB
9.5% other
Molecular Weight: 283.494 g/mol
Purity 90.5%
Physical State: solid powder
Color: white
Stability: stable
Storage Conditions: at room temperature
Expiry Date: October 2019

Details on the study design:

The in vitro KeratinoSens™ assay enables detection of the sensitising potential of a test item by addressing the second molecular key event of the adverse outcome pathway (AOP), namely activation of keratinocytes, by quantifying the luciferase activity in the transgenic cell line KeratinoSens™. The luciferase activity, assessed by luminescence measurement, compared to the respective solvent controls is used to support discrimination between skin sensitisers and non-sensitisers. In the present study the test item was dissolved in DMSO. Based on a molecular weight of 283.494 g/mol a stock solution of 200 mM was prepared. Based on the stock solution a set of twelve master solutions in 100% solvent was prepared by serial dilution using a constant dilution factor of 1:2. These master solutions were diluted 1:100 in cell culture medium. The following concentration range was tested in the assay: 2000, 1000, 500, 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91, 1.95, 0.98 µM. Cells were incubated with the test item for 48 h at 37°C. After exposure cells were lysed and luciferase activity was assessed by luminescence measurement. A blank, a negative control and a positive control were set up in parallel in order to confirm the validity of the test. A blank well with no seeded cells was included in every plate to determine the background. The well was incubated with the negative control. DMSO (AppliChem; Lot No.: 0001055932) at a final concentration of 1% (v/v) in test item exposure medium was used as negative control. Six wells were included in every testing plate. The preparation of the negative control was carried out analogous to the test item. Cinnamic aldehyde (CA, (2E)-3-phenylprop-2-enal; CAS 104-55-2; >98%; Alfa Aesar; Lot No.: 10176010) was used as positive control. CA was dissolved in DMSO (AppliChem; Lot No.: 0001055932) at a concentration of 6.4 mM and was further diluted four times with a constant dilution factor of 1:2 resulting in a concentration range of 0.4 mM – 6.4 mM. The following preparation of the positive control was carried out analogous to the preparation of the test item, resulting in a final concentration range of 4 µM – 64 µM. The final concentration of the solvent DMSO was 1% (v/v) for all wells. The test was carried out using the transgenic cell line KeratinoSens™ (Givaudan, Switzerland), a cell line derived from human keratinocytes (HaCaT) transfected with a stable insertion of the Luciferase construct. Cells from frozen stock cultures, tested routinely for mycoplasma, were seeded in culture medium at an appropriate density and were used for routine testing. Only cells at a low passage number <25 (P 6 in experiment 1; P 9 in experiment 2) were used. Cells were cultured in 75 cm2 culture flasks (Greiner) in maintenance medium at 37  1°C and 5% CO2 in a humidified incubator. For test item exposure, cells were cultured in medium for test item exposure.

The test meets acceptance criteria if:
- the luciferase activity induction of the positive control is statistically significant above the threshold of 1.5 (using a t-test) in at least one of the tested concentrations
- the average induction in the three technical replicates for the positive control at a concentration of 64 µM is between 2 and 8
- the EC1.5 value of the positive control is within two standard deviations of the historical mean
- the average coefficient of variation (CV; consisting of 6 wells) of the luminescence reading for the negative (solvent) control DMSO is <20% in each repetition.

Positive control results:

The acceptance criteria for the positive control were fulfilled.

Key result

Run / experiment:

other: 1

Parameter:

other: Induction of Luciferase Activity

Value:

1.5

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Run / experiment:

other: 2

Parameter:

other: Induction of Luciferase Activity

Value:

1.5

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Run / experiment:

other: Overall

Parameter:

other: Induction of Luciferase Activity

Value:

1.5

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Other effects / acceptance of results:

In the first experiment, no significant luciferase induction > 1.5 was found in the tested concentration range. Therefore, no EC1.5 value could be calculated. In the second experiment, no significant luciferase induction > 1.5 was found in the tested concentration range. Therefore, no EC1.5 value could be calculated. No dose response for luciferase activity induction was observed for each individual run as well as for an overall luciferase activity induction. Under the condition of this study the test item is therefore considered as non sensitiser. The controls confirmed the validity of the study.

Induction of Luciferase Activity – Overall Induction

	Concentration [µM]	Fold Induction				Significance
		Experiment 1	Experiment 2	Mean	SD
Solvent Control	-	1.00	1.00	1.00	0.00	-
Positive Control	4.00	1.02	1.07	1.04	0.03
	8.00	1.05	1.27	1.16	0.15
	16.00	1.35	1.24	1.30	0.07
	32.00	1.62	1.77	1.70	0.10	*
	64.00	3.67	2.79	3.23	0.62	*
Test Item	0.98	1.09	1.42	1.25	0.23
	1.95	0.98	1.19	1.08	0.15
	3.91	0.95	1.12	1.04	0.12
	7.81	1.01	1.02	1.02	0.01
	15.63	1.00	1.01	1.00	0.01
	31.25	0.93	0.96	0.94	0.02
	62.50	0.77	0.81	0.79	0.03
	125.00	0.69	0.94	0.81	0.18
	250.00	0.62	0.69	0.66	0.05
	500.00	0.57	0.56	0.57	0.01
	1000.00	0.52	0.74	0.63	0.16
	2000.00	0.63	0.63	0.63	0.00

* = significant induction according to Student’s t-test, p<0.05

Interpretation of results:

GHS criteria not met

Conclusions:

The test item was considered as a non-sensitizer according to theARE-Nrf2 Luciferase Test Method.

Executive summary:

In this GLP guideline study conducted according to OECD 442D,the test item did not induce the luciferase activity in the transgenic KeratinoSens™ cell line in either of the two independent experimental runs. Therefore, the test item can be considered as a non-sensitizer according to theARE-Nrf2 Luciferase Test Method.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on a weight of the evidence from three GLP guideline studies to examine in vitro the key events of the adverse outcome pathway (AOP) for skin sensitisation using the DPRA, KeratinoSens, and h-CLAT assays, no indication of skin sensitisation was observed and, therefore, the substance can be regarded as not meeting the GHS criteria for classification as a skin sensitizer. There are no data to fully assess the potential for respiratory sensitisation but there are no indications that respiratory sensitisation should be expected.