Sodium hydrogen m-sulphonatobenzoate

Endpoint summary

Description of key information

OECD 442E

An in vitro study (according to OECD 442E) was performed to assess the sensitising potential of the test item sodium 3-sulfobenzoate by quantifying changes in the expression level of the two cell surface markers CD86 and CD54 which are associated with the process of activation of monocytes and dendritic cells.

Two valid experiments with a treatment period of 24 hours were performed. For the experiments, the highest nominal applied concentration (1000 µg/mL) was chosen based on the results obtained in the pre-test and the insolubility of the test item at any higher concentration. A geometric series (factor 1.2) of 7 dilutions thereof was prepared.

As solvent control for the test item, RPMI 1640 was used in a final concentration of 1 % in culture medium.

As positive control, 2.4-dinitrochlorobenzene (DNCB. CAS n. 97-00-7. ≥ 99% purity) was used.

Prior to the study, the cells used for the experiments were checked in a reactivity check and were found to be suitable for the experiments.

All acceptance criteria were met and therefore, the study was considered valid.

In both experiments, no cytotoxic effect was detected in all tested concentrations. The viability was ≥ 90 %. In both experiments the RFI of CD86 was not ≥ 150 % and the RFI of CD54 was not ≥ 200 % at any tested concentration. Therefore, in accordance to the classification criteria the result of this study is negative.

In conclusion, it can be stated that under the experimental conditions of this study, the test item, sodium 3-sulfobenzoate was negative in the h-CLAT and is therefore considered not having the potential to activate dendritic cells and therefor is a non-sensitizer.

OECD 442C

This study is performed in order to estimate the skin sensitisation potential of sodium 3 -sulfobenzoate using a peptide model.

The direct peptide reactivity assay (DPRA) is an in chemico assay to quantify the reactivity of the test item towards cysteine and lysine containing peptides. This reactivity is related to the skin sensitisation potential.

To quantify the sensitisation potential, the depletion of the cysteine and lysine containing peptides caused by known amounts of the test item is measured using HPLC. As a results no skin sensitisation potential has been identified for sodium 3-sulfobenzoate.

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:

key study

Study period:

2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

04. Feb. 2015

Deviations:

yes

Remarks:

Please, see all deails in field "Principles of method if other than guideline".

Principles of method if other than guideline:

Deviations from the Guideline
- No historical data were available for the positive control 2,3-butanedione during the experimental phase of the study. This was considered uncritical because 2,3-butanedione is one of the proficiency chemicals and was shown to be suitable during in-house proficiency testing.
- The planned buffer concentrations for dissolution of the Cys-peptide and Lys-peptide were 25 mM instead of 100 mM. This was considered uncritical, because the buffer strength was sufficient, confirmed by the positive controls.
- The phosphate buffer used for dissolution of the Cys-peptide was prepared by dis-solving disodium hydrogen phosphate dihydrate and adjusting the pH by using NaOH instead of using sodium dihydrogen phosphate monohydrate and disodium hydrogen phosphate heptahydrate in combination. This was considered uncritical because the same ions are formed irrespective of the used phosphate salt, and the pH was adjusted to the correct value.
- Due to an error on the protocol for the co-elution controls of the Lys-peptide, there was 200 μL acetonitrile and 50 μL test item stock solution in the samples instead of only 250 μL test item stock solution. This was considered uncritical, co-elution with the test item could be excluded also with the lower amount of test item.
The deviations were assessed and signed by the study director on 03. Jun. 2019.

GLP compliance:

yes (incl. QA statement)

Type of study:

other: direct peptide reactivity assay (DPRA)

Justification for non-LLNA method:

According to Annex VII of REACH Regulation.

Specific details on test material used for the study:

Name sodium 3-sulfobenzoate
Batch no. 170103
CAS No. 17625-03-5
Purity > 99.0% (Titration with NaOH, Glass indicating electrode, Calomel reference electrode)
Expiry date 1 4. Feb. 2019
Storage Room Temperature (20 ± 5°C), keep away from humidity

Details on the study design:

The direct peptide reactivity assay (DPRA) is an in chemico assay to quantify the reactivity of the test item towards cysteine and lysine containing peptides. This reactivity is related to the skin sensitisation potential.
To quantify the sensitisation potential, the depletion of the cysteine and lysine containing peptides caused by known amounts of the test item is measured using HPLC.
The assay is used for supporting the discrimination between skin sensitizers (i.e. UN GHS Category 1) and non-sensitizers in accordance with the UN GHS. A categorization in the sub-categories 1 A and 1 Bis not possible.

Positive control results:

The mean peptide depletion and standard deviation of the three replicates of the positive control cinnamaldehyde were in the acceptable range of 60.8 – 100.0 %
and ≤ 14.9 %, respectively, for the Cys-peptide.
The mean peptide depletion and standard deviation of the three replicates of the positive control 2,3-Butanedione were in the acceptable range of 10.0 – 45.0 % and ≤ 11.6 %, respectively, for the Lys-peptide.

Run / experiment:

other: Test item Rep. 1

Parameter:

other: Lys-Peptide depletion %

Value:

0.18

Vehicle controls validity:

not applicable

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Run / experiment:

other: Test item Rep. 2

Parameter:

other: Lys-Peptide depletion %

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Run / experiment:

other: Test item Rep. 3

Parameter:

other: Lys-Peptide depletion %

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Run / experiment:

other: Test item mean value

Parameter:

other:

Value:

0.06

Vehicle controls validity:

not applicable

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Run / experiment:

other: Test item Rep. 1

Parameter:

other: Cys-Peptide depletion %

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Run / experiment:

other: Test item Rep. 2

Parameter:

other: Cys-Peptide depletion %

Value:

0.58

Vehicle controls validity:

not applicable

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Run / experiment:

other: Test item Rep. 3

Parameter:

other: Cys-Peptide depletion %

Value:

1.45

Vehicle controls validity:

not applicable

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Run / experiment:

other: Test item mean value

Parameter:

other: Cys-Peptide depletion %

Value:

0.68

Vehicle controls validity:

not applicable

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Other effects / acceptance of results:

Mean peptide depletion % = 0.37
Cysteine 1:10/lysine 1:50 reactivity class: Minimal - Prediction negative

Mean depletion of both peptides after incubation with the test item: 0.37 %

Acceptance criteria

a) The mean peptide depletion value for the positive control cinnamaldehyde should be 60.8 % - 100 % with a maximum standard deviation (SD) of < 14.9 % for the Cys-peptide.

b) The mean peptide depletion value for the positive control 2,3-butanedione should be 10 % - 45 % with a maximum standard deviation < 11.6 % for the Lys-peptide.

c) The standard deviation for the test item replicates should be < 14.9 % for the per-cent cysteine depletion and < 11.6 % for the percent lysine depletion

Assessment

a) The mean peptide depletion and standard deviation of the three replicates of the positive control cinnamaldehyde were in the acceptable range of 60.8 – 100.0 % and ≤ 14.9 %, respectively, for the Cys-peptide.

b) The mean peptide depletion and standard deviation of the three replicates of the positive control 2,3-Butanedione were in the acceptable range of 10.0 – 45.0 % and ≤ 11.6 %, respectively, for the Lys-peptide.

c) The standard deviation for the test item replicates was < 14.9 % for the percent cysteine depletion for the test item. The standard deviation for the test item replicates was < 11.6 % for the percent lysine depletion for the test item.

Interpretation of results:

GHS criteria not met

Conclusions:

The mean peptide depletion in the Lys-peptide and Cys-peptide assay was 0.37 %, there-fore the test item was classified with:
DPRA Prediction: Negative
Reactivity class: Minimal

Executive summary:

This study is performed in order to estimate the skin sensitisation potential of sodium 3 -sulfobenzoate using a peptide model.

The direct peptide reactivity assay (DPRA) is an in chemico assay to quantify the reactivity of the test item towards cysteine and lysine containing peptides. This reactivity is related to the skin sensitisation potential.

To quantify the sensitisation potential, the depletion of the cysteine and lysine containing peptides caused by known amounts of the test item is measured using HPLC. As a results no skin sensitisation potential has been identified for sodium 3 -sulfobenzoate.

The mean peptide depletion in the Lys-peptide and Cys-peptide assay was 0.37 %, there-fore the test item was classified with:

- DPRA Prediction: Negative

- Reactivity class: Minimal.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD Guideline for the Testing of Chemicals, Part 442E: In Vitro Skin Sensitisation: human Cell Line Activation Test (h-CLAT)

Version / remarks:

adopted 29. July 2016

Deviations:

yes

Remarks:

In the positive control of the pre-test only 9865 instead of 10000 viable cells were measured. The lower cell count was probably caused by a mistake during vacuuming the supernatant when washing the cells.

Qualifier:

according to guideline

Guideline:

other: OECD. (2012). The Adverse Outcome Pathway for Skin Sensitisation Initiated by Covalent Binding, Part 1: Scientific Evidence. Series on Testing and Assessment No. 168, Paris.

Deviations:

not applicable

Qualifier:

according to guideline

Guideline:

other: Bauch, C., Kolle, S. N., & Ramirez, T. (2012). Putting the parts together: combining in vitro methods to test for skin sensitizing potentials. Regulation of Toxicology and Pharmacology, 63, 489–504.

Deviations:

not applicable

Principles of method if other than guideline:

This in vitro study was performed to assess the sensitising potential of the test item sodium 3-sulfobenzoate by quantifying changes in the expression level of the two cell surface markers CD86 and CD54, which are associated with the process of activation of monocytes and dendritic cells. For this purpose the human monocytic leukaemia cell line THP-1 was used and treated with the test item for 24 h before evaluation. The measured expression levels were used to distinguish potential skin sensitizers from non-sensitizers.
The h-CLAT addresses the third key event of the skin sensitisation adverse outcome pathway (AOP) that was defined by the OECD in 2012 and is a part of the AOP-based “two out of three” skin sensitisation integrated testing strategy for hazard identification (Bauch et al., 2012).
In order to conclude on the skin sensitisation potential of the test substance, a human Cell Line Activation Test (h-CLAT) comprises a minimum of two independent and valid experiments. A categorization in the subcategories 1 A and 1 B is not possible. However, a positive result with the h-CLAT may be used on its own to classify a chemical into UN GHS category 1. For a confirmation of a negative result another in vitro skin sensitisation test has to be performed.

Deviations from the Guideline
The following deviation from the guideline was observed:
In the positive control of the pre-test only 9865 instead of 10000 viable cells were measured. The lower cell count was probably caused by a mistake during vacuuming the supernatant when washing the cells. Therefore, the size of the population that was used for the evaluation of the results was smaller than indicated by the OECD 442E. The deviation is uncritical since the measured cell population is not even 2% below target and therefore it doesn’t have any effect on the result. Furthermore the deviation is uncritical because it doesn´t have any effect on the end results of the study.
The deviation was assessed and signed by the study director on 18. October 2017.

GLP compliance:

yes (incl. QA statement)

Type of study:

other: human Cell Line Activation Test (h-CLAT)

Justification for non-LLNA method:

This test has been chosen in accordance with Annex VII ao REACH Regulation.

Specific details on test material used for the study:

Test Item
Designation in Test Facility: 17042002G
Date of Receipt: 20. Apr. 2017
Condition at Receipt: Room temperature, in proper conditions

Specification
Name sodium 3-sulfobenzoate
Batch no. 170103
Appearance White crystalline powder
Composition S odium 3-Sulfobenzoate
Purity > 99.0% (Titration with NaOH, Glass indicating electrode, Calomel reference electrode)
Homogeneity totally homogeneous
Expiry date 14. Feb. 2019
Storage Room Temperature (20 ± 5°C), keep away from humidity
CAS No. 17625-03-5
EINECS-No. 241-602-5
Molecular formula C7H5SO5Na
Molecular weight 224.17 g/mol
The Log Kow of the test item was calculated in the test facility under non-GLP-conditions and is < 3.5.

Details on the study design:

Reasons for the Choice of the THP-1 Cell Line
The OECD 442E indicates that the human monocytic leukaemia cell line, THP-1 should be used for the h-CLAT. The cells were purchased by CLS (Eppelheim, Germany).

Cell Cultures
THP-1 cells are stored in liquid nitrogen in the cell bank of LAUS GmbH to allow a continuous stock of cells, which guarantees similar parameters of the experiment and reproducible characteristics of the cells.
For the pre-test cells of passage 5 were used. For the main experiments cells of passage 8 were used. After thawing the cells were cultivated in RPMI 1640 complete culture medium in cell culture flasks at 37 ± 1 °C in a humidified atmosphere with 5.0 ± 0.5 % CO2.

Reactivity Check
Two weeks after thawing, a reactivity check of the cells was performed. For that, the two positive controls 2,4-dinitrochlorobenzene (DNCB) (CAS n. 97-00-7, ≥ 99% purity, test concentration: 4 μg/mL) and nickel sulfate (NiSO4) (CAS n. 10101-97-0, ≥ 99% purity, test concentration: 200 μg/mL) as well as the negative control, lactic acid (LA) (CAS n. 50-21-5, ≥ 85% purity, test concentration: 1000 μg/mL) were used. These substances as well as all additional information are given by the OECD 442E. The experimental procedure was identical to the experiments in this study.
The two positive controls produced a clearly positive result of the two surface markers CD86 and CD54. The negative control produced a clearly negative result of both surface markers. Therefore the cells were found to be suitable for the experiments.
For the pre-test as well as the experiments only cells which have successfully passed the reactivity check were used.

Positive control results:

Please, see all details in section "Any other information on results incl. tables".

Run / experiment:

other: RFI of CD86

Parameter:

other: Relative fluorescence intensity RFI

Value:

150

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Run / experiment:

other: RFI of CD54

Parameter:

other: Relative fluorescence intensity RFI

Value:

200

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Experiment I

In the following table, the results of experiment I are indicated.

Table. Results from experiment I

 

	Concen-tration	Viability	Antibodies	MFI Value	MFIratiotoIsotype	RFI Value
	[µg/mL]				[%]	[%]
Medium	-	96.57%	CD86	1206	165
		97.07%	CD54	1045	143
		96.77%	ISO	729
RPMI1640	-	96.72%	CD86	1201	162	96
		96.32%	CD54	1053	142	99
		96.32%	ISO	741
DMSO	-	96.80%	CD86	1240	170	107
		96.92%	CD54	1054	144	102
		97.15%	ISO	731
DNCB	4.0	90.45%	CD86	4172		653
		89.83%	CD54	1834		306
		89.02%	ISO	846
TestItem	279.1	96.09%	CD86	1063		70
		95.94%	CD54	1113		120
		95.61%	ISO	739
TestItem	334.9	96.50%	CD86	1129		80
		96.91%	CD54	1070		99
		96.10%	ISO	761
TestItem	401.9	96.34%	CD86	1170		89
		95.95%	CD54	1079		102
		95.99%	ISO	761
TestItem	482.3	96.08%	CD86	1200		91
		96.86%	CD54	1098		102
		96.53%	ISO	780
TestItem	578.7	96.40%	CD86	1173		81
		96.76%	CD54	1109		99
		96.44%	ISO	800
TestItem	694.4	96.53%	CD86	1230		93
		96.00%	CD54	1107		98
		96.00%	ISO	801
TestItem	833.3	96.16%	CD86	1110		70
		96.10%	CD54	1076		93
		96.43%	ISO	787
TestItem	1000.0	96.44%	CD86	1097		72
		96.07%	CD54	1071		97
		96.22%	ISO	768

 

A calculation of the EC150 and EC200 was not possible since none of the RFI values was above 150 (for CD86) and 200 (for CD54) at any of the tested concentrations.

 Experiment II

In the following table, the results of experiment II are indicated.

Table. Results from experiment II

 

	Concen-tration	Viability	Antibodies	MFI Value	MFIratiotoIsotype	RFI Value
	[µg/mL]				[%]	[%]
Medium	-	97.30%	CD86	1227	178
		97.83%	CD54	994	144
		98.09%	ISO	690
RPMI1640	-	97.54%	CD86	1079	153	69
		97.81%	CD54	1012	143	100
		97.85%	ISO	707
DMSO	-	97.49%	CD86	1078	155	71
		97.74%	CD54	1002	144	101
		96.97%	ISO	695
DNCB	4.0	89.81%	CD86	3963		823
		87.63%	CD54	1652		274
		87.08%	ISO	812
TestItem	279.1	97.48%	CD86	1124		111
		97.22%	CD54	1031		105
		97.23%	ISO	712
TestItem	334.9	97.07%	CD86	1093		98
		97.19%	CD54	1039		101
		96.81%	ISO	730
TestItem	401.9	97.39%	CD86	1127		105
		97.66%	CD54	1046		102
		97.02%	ISO	735
TestItem	482.3	97.02%	CD86	1128		102
		97.23%	CD54	1054		100
		97.15%	ISO	749
TestItem	578.7	97.17%	CD86	1198		112
		97.32%	CD54	1072		95
		97.26%	ISO	781
TestItem	694.4	97.02%	CD86	1154		85
		97.01%	CD54	1110		90
		96.88%	ISO	837
TestItem	833.3	96.66%	CD86	1128		91
		97.04%	CD54	1092		99
		96.79%	ISO	791
TestItem	1000.0	96.83%	CD86	1100		88
		96.83%	CD54	1094		105
		96.22%	ISO	774

 

A calculation of the EC150 and EC200 was not possible since none of the RFI values was above 150 (for CD86) and 200 (for CD54) at any of the tested concentrations.

Acceptability

The assay is considered acceptable if it meets the following criteria:

•       The cell viabilities of medium and solvent/vehicle controls are higher than 90%.

•       In the solvent/vehicle control, RFI values of both CD86 and CD54 do not exceed the positive criteria (CD86 RFI ≥ 150 % and CD54 RFI ≥ 200 %).

•       For both medium and solvent controls, the MFI ratio of both CD86 and CD54 to iso- type control should be > 105%.

•       In the positive control, RFI values of both CD86 and CD54 should meet the positive criteria (CD86 RFI ≥ 150 % and CD54 RFI ≥ 200 %) and cell viability should be more than 50 %.

•       For the test item, the cell viability should be more than 50 % in at least four tested concentrations in each run.

If any of these criteria is not met, the experiment is considered not valid and needs to be repeated.

Negative results are acceptable only for test items exhibiting a cell viability of less than 90 % at the highest concentration tested. If the cell viability at 1.2 × CV75 is equal or above 90 % the negative result should be discarded. In such a case another Pre-test has to be performed to refine the dose selection. It should be noted that when 5000 µg/mL in PBS or medium, 1000 µg/mL in DMSO or the highest soluble concentration is used as the maximal test concentration of a test item, a negative result is acceptable even if the cell viability is above 90 %.

All validity criteria were met. Therefore, the study was considered as valid.

Interpretation of results:

GHS criteria not met

Conclusions:

For CD86/CD54 expression measurement, each test item is tested in at least two independent runs to derive a single prediction. A h-CLAT prediction is considered positive if at least one of the following conditions is met in 2 of 2 or in at least 2 of 3 independent runs, otherwise the h-CLAT prediction is considered negative:
- The RFI of CD86 is ≥ than 150 % at any tested concentration with cell viability ≥ 50 %
- The RFI of CD54 is ≥ than 200 % at any tested concentration with cell viability ≥ 50 %
If the first two runs are concordant for both markers, a third experiment is not necessary. Then, the study is completed. If the first two runs are not concordant for at least one of the markers (CD54 or CD86), a third run is needed and the final prediction will be based on the majority result of the three individual runs (i.e. 2 out of 3). In this respect, it should be noted that if two independent runs are conducted and one is only positive for CD86 and the other is only positive for CD54, a third run is required. If this third run is negative for both markers, the h-CLAT prediction is considered negative. On the other hand, if the third run is positive for either marker or for both markers, the h-CLAT prediction is considered positive.

In case of a negative result, special care should be taken if the test item
- has a Log Kow > 3.5. Those results should not be considered. However, positive results obtained with those test chemicals can be used for analysis.
- is a pro-hapten or a pre-hapten
- has an autofluorescence and is emitting at the same wavelength as FITC or as PI
In accordance to these classification criteria the test item sodium 3-sulfobenzoate was negative in the h-CLAT and is therefore considered not having the potential to activate dendritic cells and therefore to be a non-sensitizer.

Executive summary:

An in vitro study according to OECD 442E was performed to assess the sensitising potential of the test item sodium 3-sulfobenzoate by quantifying changes in the expression level of the two cell surface markers CD86 and CD54 which are associated with the process of activation of monocytes and dendritic cells.

Two valid experiments with a treatment period of 24 hours were performed. For the experiments, the highest nominal applied concentration (1000 µg/mL) was chosen based on the results obtained in the pre-test and the insolubility of the test item at any higher concentration. A geometric series (factor 1.2) of 7 dilutions thereof was prepared.

As solvent control for the test item, RPMI 1640 was used in a final concentration of 1 % in culture medium.

As positive control, 2.4-dinitrochlorobenzene (DNCB. CAS n. 97-00-7. ≥ 99% purity) was used.

Prior to the study, the cells used for the experiments were checked in a reactivity check and were found to be suitable for the experiments.

All acceptance criteria were met and therefore, the study was considered valid.

In both experiments, no cytotoxic effect was detected in all tested concentrations. The viability was ≥ 90 %. In both experiments the RFI of CD86 was not ≥ 150 % and the RFI of CD54 was not ≥ 200 % at any tested concentration. Therefore, in accordance to the classification criteria the result of this study is negative.

In conclusion, it can be stated that under the experimental conditions of this study, the test item, sodium 3-sulfobenzoate was negative in the h-CLAT and is therefore considered not having the potential to activate dendritic cells and therefore is a non-sensitizer.

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

According to criteria of CLP Regulation since negative results have been obtained from two OECD Guideline tests (i.e. OECD 442E and OECD 442C), the substance is not classified for skin sensitisation and further tests are not request.

The substance is not classified for respiratory sensitisation since no data are available.