Tetraethylammonium tetrafluoroborate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 July - 12 September 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

4 February 2015

Deviations:

no

GLP compliance:

yes

Type of study:

other: Direct Peptide Reactivity Assay

Justification for non-LLNA method:

DPRA assay was performed in accordance with Section 8.3 of Annex VII of Regulation (EC) No 1907/2006 as amended in Commission Regulation (EU) 2016/1688 of 20 September 2016 and the strategy presented in ECHA Guidance on information requirements and chemical safety assessment Chapter R.7a.

Test material

Test material information

Constituent 1

Reference substance name:

tetraethylammonium tetrafluoroborate

Cas Number:

429-06-1

Molecular formula:

C8H20BF4N

IUPAC Name:

tetraethylammonium tetrafluoroborate

Test material form:

solid: crystalline

In chemico test system

Details on the study design:

The Direct Peptide Reactivity Assay (DPRA) is an in chemico method which quantifies the remaining concentration of cysteine- or lysine-containing peptide following 24 hours incubation with the test item at 25°C. The synthetic peptides contain phenylalanine to aid in the detection. The relative peptide concentration is measured by high-performance liquid chromatography (HPLC) with gradient elution and photodiode array (PDA) detection at 220 nm and 258 nm. Cysteine and lysine peptide Percent Depletion Values are calculated and used in a prediction model which allows assigning the test item to one of four reactivity classes used to support the discrimination between sensitizers and non-sensitizers.

Results and discussion

In vitro / in chemico

Results

Results 1

Key result

Parameter:

other: Cysteine Reactivity Assay

Remarks on result:

no indication of skin sensitisation

Results 2

Key result

Parameter:

other: Lysine Reactivity Assay

Remarks on result:

no indication of skin sensitisation

Other effects / acceptance of results:

Acceptability of the Cysteine Reactivity Assay:
- The correlation coefficient (r2) of the SPCC standard calibration curve was 0.998. Since the r2 was >0.99, the SPCC standard calibration curve was accepted.
- The mean peptide concentration of Reference Controls A was 0.507 ± 0.001 mM while the mean peptide concentration of Reference Controls C was 0.512 ± 0.003 mM. The means of Reference Control samples A and C were both within the acceptance criteria of 0.50 ± 0.05 mM. This confirms the suitability of the HPLC system and indicates that the solvent (ACN) used to dissolve the test item did not impact the Percent SPCC Depletion.
- The Coefficient of Variation (CV) of the peptide areas for the nine Reference Controls B and C was 1.3%. This was within the acceptance criteria (CV <15.0%) and confirms the stability of the HPLC run over time.
- The mean area ratio (A220/A258) of the Reference Control samples was 17.27. The mean A220/A258 ratio ± 10% range was 15.54-18.99. Each sample showing an A220/A258 ratio within this range gives an indication that co-elution has not occurred.
- The Percent SPCC Depletion was calculated versus the mean SPCC peak area of Reference Controls C. The mean Percent SPCC Depletion for the positive control cinnamic aldehyde was 70.8% ± 0.7%. This was within the acceptance range of 60.8% to 100% with a SD that was below the maximum (SD <14.9%).

Acceptability of the Lysine Reactivity Assay:
- The correlation coefficient (r2) of the SPCL standard calibration curve was 0.998. Since the r2 was >0.99, the SPCL standard calibration curve was accepted.
- The mean peptide concentration of Reference Controls A was 0.507 ± 0.015 mM while the mean peptide concentration of Reference Controls C was 0.509 ± 0.015 mM. The means of Reference Control samples A and C were both within the acceptance criteria of 0.50 ± 0.05 mM. This confirms the suitability of the HPLC system and indicates that the solvent (ACN) used to dissolve the test item did not impact the Percent SPCL Depletion.
- The CV of the peptide areas for the nine Reference Controls B and C was 3.4%. This was within the acceptance criteria (CV <15.0%) and confirms the stability of the HPLC run over time.
- The mean area ratio (A220/A258) of the Reference Control samples was 16.86. The mean A220/A258 ratio ± 10% range was 15.17-18.54. Each sample showing an A220/A258 ratio within this range gives an indication that co-elution has not occurred.
- The Percent SPCL Depletion was calculated versus the mean SPCL peak area of Reference Controls C. The mean Percent SPCL Depletion for the positive control cinnamic aldehyde was 49.9% ± 1.2%. This was within the acceptance range of 40.2% to 69.0% with a SD that was below the maximum (SD <11.6%).

Any other information on results incl. tables

Results Cysteine Reactivity Assay:

Preparation of a 100 mM Tetraethylammonium tetrafluoroborate stock solution in ACN showed that the test item was dissolved completely. Upon preparation and after incubation, both the co-elution control (CC) as well as the test item samples were visually inspected. No precipitate or phase separation was observed in any of the samples.

In the CC sample no peak was observed at the retention time of SPCC. This demonstrated that there was no co-elution of the test item with SPCC. For the 209638/A-cys samples, the mean SPCC A₂₂₀/A₂₅₈ area ratio was 17.27. Since this was within the 15.54-18.99 range, this again indicated that there was no co‑elution of the test item with SPCC.

The Percent SPCC Depletion was calculated versus the mean SPCC peak area of Reference Controls C. The mean Percent SPCC Depletion for the test item was 1.3% ± 1.4%.

Results Lysine Reactivity Assay for the Test Item

Preparation of a 100 mM Tetraethylammonium tetrafluoroborate stock solution in ACN showed that the test item was dissolved completely. Upon preparation and after incubation, both the CC as well as the test item samples were visually inspected. No precipitate or phase separation was observed in any of the samples.

In the CC sample no peak was observed at the retention time of SPCL. This demonstrated that there was no co-elution of the test item with SPCL. For the 209638/A-lys samples, the mean SPCL A₂₂₀/A₂₅₈ area ratio was 16.24. Since this was within the 15.17-18.54 range, this again indicated that there was no co‑elution of the test item with SPCL.

The Percent SPCL Depletion was calculated versus the mean SPCL peak area of Reference Controls C. The mean Percent SPCL Depletion for the Test Item was 2.0% ± 2.7%.

DPRA Prediction and Reactivity Classification:

Upon preparation as well as after incubation of the SPCC and SPCL test item samples, no precipitate or phase separation was observed in any of the samples.

In the cysteine reactivity assay the test item showed 1.3% SPCC depletion while in the lysine reactivity assay the test item showed 2.0% SPCL depletion. The mean of the SPCC and SPCL depletion was 1.6% and as a result the test item was negative in the DPRA and was classified in the “no or minimal reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model.  

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Tetraethylammonium tetrafluoroborate was negative in the DPRA and was classified in the “no or minimal reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model.

Executive summary:

The objective of this study was to determine the reactivity of Tetraethylammonium tetrafluoroborate towards model synthetic peptides containing either cysteine (SPCC) or lysine (SPCL). After incubation of the test item with either SPCC or SPCL, the relative peptide concentration was determined by High-Performance Liquid Chromatography (HPLC) with gradient elution and photodiode array (PDA) detection at 220 nm and 258 nm. SPCC and SPCL Percent Depletion Values were calculated and used in a prediction model which allows assigning the test item to one of four reactivity classes used to support the discrimination between sensitizers and non-sensitizers.

Acetonitrile (ACN) was found to be an appropriate solvent to dissolve the test item and was therefore used in this Direct Peptide Reactivity Assay (DPRA) study. 

The validation parameters, i.e. calibration curve, mean concentration of Reference Control (RC) samples A and C, the CV for RC samples B and C, the mean percent peptide depletion values for the positive control with its standard deviation value and the standard deviation value of the peptide depletion for the test item, were all within the acceptability criteria for the DPRA.

Upon preparation as well as after incubation of the SPCC and SPCL test item samples, no precipitate or phase separation was observed in any of the samples.

In the cysteine reactivity assay the test item showed 1.3% SPCC depletion while in the lysine reactivity assay the test item showed 2.0% SPCL depletion. The mean of the SPCC and SPCL depletion was 1.6% and as a result the test item was considered to be negative in the DPRA and classified in the “no or minimal reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model.