Amines, bis(C11-14-branched and linear alkyl), O,O-di-iso-Pr phosphorodithioates

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2011-10-26 - 2012-01-26

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: in-vitro experiment, not yet supported by guideline

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Qualifier:

no guideline available

Principles of method if other than guideline:

The reactivity of the test article towards synthetic cysteine (C)- or lysine (K)-containing peptides was evaluated in the Direct Peptide Reactivity Assay (DPRA). For this purpose the test substance is incubated with synthetic peptides for 24 hours at room temperature and the remaining non-depleted peptide concentration is determined thereafter by high performance liquid chromatography with gradient elution and UV-detection at 220 nm. The peptide depletion of test-substance incubated samples is compared to the peptide depletion of the negative control samples and expressed as relative peptide depletion.

GLP compliance:

yes (incl. QA statement)

Remarks:

BASF SE, Experimental Toxicology and Ecology

Type of study:

other: Direct Peptide Reactivity Assay (DPRA)

Test material

Test material information

Constituent 1

Reference substance name:

Bis-[N-C13-(branched and linear)-alykl]-ammonium O,O-dipropan-2-yl phosphorodithioate

EC Number:

700-768-3

Cas Number:

1285610-71-0

Molecular formula:

C26H56N.C6H14O2PS2

IUPAC Name:

Bis-[N-C13-(branched and linear)-alykl]-ammonium O,O-dipropan-2-yl phosphorodithioate

Results and discussion

Any other information on results incl. tables

Experimental Result

The test substance was soluble in acetonitrile. However when mixed with the peptide stock solution or, in the case of the test-substance control with the peptide buffers, the samples became cloudy directly after preparation. After 24 hours the samples were clear but precipitates were noticed in the samples of the C-peptide. Thus the samples of the C-peptide and the co-elution control were centrifuged or filtrated prior to HPLC analysis. In the 1st test run performed with the K-containing peptide a mean depletion of 25.4% was determined. Co-elution of the K-containing peptide and the test substance was noticed. Thus, in order to improve separation of the peaks, an alternative analytical method was run. A mean K-peptide depletion of 19.4% was determined. The mean C-peptide depletion, caused by the test substance was determined to be -6.9% using the standard analytical method. No co-elution of test substance and C-peptide was noticed.

To verify the results mentioned above, an additional test run was performed with both peptides. The alternative analytical method was used and further modified by measuring UV absorbance at 258 nm in addition. The peak area ratio 220/258 was calculated and used as a measure for peak purity. The mean C-peptide depletion, caused by the test substance was determined to be -2.7%. Peak purity was confirmed by a constant area ratio 220/258. The mean K-peptide depletion, caused by the test substance was determined to be 26.5%. Peak purity was confirmed by a constant area ratio 220/258. Negative depletions were considered to be “zero” for calculation of the mean peptide depletion, which was thus calculated to be 11.9%.

Mean Peptide Depletion

For the test substance the mean peptide depletion as average of cysteine- and lysine-peptide depletions is calculated and summarized in the table below. Negative depletions were considered to be “zero” for calculation.

	Cystein-Peptide		Lysine-Peptide
	mean depletion [%]	SD	mean depletion [%]	SD	mean of both depletions [%]
positive control	99.1	1.1	99.2	1.2	99.1
test article	-4.8	2.4	23.7	3.4	11.9

Discussion

The test substance did not cause depletion of the cysteine-containing peptide. However, depletion of the lysine-containing peptide between 19.4% and 26.5% was observed. In addition co-elution of the test substance with the lysine-containing peptide was noticed.

Nevertheless the results are considered to be valid as the three test runs performed with different analytical methods produced comparable values for peptide depletion. Additionally peak purity, determined in the 3rd test run, demonstrated that the peptide peaks could be integrated correctly.

Applicant's summary and conclusion

Conclusions:

Based on the observed results and applying the prediction model proposed in Gerberick et. al (2007) it was concluded that the test article shows a low chemical reactivity in the DPRA under the test conditions chosen.