Registration Dossier

Diss Factsheets

Toxicological information

Skin sensitisation

Currently viewing:

Administrative data

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2012-04-23 to 2012-05-09
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report date:
2013

Materials and methods

Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
There are no official national or international guidelines for the DPRA Test; however, the study is performed according to the methods described in the following publications:
- Gerberick GF, Vassallo JD, Bailey RE, Chaney JG, Morrall SW, Lepoittevin JP. Development of a Peptide Reactivity Assay for Screening Contact Allergens. Toxicological Sciences 81,332-343, 2004.
- Gerberick GF, Vassallo JD, Foertsch LM, Price BB, Chaney JG, Lepoittenvin JP. Quantificationn of Chemical Peptide Reactivity for Screening Contact Allergens: A Classification Tree Model Approach. Toxicological Sciences 97(2), 417-427, 2007.
- Bauch C, Kolle SN, Fabian E, Pachel C, Ramirez T, Wiench B, Wruck CJ, van Ravenzwaay B, Landsiedel R. Intralaboratory validation of four in vitro assays for the prediction of the skin sensitizing potential of chemicals. Toxicology in Vitro 25, 1162 – 1168, 2011.
- Maxwell G, Aeby P, Ashikaga T, Bessou-Touya S, Diembeck W, Gerberick F, Kern P, Marrec-Fairley M, Ovigne JM, Sakaguchi H, Schroeder K, Tailhardat M, Teissier S, Winkler P. Skin sensitisation: the Colipa strategy for developing and evaluating nonanimal test methods for risk assessment. ALTEX 28(1): 50-5, 2011.
GLP compliance:
yes (incl. QA statement)
Remarks:
BASF SE
Type of study:
other: Direct Peptide Reactivity Assay (DPRA)

Test material

Constituent 1
Reference substance name:
2-Propenoic acid, C16-18-alkyl esters
EC Number:
292-060-1
EC Name:
2-Propenoic acid, C16-18-alkyl esters
Cas Number:
90530-21-5
IUPAC Name:
2-Propenoic acid, C16-18-alkyl esters

In vitro test system

Details on the study design:
Skin sensitisation (In vitro test system) - Details on study design:

CONTROLS
- Negative Control (NC): vehicle control = isopropanol; acetonirile was performed as an additional vehicle control for the PC EGDMA
- Positive Control (PC): Ethylene glycol dimethacrylate (EGDMA, prepared as a 50 mM solution in isopropanol and in acetonitrile), p-Benzoquinone (puriss., prepared as a 100 mM solution in isopropanol); as EGDMA may result in different peptide depletions when formulated in the different vehicles the standard vehicle acetonitrile was used additionally to the vehicle for test substance formulation

TEST SUBSTANCE PREPARATION
- The test substance solutions were prepared within 4 hours of performing the assay (preparation of samples). The test substance was prepared as a 100 mM solution in isopropanol. After short shaking the test substance was soluble in the vehicle.
- Vehicle: isopropanol

EXPERIMENTAL PROCEDURE
- The test substance was solved in a suitable vehicle. Three samples of the test substance were incubated with each peptide. Additionally triplicates of the concurrent vehicle control (= NC) were incubated with the peptides. The remaining non-depleted peptide concentration was determined thereafter by HPLC with gradient elution and UV-detection at 220 nm. In addition calibration samples of known peptide concentration, prepared from the respective peptide stock solution used for test substance incubation, were measured in parallel with the same analytical method.
- Test substance solubility: Prior to the assay the solubility of the test substance was tested. A suitable non-reactive, water-miscible solvent which dissolves the test substance completely should be used. The preffered solvent was acetonitrile. When not soluble in acetonitrile, deionized water, methanol, propanol, isopropanol, acetone or mixtures of these solvents were tried.
- Preparation of peptide stock solutions: Peptide stock solutions in a concentration of 0.667 mM of peptide were prepared in pH 7.5 phosphate buffer (C-containing peptide) or pH 10.2 ammonium acetate buffer (K-containing peptide). The peptide stock solution was used for preparing the calibration samples and the test substance and control samples.
- Preparation of the test substance samples: The samples were prepared in triplicates for each peptide. The test substance was incubated with the C-containing peptide in a ratio of 1 : 10 (0.5 mM peptide, 5 mM test substance) and with the K-containing peptide in a ratio of 1 : 50 (0.5 mM peptide, 25 mM test substance). The samples were prepared in suitable tubes, capped tightly and incubated at room temperature in the dark for 24 +/- 2 hours. Prior to HPLC analysis the samples were visually investigated for any precipitate that may have formed during the exposure period. As the samples were visually turbid or displayed precipitates they were centrifuged (samples of the K-peptide) or centrifuged and filtrated (samples of the C-peptide) prior to injection into the HPLC in order to remove any unsolved particles. The HPLC analysis of the batch of samples started about 24 hours after sample preparation and the analysis time itself did not exceed 30 hours.
- Preparation of the vehicle controls: Several isopropanol controls were prepared in triplicates in the same way as the test substance samples described above but with isopropanol instead of the test substance: One set (set A) was analyzed together with the calibration samples without incubation and serves as a performance control. Another three sets (two sets B and set C) were prepared and incubated with the samples. Sets B were placed at the very start and ending of the sample list and serves as stability control of the peptide over the analysis time. Set C was analyzed with the samples and serves for calculation of the peptide depletion of any chemical formulated in isopropanol. As one set of the PC EGDMA was formulated in acetonitrile a respective set of vehicle controls (set C) was analyzed with the samples and was used for calculation of peptide depletion.
- Preparation of the co-elution control: The co-elution control was prepared in the same way as the test substance samples described above but without the peptide. Instead the respective peptide buffer was used. The samples were analyzed together with the calibration samples. As the samples were visually turbid or displayed precipitates they were centrifuged prior to injection into the HPLC in order to remove any unsolved particles.

Results and discussion

In vitro / in chemico

Resultsopen allclose all
Run / experiment:
mean
Parameter:
other: cysteine-peptide depletion [%]
Value:
25.5
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Run / experiment:
mean
Parameter:
other: lysine-peptide depletion [%]
Value:
0
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Run / experiment:
mean
Parameter:
other: mean peptide depletion [%]
Value:
12.8
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Other effects / acceptance of results:
When mixed with the peptide stock solutions the samples became cloudy directly after preparation. After 24 hours precipitates were noticed in the samples with the C-peptide, additionally.
Thus all samples were centrifuged prior to HPLC analysis. The samples containing the C-peptide were additionally filtered as a separation could not be achieved by centrifugation.
No co-elution of the test substance and peptides occured as demonstrated by the consistent values of the area ratios 220/258.

Any other information on results incl. tables

The test substance was soluble in isopropanol. However when mixed with the peptide stock solutions the samples became cloudy directly after preparation. After 24 hours precipitates were noticed in the samples with the C-peptide, additionally. Thus all samples were centrifuged prior to HPLC analysis. The samples containing the C-peptide were additionally filtered as a separation could not be achieved by centrifugation.

The mean C-peptide depletion, caused by the test substance was determined to be 25.5%.

The mean K-peptide depletion, caused by the test substance was determined to be -0.2%.

Negative depletions were considered to be “zero” for calculation of the mean peptide depletion, which was thus calculated to be 12.8%.

No co-elution of test substance and peptides was noticed.

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

Applicant's summary and conclusion

Interpretation of results:
other: low chemical reactivity
Conclusions:
No prediction can be made for skin sensitization according to GHS criteria based on the results of this in vitro study alone.
Based on the observed results and applying the prediction model proposed in Gerberick et al. (2007) it was concluded that the test substance shows a low chemical reactivity in the DPRA under the test conditions chosen.
Executive summary:

The reactivity of the test substance towards synthetic cysteine (C)- or lysine (K)-containing peptides was evaluated in the Direct Peptide Reactivity Assay (DPRA). For this purpose the test substance was incubated with synthetic peptides for ca. 24 hours at room temperature and the remaining non-depleted peptide concentrations were determined thereafter by high performance liquid chromatography with gradient elution and UV-detection at 220 nm.

The test substance was dissolved at a 100 mM concentration in isopropanol. Three samples of the test substance were incubated with each peptide in ratios of 1 : 10 (for C-peptide) or 1 : 50 (for K-peptide). Additionally triplicates of the concurrent vehicle control (= NC) were incubated with the peptides.

Additionally, a co-elution control was performed in order to detect possible interference of the test substance with the peptides. The samples consisted of the test substance, vehicle and the respective peptide buffer but without peptide. Moreover the samples were additionally analyzed by measuring UV absorbance at 258 nm and the area ratio 220 / 258 was calculated as a measure of peak purity.

The following results were obtained in the DPRA:

The test substance was soluble in isopropanol. However when mixed with the peptide stock solutions the samples became cloudy directly after preparation. After 24 hours precipitates were noticed in the samples with the C-peptide, additionally.

Thus all samples were centrifuged prior to HPLC analysis. The samples containing the C-peptide were additionally filtered as a separation could not be achieved by centrifugation.

The mean C-peptide depletion, caused by the test substance was determined to be 25.5 %. The mean K-peptide depletion, caused by the test substance was determined to be - 0.2 %.

Negative depletions were considered to be "zero" for calculation of the mean peptide depletion, which was thus calculated to be 12.8 %.

No co-elution of test substance and peptides was noticed.

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