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Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in chemico
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2017
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 guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
Version / remarks:
2015
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Commission Regulation (EU) 2017/735 of 14 February 2017; B.59 In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA); Official Journal of the European Union, No. L 112/1.
GLP compliance:
yes (incl. QA statement)
Remarks:
(from the competent authority) Landesamt für Umwelt Rheinland-Pfalz
Type of study:
direct peptide reactivity assay (DPRA)
Justification for non-LLNA method:
In vitro/in chemico test methods have been considered scientifically valid for the evaluation of the skin sensitization hazard of chemicals.

Test material

Constituent 1
Chemical structure
Reference substance name:
4,5,6,7-tetrahydro-1H-benzotriazole
EC Number:
229-858-6
EC Name:
4,5,6,7-tetrahydro-1H-benzotriazole
Cas Number:
6789-99-7
Molecular formula:
C6H9N3
IUPAC Name:
4,5,6,7-tetrahydro-1H-1,2,3-benzotriazole
Details on test material:
Physical state, appearance: solid, white
Storage conditions: room temperature
Specific details on test material used for the study:
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- The test substance was prepared as a 100 mM (considering a molecular weight of 123.16 g/mol and a purity/contents of 83.2%) preparation in acetonitrile. After short stirring the test substance was soluble in the vehicle.
- Reason fot the vehicle: The test diubstancve was soluble in acetonitile

In chemico test system

Details on the study design:
Details on study design:
The test substance was prepared as a 100 mM (considering a molecular weight of 123.16 g/mol and a purity/contents of 83.2%) preparation in acetonitrile. After short stirring the test substance was soluble in the vehicle. The test substance was dissolved 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.
Prior to the assay the solubility of the test substance at a concentration of 100 mM was tested. A suitable non-reactive, water-miscible solvent which dissolves the test substance completely (no visible precipitation or cloudyness of the test-susbtance preparation) should be used. The preferred solvent was acetonitrile. When not soluble in acetonitrile solutions in water, isopropanol, acetone, propanol, methanol or mixtures of these solvents were tried.
Peptide stock solutions in a concentration of 0.667 mM 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.

The test substance samples were prepared in triplicates for each peptide. The C-containing peptide was incubated with the test substance in a ratio of 1:10 (0.5 mM peptide, 5 mM test substance) and the K-containing
peptide in a ratio of 1:50 (0.5 mM peptide, 25 mM test substance).
C-peptide: 750 µL C-peptide stock-solution, 200 µL solvent (vehicle), 50 µL test substance preparation (or PC preparation or solvent (VC)).
K-peptide: 750 µL K-peptide stock-solution, 250 µL test substance preparation (or PC preparation or solvent (VC)).
The samples were prepared in suitable tubes, capped tightly and incubated at 25 °C ± 2.5 °C in the dark for 24 ± 2 h. Visual inspection for solubility was performed directly after sample preparation and prior to HPLC analysis. Unsolved samples were centrifuged or filtrated prior to injection into the HPLC in order to remove any unsolved particles. The HPLC analysis of the batch of samples started about 24 h after sample preparation and the analysis time itself did not exceed 30 h.

Several vehicle controls were prepared in triplicates in the same way as the test-substance samples described above but with the vehicle (acetonitrile) 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 serve 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 the vehicle.

Co-elution control: One sample per peptide was prepared in the same way as the test-substance samples described above but without the peptides. Instead the respective peptide buffer was used. The samples were analyzed together with the calibration samples. Samples which were visually turbid or display precipitates were centrifuged or filtrated prior to injection into the HPLC in order to remove any unsolved particles. The analyses of the samples were performed via HPLC.

For each peptide a calibration curve is generated from the measured peak areas of the calibration samples of known peptide concentration. The peptide concentration of the samples is calculated with the respective calibration curve using linear regression (b = axis intercept; m = slope). The mean peptide depletion for each of the two peptides is calculated as the mean value of the three samples conducted for each peptide and test substance. The mean peptide depletion of a test substance is calculated as the mean value of C-containing peptide depletion and K-containing peptide depletion.

Controls:
- Negative control (NC): vehicle control = acetonitrile
- Positive control (PC): Ethylene glycol dimethacrylate, prepared as a 50 mM solution in acetonitrile.
- Co-elution control: Sample prepared of the respective peptide buffer and the test substance but without peptide.

Synthetic peptides:
Cysteine- (C-) containing peptide: Ac-RFAACAA-COOH (MW=751.9 g/mol)
Lysine- (K-) containing peptide: Ac-RFAAKAA-COOH (MW=776.2 g/mol)
The peptides are custom material (Supplier: GenScript, Piscataway, NJ, USA and RS Synthesis, Louisville KY, USA) containing phenylalanine to aid in detection and either cysteine or lysine as the reactive center.

Results and discussion

Positive control results:
For cysteine containing peptides the positive control revealed a peptide depletion of 50.95, 53.62, and 58.21% in sample 1, sample 2, and sample 3, respectively, resulting in a moderate reactivity compared with the evaluation criteria. For lysine containing peptides the positive control revealed a peptide depletion of 9.19, 7.18, and 6.79% in sample 1, sample 2, and sample 3, respectively, resulting in a low reactivity compared with the evalutation criteria. The mean of both depletions of the positive control was 30.99% which is comparable to a moderate reactivity.

In vitro / in chemico

Results
Key result
Run / experiment:
mean
Parameter:
other: Peptide depletion
Value:
0
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: minimal or no chemical reactivity
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: no

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: Two test runs were performed for the C-containing peptide due to relative high standard deviation of the three values measured in the first test run and as the result was close to the evaluation threshold.

Any other information on results incl. tables

The mean C-peptide depletion, caused by the test substance was determined to be -0.65%. The mean K-peptide depletion, caused by the test substance was determined to be -0.77%. Negative depletions were considered to be “zero” for calculation of the mean peptide depletion, which was thus calculated to be 0.00%. No co-elution of the test substance and peptides occurred as demonstrated by the consistent

values of the area ratios 220 nm/258 nm. Based on the observed results and applying the cysteine 1:10 / lysine 1:50 prediction model cited in chapter 3.10 it was concluded that the test item shows minimal or no chemical reactivity in the DPRA under the test conditions chosen.

Applicant's summary and conclusion

Interpretation of results:
other: no chemical reactivity
Conclusions:
The mean peptide depletion was calculated to be 0.0 %. Based on the observed results and applying the cysteine 1:10 / lysine 1:50 prediction model it was concluded that the test substance shows minimal or no chemical reactivity in the DPRA under the test conditions chosen.
Executive summary:

The reactivity of the test item 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 ca. 25°C and the remaining non-depleted peptide concentrations were determined by high performance liquid chromatography (HPLC) with gradient elution and UV-detection at 220 nm. The test substance was dissolved at a 100 mM concentration in acetonitrile. Three samples of the test substance were incubated with each peptide in ratios of 1:10 (for C-containing peptide) or 1:50 (for K-containing peptide). Additionally, triplicates of the concurrent vehicle control (=VC) were incubated with the peptides. Further, 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 analyzed by measuring UV absorbance at 258 nm and the area ratio 220 nm / 258 nm was calculated as a measure of peak purity. The test substance was dissolved in acetonitrile at a concentration of 100 mM. The samples of the test substance with the peptides were solutions at the time of preparation. Visual observation after the 24-hour incubation time did not reveal precipitates in any samples of the test substance with the peptides. No co-elution of test substance and peptides was present. The mean C-peptide depletion, caused by the test substance was determined to be -0.65%. The mean K-peptide depletion, caused by the test substance was determined to be -0.77%. Negative depletions were considered to be “zero” for calculation of the mean peptide depletion, which was thus calculated to be 0.00%. Based on the observed results and applying the cysteine 1:10 / lysine 1:50 prediction model cited in chapter 3.10 it was concluded that 4,5,6,7-tetrahydro-1H-benzotriazole shows minimal or no chemical reactivity in the DPRA under the test conditions chosen.

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