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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in chemico
Type of information:
experimental study
Adequacy of study:
key study
Study period:
6 July 2017 - 3 Nov 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
Version / remarks:
04 February 2015
GLP compliance:
yes (incl. certificate)
Remarks:
Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht
Type of study:
direct peptide binding assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid

In chemico test system

Details on study design:
The test substance was dissolved in deionized water. Three samples of the test substance were incubated with each peptide (i.e., cysteine peptide or lysine peptide). Additionally, triplicates of the concurrent vehicle control were incubated with the peptides. These vehicle controls were prepared as three separates sets, set A, set B, and set C. Set A was analyzed together with the calibration samples without incubation and served as a performance control. Sets B and C were prepared and
incubated with the samples. Set B was placed at the very start and ending of the sample list and served as stability control of the peptide over the analysis time. Set C was analyzed with the samples and served for calculation of the peptide depletion of any chemical formulated in the vehicle.

Positive and co-elution controls also were evaluated. The positive control used was ethylene dim ethacrylate (50 mM) and the co-elution control was peptide buffer with the test substance but without peptide. The remaining non-depleted peptide concentration was determined thereafter by HPLC with gradient elution and UV-detection at 220 nm.

For the assay to be considered valid, the following criteria needed to be met:
The standard calibration curve was an r² > 0.99.
The negative control (vehicle control) samples of sets A and C were 0.50 mM +/- 0.05 mM.
The CV of the nine vehicle controls B and C were < 15%.
The variability between the mean of three single samples was low (SD < 14.9% for % cysteine de
pletion and < 11.6% for % lysine depletion).
The positive control caused depletion of both peptides comparable to historic data.

For results to be considered negative, the mean peptide depletion is ≤ 6.38%. All other results are considered positive with low (> 6.38 ≤ 22.62%), moderate (> 22.62 ≤ 42.47%), or high (> 42.47%) reactivity. In the case where mean peptide depletion could be determined, but valid C-peptide depletion was available, a C peptide depletion of ≤ 13.89% was considered negative. All other results are considered positive with low (> 13.89 ≤ 23.09), moderate (> 23.09 ≤ 98.24), or high (> 98.24) reactivity.

Results and discussion

Positive control results:
The positive control caused depletion of both peptides comparable to historic data.

In vitro / in chemico

Resultsopen allclose all
Parameter:
other: mean cysteine peptide depletion
Value:
5.02
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Parameter:
other: mean lysine peptide depletion
Value:
-0.53
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Parameter:
other: mean of both peptide depletions
Value:
2.51
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: The test substance was dissolved in de-ionized water 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.

DEMONSTRATION OF TECHNICAL PROFICIENCY: The mean peptide concentration of the three samples of set A was 0.505 ± 0.005 mM, demonstrating good performance. The mean peptide concentration of the three samples of set B, analyzed at the beginning of the sample list was calculated to be 0.485 ± 0.008 mM. The other three samples of set B, analyzed at the end of the sample list had a mean peptide concentration of 0.471 ± 0.014 mM. The CV of the 9 vehicle control samples of sets B and C was calculated to be 2.2%. Thus the peptide was considered stable over the time of analysis.

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: yes

Applicant's summary and conclusion

Interpretation of results:
other: no skin sensitising potential based on the key event “protein reactivity”
Conclusions:
Based on the observed results and applying the cysteine 1:10 / lysine 1:50 prediction model, it was concluded that Sodium 2-butoxyethyl sulphate shows minimal or no chemical reactivity in the DPRA under the test conditions chosen.