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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vitro
Remarks:
Direct Peptide Reactivity Assay (DPRA)
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
21 Jun 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 guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
Version / remarks:
2015
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
direct peptide reactivity assay (DPRA)

Test material

Constituent 1
Chemical structure
Reference substance name:
Sodium 1-hydroxyethanesulphonate
EC Number:
213-037-4
EC Name:
Sodium 1-hydroxyethanesulphonate
Cas Number:
918-04-7
Molecular formula:
C2H6O4S.Na
IUPAC Name:
sodium 1-hydroxyethane-1-sulfonate
Test material form:
liquid
Details on test material:
- Batch identification: Lab-sample from Dec 2016
- Content: 50.7 g/100 g sodium 1-hydroxyethanesulphonate
- Physical state / color: liquid / colorless to yellowish
- Water: 46.1 g/100g
- Molecular weight: 148.1 g/mol
- LOG KOW: -5.58 (calculated)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: BASF SE; batch No.: Lab-sample from Dec 2016

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability under test conditions: The stability under storage conditions over the study period was guaranteed by the sponsor.
- Solubility and stability of the test substance in the solvent/vehicle: Test test substance was soluble in the vehicle (after short stirring). Due to the use of deionized water as vehicle the verification of the stability of the test substance in the vehicle was not required.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test substance was prepared as ca. 100 mM (considering a molecular weight of 137.4 g/mol and a purity/contents of 53.9%) preparation in de-ionized water. At the time of the conduct of the DPRA preliminary information on the content of the test substance was available and used for calculation of 100 mM concentration (53.9 g/ 100 g). However, the final content of the test substance is reported to be 50.7 g /100 g. Hence, considering the final purity, a concentration of ca. 94 mM was used as stock concentration of the DPRA.

FORM AS APPLIED IN THE TEST (if different from that of starting material): diluted in de-ionized water

In vitro test system

Details on the study design:
TEST SYSTEM
- 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)
- Source: The peptides are custom material (Supplier: GenScript, Piscataway, NJ, USA and/or RS Synthesis, Louisville KY, USA and/or JPT Peptide Technologies GmbH, Berlin, Germany) containing phenylalanine to aid in detection and either cysteine or lysine as the reactive center.
- Preparation of peptide stock solutions: 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 were used for preparing the calibration samples, the test-substance and control samples.


CONTROLS
- vehicle control: de-ionized water: Set A) performance control (analyzed together with the calibration samples without incubation); Set B) Stability control (placed at the very start and ending of the sample list for HPLC analysis); Set C) for calculation of the peptide depletion (analyzed with the samples)
- Positive control: ethylene glycol dimethacrylate (EGDMA; CAS no. 97-90-5) (prepared as a 50 mM solution in de-ioinzed water)
- Co-elution control: Sample prepared of the respective peptide buffer and the test substance but without peptide

VEHICLE
- Vehicle: de-ionized water
- Reason for choice of the vehicle: The test substance was soluble in de-ionized water (tested prior to the assay)

SAMPLE PREPARATION
- Peptide stock solutions were mixed with the test substance or positive control or vehicle control at a ration of 1:10 (C-peptide) or 1:50 (K-peptide)

EXPERIMENTAL PROCEDURE
- No. of replicates: 3 (for each peptide)
- The test substance was prepared at a ca. 100 mM concentration. 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-containg peptide in a ratio of 1:50 (0.5 mM peptide, 25 mM test substance).
- Visual inspection for solubility was performed directly after sample preparation and prior to HPLC analysis
- Samples were incubated at 25°C ± 2.5°C in the dark for 24 +/- 2 hours
- The remaining non-depleted peptide concentration was determined by HPLC with gradient elution and UV-detection at 220 nm about 24 hours after sample preparation (for details on HPLC conditions see tab. 3). The analysis time itself did not exceed 30 hours.
- Calibration samples of known peptide concentration (dissolved in 20% de-ionized water in the respective buffer), prepared from the respective peptide stock solution used for test-substance incubation were measured before analysis of the test-substance samples with the same analytical method (for details see tab. 1)

DATA EVALUATION (for detailed formulas see "Any other information on material and methods")
Calculation of the peptide concentrations:
- 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).
Calculation of the peptide depletion:
- 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. When a negative value for C- or K-containing peptide depletion is obtained the value is considered zero for calculation of the mean peptide depletion. The mean peptide depletion of a test substance is calculated as the mean value of C-containing peptide depletion and K-containing peptide depletion.

ACCEPTANCE CRITERIA
- The standard calibration curve should have an r² >0.99.
- The negative control (vehicle control) samples of sets A and C should be 0.50 mM +/- 0.05 mM.
- The CV of the nine vehicle controls B and C should be < 15%.
- Since the mean peptide depletion for each peptide is determined from the mean of three single samples, the variability between these samples should be acceptably low (SD < 14.9% for % cysteine depletion and < 11.6% for % lysine depletion).
- The positive control should cause depletion of both peptides comparable to historic data.


Results and discussion

In vitro / in chemico

Results
Parameter:
other: mean peptide depletion [%]
Value:
0
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Other effects / acceptance of results:
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for vehicle control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes
- Range of historical values: see tab. 9a+b

The test substance was dissolved in de-ionized water at a concentration of ca. 100 mM.

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.

Any other information on results incl. tables

Table 6: Peptide depletion for C-peptide

 

Reaction with cysteine- peptide

 

peptide depletion [%]

sample 1

sample 2

sample 3

mean             SD

 

NC: H2O

 

-1.32

 

0.94

 

0.38

 

0.00             1.18

Test substance

 

-8.79

 

-9.60

 

-3.84

 

-7.41            3.12

 

PC: EGDMA in H2O

 

84.38

 

89.50

 

92.78

 

88.89            4.24

Table 7: Peptide depletion for K-peptide

 

 

Reaction with lysine-peptide

 

peptide depletion [%]

sample 1

sample 2

sample 3

mean             SD

 

NC: H2O

 

1.17

 

-0.65

 

-0.52

 

0.00             1.01

Test substance

 

0.21

 

1.10

 

-1.47

 

-0.05            1.31

 

PC: EGDMA in H2O

 

6.60

 

8.61

 

4.92

 

6.71             1.84

Table 8: Mean peptide depletions

 

Cysteine-Peptide

 

mean depletion

[%]          SD[%]

Lysine-Peptide

 

mean depletion

[%]          SD[%]

 

mean of both depletions[%]

Test substance

 

-7.41

 

3.12

 

-0.05

 

1.31

 

0.00

 

PC: EGDMA in H2O

 

88.89

 

4.24

 

6.71

 

1.84

 

47.80

Table 9a: Historic control data of vehicle control (de-ionized water) (not including present study)

 

C-peptide

concentration

K-peptide

concentration

[mM]

[mM]

Min

0.432

0.459

Max

0.510

0.528

Mean

0.475

0.504

SD

0.017

0.016

n

17

15

Table 9b: Historic control data of positive control (EGDMA, 50 mM in de-ionized water) (not including present study)

 

C-peptide concentration

[mM]

 

C-peptide depletion [%]

K-peptide concentration

[mM]

 

K-peptide depletion [%]

Min

0.032

44.32

0.403

5.93

Max

0.266

93.44

0.481

16.01

Mean

0.153

67.87

0.455

9.39

SD

0.063

13.34

0.020

2.58

n

13

12

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
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.
However, it should be noted, that due to the lower concentration of the stock concentration used (ca. 94 mM instead of 100 mM), the result could be under-predictive.
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 ca. 25°C and the remaining non-depleted peptide concentrations were determined by high performance liquid chromatography (HPLC) with gradient elution and UVdetection at 220 nm.

The test substance was dissolved at ca. 100 mM concentration in de-ionized water (At the time of the conduct of the DPRA preliminary information on the content of the test substance was available and used for calculation of 100 mM concentration (53.9 g/ 100 g). However, the final content of the test substance is reported to be 50.7 g /100 g. Hence, considering the final purity, a concentration of ca. 94 mM was used as stock concentration of the DPRA). 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 following results were obtained in the DPRA:

The test substance was dissolved in de-ionized water at a concentration of ca. 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 -7.41%. The mean K-peptide depletion, caused by the test substance was determined to be -0.05%. 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 it was concluded that the test substance shows minimal or no chemical reactivity in the DPRA under the test conditions chosen.

However, it should be noted, that due to the lower concentration of the stock concentration used, the result could be under-predictive.