Butylhydroxyoxostannane

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

24 July 2017 to 26 July 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

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

Type of study:

direct peptide reactivity assay (DPRA)

Justification for non-LLNA method:

The study was conducted to quantify the reactivity of the test material towards model synthetic peptides containing either lysine or cysteine. The data may be used as part of an integrated approach to testing and assessment (IATA) to support the discrimination between skin sensitisers and non-sensitisers for the purpose of hazard classification and labelling.

Test material

Test material information

Constituent 1

Reference substance name:

Butylhydroxyoxostannane

EC Number:

218-880-1

EC Name:

Butylhydroxyoxostannane

Cas Number:

2273-43-0

Molecular formula:

C4H10O2Sn

IUPAC Name:

butyl(oxo)stannanol

Test material form:

solid: particulate/powder

Details on test material:

- Appearance: white powder
- Storage: 15 to 25 °C, protected from light

Specific details on test material used for the study:

- The test material was dissolved in isopropanol. This was the first of the listed vehicles that produced a suitable, slightly hazy, solution at a concentration of 100 mM.
- Formulations were prepared shortly before testing.

In chemico test system

Details on the study design:

METHODS
Test Material Incubation
- Each test solution was prepared at ratios of 1:10 and 1:50 with the cysteine and lysine stock solutions, respectively. The preparations were placed in an incubator set at 25°C for 24 ± 2 hours. At the end of the incubation period the all samples were centrifuged at 400 g for 5 minutes, due to precipitate noted in the co-elution and test material samples.

Analytical Method
The following HPLC conditions were applied:
Column: Agilent Zorbax SB-C18 2.1 mm x 100 mm, 3.5 µm or equivalent
Wavelength: 220 nm
Guard column: Phenomenex Security Guard c18 4 mm x 2 mm
Flow rate: 0.35 mL/min
Oven temperature: 30 °C
Sample temperature: 25 °C
Injection volume: 7 µL
Mobile Phase: Phase A: 0.1 % (v/v) of trifluoroacetic acid in MilliQ water and Phase B: 0.085 % (v/v) of trifluoroacetic acid in acetonitrile.
Gradient:
0 minutes: 90 % A, 10 % B
10 minutes: 75 % A, 25 % B
11 minutes: 10 % A, 90 % B
13 minutes: 10 % A, 90 % B
13.5 minutes: 90 % A, 10 % B
20 minutes: 90 % A, 10 % B

Reference and Co-elution Controls
- Reference controls were prepared for each peptide.
- Reference control A and B for each peptide were prepared by adding 750 µL of peptide stock solution to 250 µL of acetonitrile.
- Reference control C for cysteine was prepared by adding 750 µL of peptide stock solution to 200 µL of acetonitrile and 50 µL vehicle.
- Reference control C for lysine was prepared by adding 750 µL of peptide stock solution to 250 µL vehicle.
- Reference control A (in triplicate) was used to verify the HPLC system suitability prior to the analysis. Reference control B (six replicates) was used to verify the stability of the reference controls over time and reference control C (in triplicate) was used to verify that acetonitrile did not impact the percent peptide depletion.
- Co-elution controls were prepared to detect possible co-elution of the test material with the peptides. A mixture of 750 µL of 100 mM Phosphate Buffer pH 7.5, 200 µL of acetonitrile and 50 µL of test material solution was used to detect possible co-elution of the test material with cysteine. A mixture of 750 µL of 100 mM ammonium acetate buffer pH 10.2 and 250 µL of test material solution was used to detect possible co elution of the test material with lysine.

Calibration Curves for Peptides
- Calibration curves were prepared for each peptide using a range of concentrations from approximately 0.534 mM to 0.0167 mM (Standards 1 to 6).
- Standard 1 was prepared at approximatively 0.534 mM by dilution of 1600 µL of the peptide stock solution (0.667 mM) with 400 µL of acetonitrile.
- Standards 2 to 6 for cysteine were prepared by serial dilution using dilution buffer (20% acetonitrile in 100 mM Phosphate Buffer pH 7.5).
- Standards 2 to 6 for lysine were prepared by serial dilution using dilution buffer (20% acetonitrile in 100 mM ammonium acetate buffer pH 10.2).
- Samples of dilution buffer alone were also prepared.

Sample Analysis Sequence
The analysis sequence for each peptide was as follows:
- System suitability: Standard 1, Dilution buffer.
- Calibration standards and reference controls: Standard 1, Standard 2, Standard 3, Standard 4, Standard 5, Standard 6, Dilution Buffer, Reference Control A, rep 1, Reference Control A, rep 2, Reference Control A, rep 3.
- Co-elution controls: Co-elution control for test material.
- Reference controls: Reference Control B, rep 1, Reference Control B, rep 2, Reference Control B, rep 3.
- First set of replicates: Reference Control C (positive control), rep 1, Reference Control C (test material), rep 1, Positive Control, rep 1, Test sample, rep 1.
- Second set of replicates: Reference Control C (positive control), rep 2, Reference Control C (test material), rep 2, Positive Control, rep 2, Test sample, rep 2.
- Third set of replicates: Reference Control C (positive control), rep 3, Reference Control C (test material), rep 3, Positive Control, rep 3, Test sample, rep 3.
- Reference controls: Reference Control B, rep 4, Reference Control B, rep 5, Reference Control B, rep 6.

DATA EVALUATION
Data Analysis and Calculations
- The chromatographic data were collected and processed using Chromeleon, a validated data capture system.
- A calibration curve was generated based on the concentration of standards and the peak area. All the peaks of the samples were integrated (standards, samples and controls) “valley to valley” (when possible) and Cysteine and Lysine peptides concentrations were determined from absorbance at 220 nm using the respective calibration curves. Areas were determined for all the samples.
- The PPD for each individual test material and positive control samples was calculated as follows:
PPD = 1 – [(Peptide peak area in replicate injection) / (Mean peptide peak area in reference controls C)] x 100
- The mean peptide peak areas for the nine reference controls B and C, Standard Deviation (SD) and Coefficient of Variation (CV) were calculated.
- The mean peptide peak areas and the mean peptide concentrations (mM) for the three reference controls C were calculated.
- The chromatograms of co-elution controls were compared to the chromatograms of reference controls C.
- For the positive control and for the test material, the PPD in each replicate was calculated from the peptide area of the replicate injection and the mean peptide peak for reference control C. The PPD of every injected positive control and test chemical replicate was calculated. The mean PPD of the three replicate determinations and SD were calculated.
- The mean percent cysteine and percent lysine depletion values were calculated for each test chemical (negative depletion is considered as “0” when calculating the mean).

Assay Acceptance Criteria
The following criteria should be met for a run to be considered valid:
- The standard calibration curve should have a r² >0.99.
- The mean peptide concentration for reference controls A should be 0.50 ± 0.05 mM and the coefficient of variation (CV) of peptide peak areas for the nine reference controls B and C should be < 15.0 %.
- The mean PPD value (%) of the three replicates for the positive control and maximum standard deviation (SD) must fall within the following ranges:
Cysteine: 60.8 to 100% (SD < 14.9)
Lysine: 40.2 to 69.0% (SD < 11.6)

The following criteria should be met for the test material’s result to be considered valid:
- The maximum standard deviation for the test material replicates should be < 14.9 for the percent cysteine depletion and < 11.6 for the percent lysine depletion.
- The mean peptide concentration of the three reference controls C in the appropriate should be 0.50 ± 0.05 mM.

Prediction Model
The mean percent cysteine and percent lysine depletion value was calculated. By using the cysteine 1:10/lysine 1:50 prediction model below, the threshold of 6.38% average peptide depletion was used to support the discrimination between skin sensitisers and non-sensitisers in the framework of an IATA:

Negative DPRA Prediction:
0 % ≤ mean % depletion ≤ 6.38% (No or minimal reactivity)

Negative DPRA Prediction:
6.38 % < mean % depletion ≤ 22.62% (Low reactivity)
22.62 % < mean % depletion ≤ 42.47% (Moderate reactivity)
42.47 % < mean % depletion ≤ 100% (High reactivity)

Results and discussion

In vitro / in chemico

Results

Results 1

Key result

Run / experiment:

other: Lysine Depletion

Parameter:

other: Mean PPD

Value:

0.45

Vehicle controls validity:

not applicable

Negative controls validity:

not applicable

Positive controls validity:

valid

Results 2

Key result

Run / experiment:

other: Cysteine Depletion

Parameter:

other: mean PPD

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

not applicable

Positive controls validity:

valid

Other effects / acceptance of results:

Lysine Depletion
- The percentage peptide depletion values are shown in Table 1.
- The r² value for the standard calibration curve was 0.9999.
- The mean peptide concentrations for the reference controls A and C were: A: 0.48 mM, C (Positive control): 0.47 mM and C (Test material): 0.34 mM.
- The mean peak area result for reference controls B and C (Positive control) was 32.02 (SD 1.34, CV 4.19).
- The mean peak area result for reference controls B and C (Test material) was 29.27 (SD 5.02, CV 17.16).

Cysteine Depletion
- The percentage peptide depletion values are shown in Table 2.
- The r² value for the standard calibration curve was 0.9951.
- The mean peptide concentrations for the reference controls A and C were: A: 0.53 mM, C (Positive control): 0.51 mM and C (Test material): 0.50 mM.
- The mean peak area result for reference controls B and C (Positive control) was 30.59 (SD 0.96, CV 3.14).
- The mean peak area result for reference controls B and C (Test material) was 30.54 (SD 1.04, CV 3.39).

Any other information on results incl. tables

Table 1: Lysine Depletion Percentage Peptide Depletion Values

Substance	Replicate Peptide Peak Areas	Reference Control C Mean Peptide Peak Area	PPD	Mean PPD	SD
Test Material	23.75	22.750	0.00	0.45	0.8
	23.26		0.00
	22.44		1.36
Positive Control	11.50	31.020	62.93	57.54	4.7
	14.18		54.29
	13.83		55.42

Table 2: Cysteine Depletion Percentage Peptide Depletion Values

Substance	Replicate Peptide Peak Areas	Reference Control C Mean Peptide Peak Area	PPD	Mean PPD	SD
Test Material	32.820	30.200	0.00	0.0	-
	31.570		0.00
	31.340		0.00
Positive Control	8.780	30.370	71.09	71.53	0.4
	8.600		71.68
	8.560		71.81

Applicant's summary and conclusion

Interpretation of results:

other: Negative in the Direct Peptide Reactivity Assay.

Conclusions:

Under the conditions of this study, the test material was considered to be negative in the Direct Peptide Reactivity Assay.

Executive summary:

The skin sensitisation potential of the test material cause investigated in accordance with the standardised guideline OECD 442C, under GLP conditions.

The study was conducted to quantify the reactivity of the test material towards model synthetic peptides containing either lysine or cysteine. The data may be used as part of an integrated approach to testing and assessment (IATA) to support the discrimination between skin sensitisers and non-sensitisers for the purpose of hazard classification and labelling.

The test material was dissolved in isopropanol at a concentration of 100 mM. The test solutions were incubated at 1:10 and 1:50 ratios with the cysteine and lysine peptides, respectively, for 24 ± 2 hours in glass autosampler vials, protected from light and set at 25°C.

The remaining concentration of cysteine- or lysine-containing peptides following the 24 hour incubation period was measured by high performance liquid chromatography (HPLC) with gradient elution and UV detection at 220 nm.

The cysteine depletion value was 0.00 %, the lysine depletion value was 0.45 % and the mean of the cysteine and lysine depletion values was 0.23 %.

Under the conditions of this study, the test material was considered to be negative in the Direct Peptide Reactivity Assay.