Isobutyl salicylate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

key study

Study period:

01.06.2017-08.02.2018

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:

February 04, 2015

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Rathausgasse 4, 91126 Schwabach, Germany

Type of study:

direct peptide reactivity assay (DPRA)

Test material

Test material information

Constituent 1

Reference substance name:

Isobutyl salicylate

EC Number:

201-729-9

EC Name:

Isobutyl salicylate

Cas Number:

87-19-4

Molecular formula:

C11H14O3

IUPAC Name:

2-methylpropyl 2-hydroxybenzoate

In chemico test system

Details on the study design:

TEST-SUBSTANCE PREPARATION
- Stock solution: 100 mM
- Vehicle: Acetonitrile main experiment 1 (exp. 1) and methanol main experiment 2 (exp. 2).
- Reason for the vehicle: The test item was not soluble in dist. water and dist. water : acetonitrile 1:1 (v/v).

CONTROLS
- Reference controls (RCs) were set up in parallel to sample preparation in order to verify the validity of the test run.
- Co-elution control: buffer and test substance without the peptide
- Positive control: Cinnamic aldehyde in acetonitrile

PEPTIDES
- Synthetic peptides:
-- Cysteine- (C-) containing peptide: 22.61 mg (exp. 1) and 20.15 mg (exp. 2); amino acid sequence: Ac-RFAACAA; dissolved in a defined volume (43.9 mL exp. 1 / 39.18 mL exp. 2) of a phosphate buffer with pH 7.5 to reach a concentration of 0.667 mM
-- Lysine- (K-) containing peptide: 20.94 mg (exp. 1) and 19.32 mg (exp. 2); amino acid sequence: Ac RFAAKAA; dissolved in a defined volume of ammonium acetate buffer with pH 10.2 (36.69 mL exp. 1 / 39.74 mL exp. 2) to reach a concentration of 0.667 mM

EXPERIMENTAL PROCEDURE
- Replicates: 3 for each peptide
- Determination remaining non-depleted peptide concentration: HPLC at 220nm: HPLC analysis started 22 to 26 hours after sample preparation and the analysis time was less than 30 hours.
- Calibration samples: samples of a known peptide concentration are measured in parallel

PREPARATIONS SAMPLES
- Calibration sample was prepared from the peptide stock solution in 20% acetonitrile in the respective buffer using serial concentration: 0.534, 0.267, 0.134, 0.067, 0.033, 0.017 or 0.000 mM peptide
- Test-substance samples: samples were incubated at 25°C +/- 2.5°C in the dark for 24 +/- 2 hours
- Reference controls, co-elution controls as well as the positive control were set up in parallel (see table below)

MEASUREMENT PEPTIDE CONCENTRATIONS
- Method: HPLC Agilent 1200 series
- Wavelength: 220 nm and 258 nm
- Detector: UV detector

DATA EVALUATION
- Concentration of the cysteine and lysine peptide determined from absorbance at λ = 220 nm, measuring the area of the appropriated peaks (peak area (PA)) and calculating the concentration of peptide using the linear calibration curves.
- The percent peptide depletion (PPD) was calculated according to the following formula: PPD = [ 1 – ( Peptide Peak Area in the Replicate Injection / Mean Peptide Peak Area in the Reference Control C)] * 100

ACCEPTANCE CRITERIA
The run meets the acceptance criteria if:
- the standard calibration curve has a r² > 0.99,
- the mean percent peptide depletion (PPD) value of the three replicates for the positive control is between 60.8% and 100% for the cysteine peptide and the maximum standard deviation (SD) for the positive control replicates is < 14.9%,
- the mean percent peptide depletion (PPD) value of the three replicates for the positive control is between 40.2% and 69.0% for the lysine peptide and the maximum SD for the positive control replicates is < 11.6%,
- the mean peptide concentration of the three reference controls A replicates is 0.50 ± 0.05 mM,
- the coefficient of variation (CV) of peptide peak areas for the six reference control B replicates and three reference control C replicates in acetonitrile is < 15.0%

The results of the test item meet the acceptance criteria if:
- the maximum standard deviation (SD) for the test chemical replicates is < 14.9% for the cysteine percent depletion (PPD),
- the maximum standard deviation (SD) for the test chemical replicates is < 11.6% for the lysine percent depletion (PPD),
- the mean peptide concentration of the three reference controls C replicates in the appropriate solvent is 0.50 ± 0.05 mM

EVALUATION RESULTS
Cysteine 1:10/ Lysine 1:50 Prediction Model:
- Chemical reactivity was determined by mean peptide depletion [%] and was rated as
-- high: mean peptide depletion > 42.47
-- moderate: mean peptide depletion > 22.62 ≤ 42.47
-- low: mean peptide depletion > 6.38 ≤ 22.62
-- minimal: mean peptide depletion ≤ 6.38
High, moderate and low reactivity are evaluated as positive.
In case of co-elution of the test item with a peptide peak, the peak cannot be integrated correctly and the calculation of the PPD is not possible.
If severe co-elution occurs with both peptides then the analysis was reported as "inconclusive".

- In cases where the co-elution occurs only with the lysine peptide prediction model 2 can be applied (cysteine 1:10 prediction model) :
-- high: mean peptide depletion > 98.24
-- moderate: mean peptide depletion > 23.09 ≤ 98.24
-- low: mean peptide depletion > 13.89 ≤ 23.09
-- minimal: mean peptide depletion ≤ 13.89
High, moderate and low reactivity are evaluated as positive.

DEVIATIONS FROM THE STUDY PLAN
change of supplier for some reagents: the deviation did not influence the quality or integrity of the present study.

Results and discussion

Positive control results:

Since the acceptance criteria for the linearity of the standard curve as well as for the depletion range of the positive control (Positive control values for cystein: 72.27 %, SD 0.27 %; positive control values for lysine: 63.51 %, SD 0.86 %) were fulfilled, the observed precipitations and phase separation were regarded as insignificant.

In vitro / in chemico

Results

Results 1

Run / experiment:

other: Experiment 1, both peptides

Parameter:

other: mean depletion % of peptide

Value:

0.32

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable

Remarks:

no prediction can be made (tested in concentration < 100 mM)

Results 2

Key result

Run / experiment:

other: Experiment 2, cysteine peptide

Parameter:

other: mean depletion % of peptide

Value:

0.3

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

Based on the prediction model 2

Results 3

Run / experiment:

other: Experiment 2, lysine peptide

Parameter:

other: mean depletion % of peptide

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable

Remarks:

Phase separation

Other effects / acceptance of results:

Exp. 1: Since the acceptance criteria for the linearity of the standard curve as well as for the depletion range of the positive control were fulfilled, the observed precipitations and phase separation were regarded as insignificant.

Exp. 2: Since the acceptance criteria for the depletion range of the positive control were fulfilled, the observed phase separation was regarded as insignificant.

Any other information on results incl. tables

Results of the Cysteine Peptide Depletion

Depletion of the Cysteine Peptide (Experiment1)

Sample	Peptide concentration (mM)	Peptide Depletion (%)	Mean Peptide Depletion (%)	SD of Peptide Depletion (%)	CV (%)
Positive control	0.1438	71.3	71.38	0.25	0.35
	0.1444	71.18
	0.1420	71.66
Test item	0.5005	0.30	0.64	0.32	50.31
	0.4986	0.68
	0.4976	0.93

Depletion of the Cysteine Peptide (Experiment2)

Sample	Peptide concentration (mM)	Peptide Depletion (%)	Mean Peptide Depletion (%)	SD of Peptide Depletion (%)	CV (%)
Positive control	0.1446	72.07	72.27	0.27	0.38
	0.1441	72.17
	0.1419	72.58
Test item	0.5155	0.00	0.30	0.52	173.21

Results of the Lysine Peptide Depletion

Depletion of the Lysine Peptide (Experiment1)

Sample	Peptide concentration (mM)	Peptide Depletion (%)	Mean Peptide Depletion (%)	SD of Peptide Depletion (%)	CV (%)
Positive control	0.2081	58.68	57.65	1.11	1.93
	0.2125	57.80
	0.2193	56.47
Test item	0.5085	0.00	0.00	0.00	#DIV/0 !
	0.5072	0.00
	0.5074	0.00

Depletion of the Cysteine Peptide (Experiment2)

Sample	Peptide concentration (mM)	Peptide Depletion (%)	Mean Peptide Depletion (%)	SD of Peptide Depletion (%)	CV (%)
Positive control	0.1777	64.50	68.51	0.86	1.35
	0.1848	63.08
	0.1854	62.95
Test item	0.4932	3.47	3.87	0.39	10.08
	0.4911	3.89
	0.4893	4.25

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In the first experiment using acetonitrile as solvent under the given conditions, the test item showed minimal reactivity towards the peptides. Due to the observed precipitation in both experiments and phase separations in the lysine run, the prediction model does not apply and a prediction cannot be made.

In the second experiment using methanol as solvent under the given conditions, the test item showed minimal reactivity towards the cysteine peptide. The test item can be considered as “non-sensitiser”.

Executive summary:

The in chemico direct peptide reactivity assay (DPRA) enables detection of the sensitising potential of a test item by quantifying the reactivity of test chemicals towards synthetic peptides containing either lysine or cysteine. The test was performed according to OECD TG 442C and in compliance to GLP.

Experiment 1

In the present study the test item was dissolved in acetonitrile based on the results of the pre-experiments. Based on a molecular weight of 194.23 g/mol a 100 mM stock solution was prepared. The test item solutions were tested by incubating the samples with the peptides containing either cysteine or lysine for 24 ± 2 h at 25 ± 2.5 °C. Subsequently samples were analysed by HPLC.

For the 100 mM stock solution of the test item no turbidity or precipitation was observed when diluted with the cysteine peptide solution. After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples were inspected for precipitation, turbidity or phase separation. A slight turbidity was observed for the standard 1 (0.534 mM). Precipitation at the bottom was noted in the samples of the positive control (excluding the co-elution control) as well as for the samples and co-elution control of the test item. Samples were not centrifuged prior to the HPLC analysis.

For the 100 mM stock solution of the test item, turbidity was observed when diluted with the lysine peptide solution. After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples were inspected for precipitation, turbidity or phase separation. Phase separation was observed for the samples of the positive controls. However, the turbity noted for the test item sample 24 h before was gone. Instead phase separation was observed for the samples of the test item and their co-elution control. Samples were not centrifuged prior to the HPLC analysis.

Since the acceptance criteria for the linearity of the standard curve as well as for the depletion range of the positive control were fulfilled, the observed precipitations and phase separation were regarded as insignificant.

The stock solution of the test item showed minimal reactivity towards the synthetic peptides. The mean depletion of both peptides was < 6.38% (0.32%). However, due to the observed precipitation in both experiments and phase separations in the lysine run prior to the HPLC analysis, a test item concentration of 100 mM as well as the full contact of peptide and test item is not guaranteed. According to the evaluation criteria in the guideline, no firm conclusion on the lack of reactivity should be drawn from a negative result, if a test chemical is tested in concentration < 100 mM.

Therefore, no prediction can be made.

The 100 mM stock solution of the positive control (cinnamic aldehyde) showed high reactivity towards the synthetic peptides. The mean depletion of both peptides was 64.52%.

Experiment 2:

In the present study the test item was dissolved in methanol based on the results of the pre-experiments. Based on a molecular weight of 194.23 g/mol a 100 mM stock solution was prepared. The test item solutions were tested by incubating the samples with the peptides containing either cysteine or lysine for 24 ± 2 h at 25 ± 2.5 °C. Subsequently samples were analysed by HPLC.

For the 100 mM stock solution of the test item turbidity was observed when diluted with the cysteine peptide solution. After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples were inspected for precipitation, turbidity or phase separation. No precipitation, turbidity or phase separation was observed for any of the samples.

For the 100 mM stock solution of the test item no turbidity or precipitation was observed when diluted with the lysine peptide solution. After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples were inspected for precipitation, turbidity or phase separation. Phase separation was observed for the samples of the test item (including co-elution control) and the co-elution of the positive control. Samples were not centrifuged prior to the HPLC analysis.

Since the acceptance criteria for the depletion range of the positive control were fulfilled, the observed phase separation was regarded as insignificant.

Phase separation in the lysine experiment was observed. Therefore, the given peak areas and corresponding lysine peptide values can only be considered as an estimation of the peptide depletion and cannot be used for evaluation.

Sensitising potential of the test item was predicted from the mean peptide depletion of the cysteine peptide by comparing the peptide concentration of the test item treated samples to the corresponding reference control C (RC Cmethanol). The 100 mM stock solution of the test item showed minimal reactivity towards the synthetic peptide. The mean depletion of the cysteine peptide was ≤ 13.89% (0.30%). Based on the prediction model 2 the test item can be considered as non-sensitiser.

The 100 mM stock solution of the positive control (cinnamic aldehyde) showed high reactivity towards the synthetic peptides. The mean depletion of both peptides was 67.89%.

Conclusions:

In the first experiment using acetonitrile as solvent under the given conditions, the test item showed minimal reactivity towards the peptides. Due to the observed precipitation in both experiments and phase separations in the lysine run, the prediction model does not apply and a prediction cannot be made.

In the second experiment using methanol as solvent under the given conditions, the test item showed minimal reactivity towards the cysteine peptide in the second experiment. The test item can be considered as “non-sensitiser”.

The data generated with this test should be considered in the context of integrated approached such as IATA, combining the result with other complementary information, e.g. derived from in vitro assays addressing other key events of the skin sensitisation AOP.