Reaction mass of m,alpha-dimethylstyrene and p,alpha-dimethylstyrene

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

10 July 2018 - 12 July 2018

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:

4 February 2015

Deviations:

no

GLP compliance:

yes

Type of study:

direct peptide reactivity assay (DPRA)

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of m,alpha-dimethylstyrene and p,alpha-dimethylstyrene (IPT)

Cas Number:

Not available.

Molecular formula:

No data available.

IUPAC Name:

Reaction mass of m,alpha-dimethylstyrene and p,alpha-dimethylstyrene (IPT)

Test material form:

liquid

Details on test material:

Manufacturer: Mitsui Chemicals Inc.
Storage conditions: at room temperature
Appearance: clear colourless liquid

Specific details on test material used for the study:

No purity correction factor was required.

In chemico test system

Details on the study design:

TEST ITEM PREPERATION
No correction for the purity/composition of the test item was performed.
Solubility of the test item in an appropriate solvent was assessed before performing the DPRA. The test item should dissolve completely in an appropriate solvent, i.e. by visual inspection the solution has to be not cloudy nor have noticeable precipitate. The following solvents were evaluated: acetonitrile (ACN), Milli-Q water (MQ), ACN:MQ (1:1, v/v), isopropanol, acetone:ACN (1:1, v/v), dimethylsulfoxide (DMSO):ACN (1:9, v/v), methanol (MeOH) and ethanol (EtOH).
Test item stock solutions were prepared freshly for each reactivity assay. For both the cysteine and lysine reactivity assay 25.65 mg of test item was pre-weighed into a clean amber glass vial and dissolved, just before use, in 1940 μL ACN after vortex mixing to obtain a 100 mM solution. Visual inspection of the forming of a clear solution was considered sufficient to ascertain that the test item was dissolved. The test item, positive control and peptide samples were prepared less than 4 hours before starting the incubation of the cysteine (cys) or lysine (lys) reactivity assay, respectively.

TEST SYSTEM: Synthetic peptides containing cysteine (SPCC) (Ac- RFAACAA-COOH) or synthetic peptides containing lysine (SPCL) (Ac-RFAAKAA-COOH). The molecular weight of SPCC is 750.9 g/mol, and 775.9 g/mol for SPCL. The peptides were stored in the freezer (<-15°C) for a maximum of 6 months.
- Source: JPT Peptide Technologies GmbH, Berlin, Germany.
- Rationale: Recommended test system in the international OECD guideline for DPRA studies.
- Calibration curve SPCC and SPCL: according to guideline
- Incubation: After preparation, the samples (reference controls, calibration solutions, co-elution control, positive controls and test item samples) were placed in the autosampler in the dark and incubated at 25±2.5°C. The incubation times between placement of the samples in the autosampler and analysis of the first RCcysB- or RClysB-sample were 23.5 hours and 23 hours, respectively. The time between the first RCcysB- or RClysB-injection and the last injection of a cysteine or lysine sequence, respectively, did not exceed 30 hours. Prior to HPLC-PDA analysis the samples were visually inspected for precipitation. The samples that showed a phase separation were centrifuged (at 400 g) for 5 minutes at room temperature.
- Analysis: All samples were analyzed according to the HPLC-PDA method presented in Table 1 ('Other information on methods and materials'). The HPLC sequences of the cysteine and lysine reactivity assay for the test item are presented in Table 2 ('Other information on materials and methods').

POSITIVE CONTROL: Cinnamic aldehyde
- Purity: 99.1%

DATA EVALUATION
The concentration of SPCC or SPCL was photometrically determined at 220 nm in each sample by measuring the peak area of the appropriate peaks by peak integration, and by calculating the concentration of peptide using the linear calibration curve derived from the standards.

The Percent Peptide Depletion was determined in each sample by measuring the peak area and dividing it by the mean peak area of the relevant reference controls C according to the following formula:
Percent Peptide Depletion = [1-(Peptide Peak Area in Replicate Injection (at 220 nm)/Mean Peptide Peak Area in Reference Controls (at 220 nm))]*100

In addition, the absorbance at 258 nm was determined in each sample by measuring the peak area of the appropriate peaks by peak integration. The ratio of the 220 nm peak area and the 258 nm peak was used as an indicator of co-elution. For each sample a ratio in the range of 90%< mean area ratio of control samples <110% gives a good indication that co-elution has not occurred.

DATA INTERPRETATION (see also 'Other information on materials and method')
The mean Percent Cysteine Depletion and Percent Lysine Depletion were calculated for the test item. Negative depletion was considered as “0” when calculating the mean. By using the Cysteine 1:10 / Lysine 1:50 prediction model, the threshold of 6.38% average peptide depletion was used to support the discrimination between a skin sensitiser and a non-sensitiser.

Results and discussion

Positive control results:

The positive control had a mean SPCC depletion of 70.8% ± 2.2% and a mean SPCL depletion of 54.7% ± 2.9%.

In vitro / in chemico

Results

Results 1

Key result

Run / experiment:

other: Cysteine Reactivity Assay

Parameter:

other: SPCC mean depletion (%)

Value:

0.3

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: SD: 0.2%; Upon preparation a precipitate was observed in the CC and test item samples while after incubation a phase separation was observed.

Results 2

Key result

Run / experiment:

other: Lysine Reactivity Assay

Parameter:

other: SPCL mean depletion (%)

Value:

0

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: SD: 0.0%; Upon preparation a precipitate was observed in the CC and test item samples while after incubation a phase separation was observed.

Other effects / acceptance of results:

Solubility of the test item:
- The test item showed to be soluble in ACN, isopropanol, acetone:ACN (1:1, v/v), DMSO:ACN (1:9, v/v), ethanol and methanol. Since ACN is the preferred solvent for the DPRA, this solvent was used to dissolve the test item in this study. In the SPCC assay, solubility of the test item was assessed in the above mentioned solvents by mixing 50 μL of the 100 mM test item solution with 750 μL phosphate buffer pH 7.5 and 200 μL ACN followed by vortex mixing. For each of the selected solvents, formation of a precipitate was observed. In the SPCL assay, solubility of the test item was assessed in the above mentioned solvents by mixing 250 μL of the 100 mM test item solution with ammonium acetate buffer pH 10.2 followed by vortex mixing. For each of the selected solvents, formation of a precipitate was observed.

Co-elution and precipitation:
In both the SPCC and the SPCL assay, the co-elution control (CC) as well as the test item samples were visually inspected upon preparation and after incubation. Upon preparation a precipitate was observed in the CC and test item samples while after incubation a phase separation was observed. In this case one cannot be sure how much test item remained in the solution to react with the peptide.

For details on results see tables included in 'Any other information on results incl. tables'.

Any other information on results incl. tables

SPCC and SPCL depletion and reactivity classification for the test item

Test item	SPCC depletion		SPCL depletion		Mean of SPCC and SPCL depletion	Reactivity class
	Mean	± SD	Mean	± SD		Cysteine 1:10 / Lysine 1:50 prediction model
m, alpha-dimethylstyrene and p, alpha-dimethylstyrene	0.3%	±0.2%	0.0%	±0.0%	0.1%	Negative: no or minimal reactivity

Acceptability of the DPRA assay

	Cysteine reactivity assay		Lysine reactivity assay
	Acceptability criteria	Results for SPCC	Acceptability criteria	Results for SPLC
Correlation coefficient (r^2) standard calibration curve	>0.99	0.995	>0.99	0.995
Mean peptide concentration RC-A samples (mM)	0.50 ± 0.05	0.505 ± 0.002	0.50 ± 0.05	0.523±0.020
Mean peptide concentration RC-C samples (mM)	0.50 ± 0.05	0.506 ± 0.001	0.50 ± 0.05	0.516±0.005
CV (%) for RC samples B and C	<15.0	0.5	<15.0	4.1
Mean peptide depletion cinnamic aldehyde (%)	60.8-100	70.8	40.2-69.0	54.7
SD of peptide depletion cinnamic aldehyde (%)	<14.9	2.2	<11.6	2.9
SD of peptide depletion for the test item (%)	<14.9	0.2	<11.6	0.0

Applicant's summary and conclusion

Interpretation of results:

other: Study is part of a weight of evidence approach and is not used for classification on its own.

Conclusions:

Reaction mass of m, alpha-dimethylstyrene and p, alpha-dimethylstyrene was negative in the DPRA, performed according to OECD 442C and GLP principles, and was classified in the “no or minimal reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model. However, since a phase separation was observed after the incubation period for both SPCC and SPCL, one cannot be sure how much test item remained in the solution to react with the peptides. Consequently, this negative result is uncertain and should be interpreted with due care.