3,4-dihydroxybenzonitrile

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

28 November - 07 December 2016

Reliability:

2 (reliable with restrictions)

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)

Justification for non-LLNA method:

In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimize the need of in vivo testing. DPRA is one of the recommended in chemico test systems in the international OECD guidelines.

Test material

Test material information

Constituent 1

Reference substance name:

3,4-dihydroxybenzonitrile

EC Number:

241-367-9

EC Name:

3,4-dihydroxybenzonitrile

Cas Number:

17345-61-8

Molecular formula:

C7H5NO2

IUPAC Name:

3,4-dihydroxybenzonitrile

Test material form:

solid: particulate/powder

Remarks:

white to brownish

Details on test material:

Batch 151222

In chemico test system

Details on the study design:

Principle of the assay
The Direct Peptide Reactivity Assay (DPRA) is an in chemico method which quantifies the remaining concentration of cysteine- or lysine-containing peptide following 24 hours incubation with the test item at 25°C. The synthetic peptides contain phenylalanine to aid in the detection. The relative peptide concentration was measured by high-performance liquid chromatography (HPLC) with gradient elution and photodiode array (PDA) detection at 220 nm and 258 nm. Cysteine- and lysine peptide Percent Depletion Values were calculated and used in a prediction model which allows assigning the test chemical to one of four reactivity classes used to support the discrimination between sensitisers and non-sensitisers.

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.
Source and batch
JPT Peptide Technologies GmbH, Berlin, Germany.
Batch No.280916K-44
Storage: The peptides were stored in the freezer (<-15°C) for a maximum of 6 months.

Positive control
Cinnamic aldehyde, kosher (104-55-2)

Reagents
Acetonitrile (ACN) HPLC-grade, Fisher Chemicals, Loughborough, England
Ammonium acetate (CH3COONH4) Fractopur, Biosolve, Valkenswaard, The Netherlands
Ammonium hydroxide (NH4OH) 25.0% Merck, Darmstadt, Germany
Disodiumhydrogenphosphate Emsure, Merck
(Na2HPO4.12H2O)
Milli-Q water (MQ) Tap water purified by reversed osmosis and subsequently passed over activated carbon and ion-exchange cartridges Sodiumdihydrogenphosphate Emsure, Merck
(NaH2PO4.H2O)
Trifluoroacetic acid, >99% (TFA) Sigma Aldrich, Zwijndrecht, The Netherlands

Test item preparation
CH02906 stock solutions were prepared freshly for each reactivity assay. For the cysteine and lysine reactivity assay respectively 19.78 mg and 13.49 mg of CH02906 were pre-weighed into a clean amber glass vial and dissolved, just before use, in respectively 1464 µL and 998 µL acetonitryl (ACN) 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.

HPLC-PDA analysis
The concentration of SPCC or SPCL in the samples was measured by HPLC-PDA. Sample analysis was performed using the following system:
• Surveyor MS HPLC pump (Thermo Scientific, Breda, The Netherlands)
• HTC PAL autosampler (DaVinci, Rotterdam, The Netherlands)
• LC Column oven 300 (Thermo Scientific)
• Surveyor PDA detector (Thermo Scientific)

Cysteine reactivity assay
The reactivity of CH02906 towards SPCC was determined by quantification of the remaining concentration of SPCC using HPLC-PDA analysis following 24±2 hours of incubation at 25±2.5°C.

Lysine reactivity assay
The reactivity of CH02906 towards SPCL was determined by quantification of the remaining concentration of SPCL using HPLC-PDA analysis following 24±2 hours of incubation at 25±2.5°C.

The following criteria had to be met for a run to be considered valid:
a) The standard calibration curve had to have an r2>0.99.
b) The mean Percent Peptide Depletion value of the three replicates for the positive control cinnamic aldehyde had to be between 60.8% and 100% for SPCC and between 40.2% and 69.0% for SPCL.
c) The maximum standard deviation (SD) for the positive control replicates had to be <14.9% for the Percent Cysteine Peptide Depletion and <11.6% for the Percent Lysine Peptide Depletion.
d) The mean peptide concentration of Reference Controls A had to be 0.50±0.05 mM.
e) The Coefficient of Variability (CV) of peptide areas for the nine Reference Controls B and C in ACN had to be <15.0%.
The following criteria had to be met for a test item’s results to be considered valid:
a) The maximum standard deviation for the test item replicates had to be <14.9% for the Percent Cysteine Depletion and <11.6% for the Percent Lysine Depletion.
b) The mean peptide concentration of the three Reference Controls C in the appropriate solvent had to be 0.50±0.05 mM.

Results and discussion

In vitro / in chemico

Results

Results 1

Run / experiment:

other: Cysteine

Parameter:

other: mean SPCC A220/A258 area ratio

Value:

14.03

Positive controls validity:

valid

Results 2

Key result

Run / experiment:

other: Cysteine

Parameter:

other: mean Percent SPCC Depletion

Remarks:

%

Value:

13.7

Remarks on result:

no indication of skin sensitisation

Remarks:

SD ± 5.1%. the Cysteine 1:10 prediction model was used for classification due to co-elution of CH02906 with SPCL

Results 3

Run / experiment:

other: lysine

Parameter:

other: mean SPCL A220/A258

Remarks on result:

not determinable

Remarks:

the mean SPCL A220/A258 could not be calculated since co-elution of CH02906 with SPCL occurred

Results 4

Run / experiment:

other: lysine

Parameter:

other: Percent SPCL Depletion

Remarks on result:

not determinable

Remarks:

As a result of co-elution, the Percent SPCL Depletion could not be calculated for CH02906

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
Acceptability of the cysteine reactivity assay
The correlation coefficient (r2) of the SPCC standard calibration curve was 0.992. Since the r2 was >0.990, the SPCC standard calibration curve was accepted.
The mean peptide concentration of Reference Controls A was 0.498±0.011 mM. The mean peptide concentration of Reference Controls C was 0.526±0.011 mM. The mean Reference Control samples A and C were both within the acceptance criteria of 0.50±0.05 mM. This confirms the suitability of the HPLC system and indicates that the solvent (ACN) used to dissolve CH02906 did not impact the Percent SPCC Depletion.
The SPCC peak areas for Reference controls B and C are presented in Table 9. The CV of the peptide areas for the nine Reference Controls B and C was 2.9%. This was within the acceptance criteria (CV <15.0%) and confirms the stability of the HPLC run over time.
The mean area ratio (A220/A258) of the Reference Control samples was 14.55. The mean A220/A258 ratio ± 10% range was 13.10 - 16.01. Each sample showing an A220/A258 ratio within this range gives an indication that co-elution has not occurred.
The Percent SPCC Depletion was calculated versus the mean SPCC peak area of Reference Controls C. The mean Percent SPCC Depletion for the positive control cinnamic aldehyde was 76.7% ± 1.0%. This was within the acceptance range of 60.8% to 100% with a SD that was below the maximum (SD <14.9%).

Acceptability of the lysine reactivity assay
The mean peptide concentration of Reference Controls A was 0.470±0.017 mM. The mean peptide concentration of Reference Controls C was 0.465±0.015 mM. The mean Reference Control samples A and C were both within the acceptance criteria of 0.50±0.05 mM. This confirms the suitability of the HPLC system and indicates that the solvent (ACN) used to dissolve CH02906 did not impact the Percent SPCL Depletion.
The CV of the peptide areas for the nine Reference Controls B and C was 3.9%. This was within the acceptance criteria (CV <15.0%) and confirms the stability of the HPLC run over time.
The mean area ratio (A220/A258) of the Reference Control samples was 14.81. The mean A220/A258 ratio ± 10% range was 13.33 - 16.29. Each sample showing an A220/A258 ratio within this range gives an indication that co-elution has not occurred.
The results of the positive control cinnamic aldehyde are presented in Table 16. The Percent SPCL Depletion was calculated versus the mean SPCL peak area of Reference Controls C. The mean Percent SPCL Depletion for the positive control cinnamic aldehyde was 44.6% ± 3.1%. This was within the acceptance range of 40.2 - 69%, with a SD that was below the maximum criteria (SD <11.6%).

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

It can be concluded that this DPRA test is valid, and that CH02906 was negative in the DPRA, and is classified in the “no or minimal reactivity class” when using the Cysteine 1:10 prediction model.

Executive summary:

In this study the reactivity of CH02906 towards model synthetic peptides containing either cysteine (SPCC) or lysine (SPCL) was determined. Following 24 hours of incubation of CH02906 with either SPCC or SPCL, the relative peptide concentration was determined by High-Performance Liquid Chromatography (HPLC) with gradient elution and photodiode array (PDA) detection at 220 nm and 258 nm. SPCC and SPCL Percent Depletion Values were calculated and used in a prediction model which allows assigning the test chemical to one of four reactivity classes used to support the discrimination between sensitisers and non-sensitisers.

The study procedures described in this report were based on the most recent OECD guidelines.

Acetonitrile (ACN) was found to be an appropriate solvent to dissolve CH02906, and was therefore used in this DPRA study.

The correlation coefficient (r²) of the SPCC and SPCL standard calibration curves were 0.992 and 0.996, respectively. The calibration curves were within the acceptance criteria, since the r²for both curves was >0.990.

For the cysteine reactivity assay, the mean peptide concentration of Reference Controls A was 0.498±0.011mM while the mean peptide concentration of Reference Controls C was 0.526±0.011mM. The means of Reference Control samples A and C were within the acceptance criteria of 0.50±0.05 mM. This confirms the suitability of the HPLC system, and indicates that the solvent (ACN) used to dissolve the test item did not impact the Percent SPCC Depletion.

The Coefficient of Variability (CV) of peptide areas for the nine Reference Controls B and C was 2.9%. This was within the acceptance criteria (CV<15.0%), and confirms the stability of the HPLC run over time. The mean area ratio (A₂₂₀/A₂₅₈) of the Reference Control samples was 14.55. The mean A₂₂₀/A₂₅₈ratio± 10% range was13.10-16.01. Each sample showing an A₂₂₀/A₂₅₈ratio within this range gives an indication that co-elution has not occurred.

The mean Percent SPCC Depletion for the positive control cinnamic aldehyde was 76.7%± 1.0%. This was within the acceptance range of 60.8 to 100% with a standard deviation (SD) that was below the maximum (SD <14.9%).

For CH02906, the mean SPCCA₂₂₀/A₂₅₈area ratio was 14.03. Since this is within the 13.10-16.01 range this indicates that there is no co-elution ofCH02906 with SPCC. The mean Percent SPCC depletion for CH02906 was 13.7%± 5.1%.

For the lysine reactivity assay, the mean peptide concentration of Reference Controls A was             0.470±0.017mM while the mean peptide concentration of Reference Controls C was 0.465±0.015mM. The means of Reference Control samples A and C were within the acceptance criteria of 0.50±0.05 mM. This confirms the suitability of the HPLC system, and indicates that the solvent (ACN) used to dissolve the test item did not impact the Percent SPCL Depletion.

The CV of peptide areas for the nine Reference Controls B and C was 3.9%. This was within the acceptance criteria (CV<15.0%) and confirms the stability of the HPLC run over time. The mean area ratio (A₂₂₀/A₂₅₈) of the Reference Control samples was 14.81. The mean A₂₂₀/A₂₅₈ratio± 10% range was13.33 - 16.29. Each sample showing an A₂₂₀/A₂₅₈ratio within this range gives an indication that co-elution has not occurred.

The mean Percent SPCL Depletion for cinnamic aldehyde was 44.6% ± 3.1%. This was within the acceptance range of 40.2 to 69.0%, with a SD that was below the maximum (SD <11.6%).

ForCH02906, the mean SPCL A₂₂₀/A₂₅₈could not be calculated since co-elution ofCH02906with SPCL occurred. As a result of this co-elution, the Percent SPCL Depletion could not be calculated for CH02906.

An overview of the individual results of the cysteine and lysine reactivity assays are presented in Table1. In the cysteine reactivity assay, CH02906 showed 13.7% SPCC depletion while in the lysine reactivity assay co-elution of CH02906 with the lysine peptide occurred. CH02906 was classified in the “no or minimal reactivityclass” when using the Cysteine 1:10 prediction model. Therefore, CH02906 was considered to be negative in the DPRA.

Table1: SPCC and SPCL depletion and reactivity classification forCH02906

Test item	SPCC depletion		SPCL depletion		Reactivity class
	Mean	± SD	Mean	± SD	Cysteine 1:10 prediction model
CH02906	13.7%	5.1%	NA, co-elution		No or minimal reactivity

                                   SD = Standard Deviation, NA = Not Applicable.

It can be concluded that this DPRA test was valid and that CH02906 was negative in the DPRA, and is classified in the “no or minimal reactivityclass” when using the Cysteine 1:10 prediction model.