p-benzoquinone dioxime

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

13-09-2018 to 26-09-2018

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Guideline study performed under GLP. All relevant validity criteria were met. Test method considered to cover Key Event-1 under OECD 168 (2012) and OECD 255 (2016) and OECD 256 (2016).

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))

Deviations:

no

GLP compliance:

yes

Remarks:

uideline study performed under GLP. All relevant validity criteria were met. Test method considered to cover Key Event-1 under OECD 168 (2012) and OECD 255 (2016) and OECD 256 (2016).

Type of study:

direct peptide reactivity assay (DPRA)

Test material

Test material information

Constituent 1

Reference substance name:

p-benzoquinone dioxime

EC Number:

203-271-5

EC Name:

p-benzoquinone dioxime

Cas Number:

105-11-3

Molecular formula:

C6H6N2O2

IUPAC Name:

N-[(1Z,4Z)-4-(hydroxyimino)cyclohexa-2,5-dien-1-ylidene]hydroxylamine

Test material form:

solid

Details on test material:

- Physical state: Solid
- Storage condition of test material: In refrigerator (2-8°C) container flushed with nitrogen
- Other: Brown powder

In chemico test system

Details of test system:

cysteine peptide, (Ac-RFAACAA-COOH)

lysine peptide (Ac-RFAAKAACOOH)

Details on the study design:

Skin sensitisation (In chemico test system) - Details on study design:
- The study was conducted in accordance with the OECD TG 422C – In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA)
- The study protocol was validated with the proficiency chemicals prescribed in the OECD test guideline 442C. The results of the testing on the proficiency chemicals at the test facility is in the public domain (refer to study references provided in the full study report at the relevant test facility). All ten proficiency chemicals described in OECD TG 442C: Annex 2, were according to the test facility correctly predicted in a study conducted outside the present study. This information is in the public domain.
- Solubility of the test item in an appropriate solvent was assessed before performing the DPRA. An appropriate solvent dissolved the test item completely, i.e. by visual inspection the solution had 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) and isopropanol (IPA). At a concentration of 100 mM, the test item was not soluble in ACN, MQ or ACN:MQ (1:1, v/v), but was soluble in IPA. Therefore, IPA solvent was used to dissolve the test item. 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 (UV) methodology are reported in the full study report.
- Preparation of synthetic peptide solutions [Synthetic Peptide Containing Cysteine (SPCC) and Synthetic Peptide Containing Lysine (SPCL)]
1. Cysteine: A stock solution of 0.667 mM SPCC (0.501 mg SPCC/mL) was prepared by dissolving 10.2 mg of SPCC in 20.36 mL phosphate buffer pH 7.5. The mixture was stirred for 5 minutes followed by 5 minutes sonication. SPCC Reference Control solutions: Three 0.5 mM SPCC reference control (RC) solutions (RCcysA, RCcysB and RCcysC) were prepared in amber vials by mixing 750 µL of the 0.667 mM SPCC stock solution with 250 µL ACN. In addition, a RCcysCIPA sample was included to evaluate the effect of the solvent that was used to dissolve the test item on the Percent Peptide Depletion. The RCcysCIPA sample was prepared by mixing 750 µL of the 0.667 mM SPCC stock solution with 200 µL ACN and 50 µL IPA. The SPCC was subsequently calibrated at multiple concentrations. Co-elution control, test item and positive control samples were also prepared (full details on calibration and sample preparation available in the full study report).
2. Lysine: A stock solution of 0.667 mM SPCL (0.518 mg SPCL/mL) was prepared by dissolving 10.6 mg of SPCL in 20.46 mL of ammonium acetate buffer pH 10.2 followed by stirring for 5 minutes. SPCL Reference Control solutions: Three 0.5 mM SPCL reference control (RC) solutions (RClysA, RClysB and RClysC) were prepared in amber vials by mixing 750 µL of the 0.667 mM SPCL stock solution with 250 µL ACN. In addition, a RClysCIPA sample was included to evaluate the effect of the solvent that was used to dissolve the test item on the Percent Peptide Depletion. The RClysCIPA sample was prepared by mixing 750 µL of the 0.667 mM SPCL stock solution with 250 µL IPA. The SPLC was subsequently calibrated at multiple concentrations. Co-elution control, test item and positive control samples were also prepared (full details on calibration and sample preparation available in the full study report).
- Sample incubations: 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 time between placement of the samples in the autosampler and analysis of the first RCcysB- or RClysB-sample was 24.5 hours. 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.
- Acceptability criteria:
(i) standard calibration curve(s) are to have an r2 > 0.99. (Actual: SPCC r2 = 0.998 and SPLC r2 = 0.9942)
(ii) mean Percent Peptide Depletion value of the three replicates for the positive control cinnamic aldehyde are to be between 60.8% and 100% for SPCC and between 40.2% and 69.0% for SPCL. (Actual: SPCC 70.4% ± 1.1% and 50.5% ± 3.8%)
(iii) maximum standard deviation (SD) for the positive control replicates are to be <14.9% for the Percent Cysteine Peptide Depletion and <11.6% for the Percent Lysine Peptide Depletion. (Actual SPCC PC : SD = 1.1% and SPCL PC : SD = 3.8%)
(iv) mean peptide concentration of Reference Controls A is to be 0.50 ± 0.05 mM. (Actual: Cysteine A, C and CIPA reference controls: 0.514 ± 0.022 mM, 0.504 ± 0.002 mM and 0.489 ± 0.022 mM ; Lysine A, C and CIPA reference controls: 0.515 ± 0.015 mM, 0.529 ± 0.009 mM and 0.523 ± 0.012 mM, respectively).
(v) Coefficient of Variation (CV) of peptide areas for the nine Reference Controls B and C in ACN are to be <15.0%. (Actual: Cysteine Reference Controls B and C : CoV = 2.3% ; Lysine: Reference Controls B and C was 3.2%)
Other: Within the Cysteine Reactivity Assay: In the CC sample no peak at 220 nm was observed at the retention time of SPCC, however, at 258 nm a peak was observed. This demonstrated that at a wavelength of 220 nm, which is used to determine the SPCC depletion, there was no interference of the test item with SPCC. It was not possible to calculate the mean SPCC A220/A258 area ratio since the test item displayed high reactivity towards SPCC. Overall, it was concluded that the test item did not interfere with SPCC at a wavelength of 220 nm for quantitative analysis purposes. Within the Lysine Reactivity Assay: In the CC sample no peak at 220 nm was observed at the retention time of SPCL whereas a small response at 258 nm was observed. This demonstrated that at a wavelength of 220 nm, which is used to determine the SPCL depletion, there was no interference of the test item with SPCL. For the A-lys samples, the mean SPCL A220/A258 area ratio was 6.13. This was outside the 15.16-18.52 range, however, since the test item did not interfere with SPCL at 220 nm, it can be concluded that the responses at a wavelength of 220 nm were suitable for quantitative analysis purposes.
All acceptance criteria were fulfilled for the PC (cinnamic aldehyde).
All relevant acceptability criteria were met.
- Synthetic peptides:
Cysteine- containing peptide: Ac-RFAACAA-COOH (MW=750.9) – full details on source provided in full study report.
Lysine-containing peptide: Ac-RFAAKAA-COOH (MW=775.9) – full details on source provided in full study report.
- Controls:
Positive control (PC): Cinnamic aldehyde (CAS 104-55-2; 99.1%) – full details on source provided in full study report.
Negative control (NC): Vehicle = Isopropanol

Evaluation of results: In accordance with OECD TG 442C – Table 1.
Test item reactivity was determined by mean peptide depletion and was rated as high, moderate, low, or minimal:
Mean peptide depletion [%] Reactivity
> 42.47 high reactivity (Positive)
> 22.62 < 42.47 moderate reactivity (Positive)
> 6.38 < 22.62 low reactivity (Positive)
< 6.38 minimal reactivity (Negative)

Vehicle / solvent:

isopropanol

Positive control:

cinnamic aldehyde

Results and discussion

Positive control results:

- All PC acceptability criteria were met.
- PC CYS-peptide depletion (mean): 70.4% ± 1.1% (high reactivity)
- PC LYS-peptide depletion (mean): 50.5% ± 3.8% (moderate reactivity)

In vitro / in chemico

Results

Results 1

Key result

Run / experiment:

mean

Parameter:

mean cystein depletion

Value:

100 %

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: HIGH REACTIVITY CLASS

Remarks:

n = 3 ; See 'any other information on results incl. tables' for further information

Results 2

Key result

Run / experiment:

mean

Parameter:

mean lysine depletion

Value:

0.6 %

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other:

Remarks:

n = 3 ; Test item appears to have no or minimal reactivity to Lys peptide ; See 'any other information on results incl. tables' for further information

Outcome of the prediction model:

high reactivity [in chemico]

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: None reported.

DEMONSTRATION OF TECHNICAL PROFICIENCY: The study protocol was validated with the proficiency chemicals prescribed in the OECD test guideline 442C. The results of the testing on the proficiency chemicals at the test facility is in the public domain (refer to study references provided in the full study report at the relevant test facility). All ten proficiency chemicals described in OECD TG 442C: Annex 2, were according to the test facility correctly predicted in a study conducted outside the present study. This information is in the public domain.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: All criteria met.
- Acceptance criteria met for positive control: All criteria met.
- Acceptance criteria met for variability between replicate measurements: All criteria met.
- Range of historical values if different from the ones specified in the test guideline: Not applicable.

- Acceptability criteria:
(i) standard calibration curve(s) are to have an r2 > 0.99. (Actual: SPCC r2 = 0.998 and SPLC r2 = 0.9942)
(ii) mean Percent Peptide Depletion value of the three replicates for the positive control cinnamic aldehyde are to be between 60.8% and 100% for SPCC and between 40.2% and 69.0% for SPCL. (Actual: SPCC 70.4% ± 1.1% and 50.5% ± 3.8%)
(iii) maximum standard deviation (SD) for the positive control replicates are to be <14.9% for the Percent Cysteine Peptide Depletion and <11.6% for the Percent Lysine Peptide Depletion. (Actual SPCC PC : SD = 1.1% and SPCL PC : SD = 3.8%)
(iv) mean peptide concentration of Reference Controls A is to be 0.50 ± 0.05 mM. (Actual: Cysteine A, C and CIPA reference controls: 0.514 ± 0.022 mM, 0.504 ± 0.002 mM and 0.489 ± 0.022 mM ; Lysine A, C and CIPA reference controls: 0.515 ± 0.015 mM, 0.529 ± 0.009 mM and 0.523 ± 0.012 mM, respectively).
(v) Coefficient of Variation (CV) of peptide areas for the nine Reference Controls B and C in ACN are to be <15.0%. (Actual: Cysteine Reference Controls B and C : CoV = 2.3% ; Lysine: Reference Controls B and C was 3.2%)
Other: Within the Cysteine Reactivity Assay: In the CC sample no peak at 220 nm was observed at the retention time of SPCC, however, at 258 nm a peak was observed. This demonstrated that at a wavelength of 220 nm, which is used to determine the SPCC depletion, there was no interference of the test item with SPCC. It was not possible to calculate the mean SPCC A220/A258 area ratio since the test item displayed high reactivity towards SPCC. Overall, it was concluded that the test item did not interfere with SPCC at a wavelength of 220 nm for quantitative analysis purposes. Within the Lysine Reactivity Assay: In the CC sample no peak at 220 nm was observed at the retention time of SPCL whereas a small response at 258 nm was observed. This demonstrated that at a wavelength of 220 nm, which is used to determine the SPCL depletion, there was no interference of the test item with SPCL. For the A-lys samples, the mean SPCL A220/A258 area ratio was 6.13. This was outside the 15.16-18.52 range, however, since the test item did not interfere with SPCL at 220 nm, it can be concluded that the responses at a wavelength of 220 nm were suitable for quantitative analysis purposes. All acceptance criteria were fulfilled for the PC (cinnamic aldehyde).
All relevant acceptability criteria were met.

Any other information on results incl. tables

Table 1.0 – Acceptability of the DPRA

	Cysteine reactivity assay		Lysine reactivity assay
	Acceptability criteria	Results for SPCC	Acceptability criteria	Results for SPCL
Correlation coefficient (r2) standard calibration curve	>0.99	0.998	>0.99	0.994
Mean peptide concentration RC-A samples (mM)	0.50 ± 0.05	0.514 ± 0.022	0.50 ± 0.05	0.515 ± 0.015
Mean peptide concentration RC-C samples (mM)	0.50 ± 0.05	0.504 ± 0.002	0.50 ± 0.05	0.529 ± 0.009
Mean peptide concentration RC-CIPA samples (mM)	0.50 ± 0.05	0.489 ± 0.022	0.50 ± 0.05	0.523 ± 0.012
CV (%) for RC samples B and C	<15.0	2.3	<15.0	3.2
Mean peptide depletion PC (cinnamic aldehyde) (%)	60.8-100	70.4	40.2-69.0	50.5
SD of peptide depletion PC (cinnamic aldehyde) (%)	<14.9	1.1	<11.6	3.8
SD of peptide depletion for the test item (%)	<14.9	0.0	<11.6	0.5

Where: RC = Reference Control; CV = Coefficient of Variation; SD = Standard Deviation

 

Table 2.0 – Results of the DPRA with the test item

	SPCC depletion (CYSTEINE)		SPCL depletion (LYSINE)		Mean of SPCC and SPCL depletion
	Mean	± SD	Mean	± SD
Test item	100.0%	±0.0%	0.6%	±0.5%	50.3%

Applicant's summary and conclusion

Interpretation of results:

other: The test item gave a positive in DPRA and was classified in the “high reactivity class” using the Cysteine 1:10 / Lysine 1:50 prediction model. The result will be considered within a weight of evidence assessment for Classification and Labelling purposes

Conclusions:

Under the condition of this study, the test item is considered to be sensitising to the skin. The test item indicated a positive in the DPRA and was classified in the “high reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model.

Executive summary:

The study was performed to the OECD TG 442C in chemico Direct peptide reactivity Assay (DPRA) guideline under GLP. The test item was assessed for reactivity to model synthetic peptides containing either cysteine (SPCC) or lysine (SPCL). After incubation of the test item 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 item to one of four reactivity classes used to support the discrimination between sensitizers and non-sensitizers. Isopropanol (IPA) was found to be an appropriate solvent to dissolve the test item. Upon preparation of the SPCC and SPCL test item samples, no precipitate or phase separation was observed in any of the samples. After incubation of the SPCC and SPCL test item samples, a precipitate was observed. In the cysteine reactivity assay the test item showed 100.0% SPCC depletion while in the lysine reactivity assay the test item showed 0.6% SPCL depletion. The mean of the SPCC and SPCL depletion was 50.3% and as a result the test item was considered to be positive in the DPRA and classified in the “high reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model. All relevant test acceptability criteria were met.