2,4-dihydroxybenzoic acid

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12.3.2018 - 16.3.2018

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))

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Justification for non-LLNA method:

non-animal test

Test material

Test material information

Constituent 1

Reference substance name:

2,4-dihydroxybenzoic acid

EC Number:

201-946-9

EC Name:

2,4-dihydroxybenzoic acid

Cas Number:

89-86-1

Molecular formula:

C7H6O4

IUPAC Name:

2,4-dihydroxybenzoic acid

Test material form:

solid

In chemico test system

Details on the study design:

HPLC conditions
Column: Agilent Zorbax SB-C18, 100x2.1 mm, 3.5µm
Precolumn: Phenomex security guard C18, 4.0 x 2.0 mm
Mobile phase A: 0.1% Trifluoracetic acid in water
Mobile phase B: 0.085% Trifluoracetic acid in acetonitrile
Time programmer: 0 min 10 % B
10 min 25 % B
11 min 90 % B
13 min 90 % B
13.5-20 min 10 % B
Column temperature: 30 °C
Sample temperature: 25°C
Flow rate: 0.35 mL/min
Injection volume: 10 μL
Detection: 220 nm (258 nm)

Results and discussion

In vitro / in chemico

Results

Results 1

Key result

Run / experiment:

other: Replicate 1

Parameter:

other: % cystein depletion

Value:

1.4

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Key result

Run / experiment:

other: Replicate 2

Parameter:

other: % cystein depletion

Value:

8.3

Negative controls validity:

valid

Positive controls validity:

valid

Results 3

Key result

Run / experiment:

other: Replicate 3

Parameter:

other: % cystein depletion

Value:

13.5

Negative controls validity:

valid

Positive controls validity:

valid

Results 4

Key result

Run / experiment:

other: Replicate 1

Parameter:

other: % lysine depletion

Value:

0

Negative controls validity:

valid

Positive controls validity:

valid

Results 5

Key result

Run / experiment:

other: Replicate 2

Parameter:

other: % lysine depletion

Value:

0.6

Negative controls validity:

valid

Positive controls validity:

valid

Results 6

Key result

Run / experiment:

other: Replicate 3

Parameter:

other: % lysine depletion

Value:

0.5

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

System Suitability
A standard calibration curve was generated for both the Cysteine and the Lysine peptides.
Linearity was determined by mathematical treatment of results obtained by analysis of calibration solutions. These solutions were prepared from series dilution. The concentration interval of the calibration solutions was 0.534 – 0.01669 mM for each peptide.
Each prepared calibration solution was injected and analysed by HPLC with DAD detection.

Any other information on results incl. tables

Using 1 ml autosampler vials as container, was prepare the sample by adding the reagents in the quantity and order listed in Table No.1 and Table No.2.:

 

Table No.1: Cystein peptide, 1:10 Ratio

1:10 ratio, Cysteine peptide (0.5 mM Peptide, 5 mM test chemical)

750 µL Cysteine peptide solution (or 750 µL Phosphate buffer, pH= 7.5 for co-elution controls)

200 µL Acetonitrile

50 µL test chemical solution or 50µL solvent (ACN) for reference controls or 50 µL positive (negative) control solution for positive (negative) controls

 

Table No.2: Lysine peptide, 1:50 Ratio

1:50 ratio, Lysine peptide (0.5 mM Peptide, 25 mM test chemical)

750 µL Lysine peptide solution (or 750 µL Ammonium acetate buffer, pH= 10.2 for co-elution controls)

250 µL test chemical solution or 250µL solvent (ACN) for reference controls or 250 µL positive (negative) control solution for positive (negative) controls

 

The vials were closed, mixed and placed in the HPLC autosampler (dark) at 25 °C for 24 hours. Then, samples were visually inspected prior to HPLC analysis. Each test chemical was analysed in triplicate for both peptides.

Table No.4: Measured data of Reference control A

Reference control A
	Cysteine peptide peak area at 220 nm	Cysteine peptideconcentration(mM)	Lysine peptide peak area at 220 nm	Lysine peptideconcentration(mM)
Replicate1	2784163	0.495	2940878	0.513
Replicate2	2905381	0.516	2993258	0.522
Replicate3	2897757	0.514	2979549	0.520
Mean	2862434	0.508	2971228	0.518
SD	67891	0.012	27163	0.005
CV	2.37	2.280	0.91	0.912

 

As a negative control, 1-butanolat a concentration of 100 mM in acetonitrilewas used. It was evaluated by peptide peak area at 220 nm of each control, peptide concentration (mM) and mean peptide concentration, SD and CV. The mean Percent peptide depletion value of three replicates for Cinnamic aldehyd and 1-butanol was calculated.

 

Table No.5: Measured data of Positive control

Positive control – Cynnamic aldehyd
	Cysteine peptide peak area at 220 nm	Percent Cystein depletion %	Lysine peptide peak area at 220 nm	Percent Lysine depletion %
Replicate 1	810317	69.4	1070054	63.6
Replicate 2	700955	73.5	1048930	64.3
Replicate 3	717294	72.9	995839	66.1
Mean	742855	71.9	1038274	64.7
SD	58992	2.23	38238	1.30
CV	7.94	3.10	3.68	2.01

 

 Table No.6: Measured data of Negative control

Negative control – 1-butanol
	Cysteine peptide peak area at 220 nm	Percent Cystein depletion %	Lysine peptide peak area at 220 nm	Percent Lysine depletion %
Replicate 1	2684674	0.00	2924106	0.48
Replicate 2	2672111	0.00	2930251	0.28
Replicate 3	2506260	5.20	2897810	1.38
Mean	2621015	1.73	2917389	0.71
SD	99579	3.00	17232	0.59
CV	3.80	173.21	0.59	82.22

 

Reference control B was made with acetonitrile and its replicates was injected in the beginning and in the end of experimental run to verify the stability of the peptide over analysis time.

Reference control C was made with acetonitrile (test item and positive, negative control were soluble in acetonitrile). Reference control C was used to calculate Percent peptide depletion to according formula.

It was evaluated by peptide peak area at 220 nm of each Reference control B and C in replicate, mean peptide peak area of the nine (in sum) reference controls B and C in acetonitrile, SD and CV, peptide concentration (mM) and mean peptide concentration, SD and CV.

Reference control C was made with mixture of acetonitrile and ultra pure water (1:1) (for test item; was soluble in this mixture). Reference control C was used to calculated Percent peptide depletion to according formula. It was evaluated by peptide peak area at 220 nm of each Reference C in replicate, mean peptide peak area, SD and CV, peptide concentration (mM) and mean peptide concentration, SD and CV.

 

Table No.7: Measured data of Reference control B

Reference control B
	Cysteine peptide peak area at 220 nm	Cysteine peptideconcentration(mM)	Lysine peptide peak area at 220 nm	Lysine peptideconcentration(mM)
Replicate 1	2803587	0.498	2907195	0.507
Replicate 2	2741476	0.488	2980060	0.520
Replicate 3	2770753	0.493	2924438	0.510
Replicate 4	2611516	0.466	2938420	0.512
Replicate 5	2554622	0.456	2939514	0.513
Replicate 6	2629747	0.469	2978208	0.519
Mean	2685284	0.478	2944639	0.514
SD	100057	0.017	29177	0.005
CV	3.73	3.54	0.99	0.99

 

 Table No.8: Measured data of Reference control C with Acetonitrile

Reference control C
	Cysteine peptide peak area at 220 nm	Cysteine peptideconcentration(mM)	Lysine peptide peak area at 220 nm	Lysine peptideconcentration(mM)
Replicate 1	2676251	0.477	2935475	0.512
Replicate 2	2670553	0.476	2957180	0.516
Replicate 3	2584788	0.461	2922384	0.510
Mean	2643864	0.471	2938346	0.513
SD	51241	0.009	17575	0.003
CV	1.94	1.90	0.60	0.60

 

CV of Cysteine peptide peak areas for nine Reference control B and C in acetonitrile is 3.21. CV of Lysine peptide peak areas for nine Reference control B and C in acetonitrile is 0.85.

 

Table No.9: Measured data of Reference control C with mixture of Acetonitrile and Ultra pure water (1:1)

Reference control C - Mixture
	Cysteine peptide peak area at 220 nm	Cysteine peptideconcentration(mM)	Lysine peptide peak area at 220 nm	Lysine peptideconcentration(mM)
Replicate 1	2726192	0.485	2912707	0.508
Replicate 2	2617440	0.467	2924706	0.510
Replicate 3	2612946	0.466	2872484	0.501
Mean	2652193	0.473	2903299	0.506
SD	64125	0.011	27353	0.005
CV	2.42	2.26	0.94	0.93

 

  Table No.10: Measured data of peak purity indicator: area ratio 220/258

Reference control C - Mixture				2.4-Dihydroxybenzoic acid
	Peak Area at 220 nm	Peak Area at 258 nm	Area ratio of 220/258	Peak Area at 220 nm	Peak Area at 258 nm	Area ratio of 220/258
Replicate 1	2726192	76913	35.4	2615626	129256	20.2
Replicate 2	2617440	73915	35.4	2430831	123488	19.7
Replicate 3	2612946	72583	36.0	2294237	113882	20.1
Mean			35.6			20.0

 

As can be seen from the above results of the co-elution between the test item and the Cysteine peptide, it has been demonstrated. 

Table No.12: Measured data of test item 2.4-Dihydroxybenzoic acid

2.4-Dihydroxybenzoic acid
	Cysteine peptide peak area at 220 nm	Percent Cystein  depletionestimated%	Lysine peptide peak area at 220 nm	Percent Lysine depletion %
Replicate 1	2615626	1.4	2910999	0.0
Replicate 2	2430831	8.3	2886768	0.6
Replicate 3	2294237	13.5	2888139	0.5
Mean	2446898	7.7*	2895302	0.4
SD	161296	6.08	13611	0.32
CV	6.59	78.57	0.47	86.85

* Co-elution – Mean percent depletion estimated

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Conclusions:

Test item 2.4-Dihydroxybenzoic acid coelutes with cysteine peptide. Data from the testing are inconclusive, DPRA prediction cannot be made.

Executive summary:

In a Direct Peptide Reactivity Assay (DPRA) according to OECD TG 442C all study acceptance criteria have not been successfully met.

 

The result of the assay is affected by the co-elution between the test item and Cysteine peptide.

Co-elution of chemical and peptide was explored by looking at the UV spectrum at 258 nm and was calculated the area ratio of 220/258. This value should be consistent over all samples and standards for pure peptide peak and thus gives a measure of peak purity. In this case, the value disables the possible co-elution. In cases where the test chemical co-elutes with the cysteine peptide and percent depletion can not be estimated, a determination of reactivity can not be made based on the percent depletion data from the lysine reaction alone. The data should be reported as "inconclusive". The lysine reactivity alone does not carry enough weight to drive a lysine-only prediction model.