4-hydroxybenzaldehyde

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-10-19 to 2018-01-18

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

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Direct Peptide Reactivity Assay (DPRA) for Skin Sensitization Testing, DB-ALM Protocol n°154, January 12, 2013

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany

Type of study:

direct peptide reactivity assay (DPRA)

Test material

Test material information

Constituent 1

Reference substance name:

4-hydroxybenzaldehyde

EC Number:

204-599-1

EC Name:

4-hydroxybenzaldehyde

Cas Number:

123-08-0

Molecular formula:

C7H6O2

IUPAC Name:

4-hydroxybenzaldehyde

Test material form:

solid

In chemico test system

Details on the study design:

Skin sensitisation (In chemico test system)
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. In the present study the test material was dissolved in acetonitrile, based on the results of the pre-experiments. Based on a molecular weight of 122.12 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.

Preparation of the cysteine or lysine-containing peptides:
Stock solutions of cysteine (Ac-RFAACAA-COOH) and lysine (Ac-RFAAKAA-COOH) containing synthetic peptides of purity higher than 95% were freshly prepared just before their incubation with the test item. The final concentration of the cysteine peptide was 0.666 mM in pH 7.5 phosphate buffer, whereas the final concentration of the lysine peptide was 0.668 mM in pH 10.2 ammonium acetate buffer

Positive control
Cinnamic aldehyde (CAS no. 14371-10-9) was used as positive control (PC) at a concentration of 100 mM in acetonitrile.

HPLC measurement
see "Other information on material and methods"

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.


 

Results and discussion

Positive control results:

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 63.81% (experiment 1) and 68.63% (experiment 2).

In vitro / in chemico

Results

Results 1

Key result

Run / experiment:

other: cysteine run (experiment 1)

Parameter:

other: mean peptide depletion [%]

Value:

0.12

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Key result

Run / experiment:

other: lysine run (experiment 1)

Parameter:

other: mean peptide depletion [%]

Value:

13.2

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Results 3

Key result

Run / experiment:

other: cysteine run (experiment 2)

Parameter:

other: mean peptide depletion [%]

Value:

0.3

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Results 4

Key result

Run / experiment:

other: lysine run (experiment 2)

Parameter:

other: mean peptide depletion [%]

Value:

4.43

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes

Any other information on results incl. tables

Cysteine and Lysine Values of the Calibration Curve Experiment 1

Sample	Cysteine Peptide		Lysine Peptide
	Peak Area at 220 nm	Peptide Concentration [mM]	Peak Area at 220 nm	Peptide Concentration [mM]
STD1	4523.9644	0.5340	4169.8604	0.5340
STD2	2255.1311	0.2670	2102.4409	0.2670
STD3	1081.2599	0.1335	1026.2726	0.1335
STD4	538.3588	0.0667	515.4506	0.0667
STD5	264.6839	0.0334	262.0216	0.0334
STD6	131.3584	0.0167	131.2270	0.0167
STD7	0.0000	0.0000	0.0000	0.0000

Cysteine and Lysine Values of the Calibration Curve Experiment 2

Sample	Cysteine Peptide		Lysine Peptide
	Peak Area at 220 nm	Peptide Concentration [mM]	Peak Area at 220 nm	Peptide Concentration [mM]
STD1	17.870	0.5340	4663.5073	0.5340
STD2	9.012	0.2670	2361.9275	0.2670
STD3	4.518	0.1335	1192.6749	0.1335
STD4	2.252	0.0667	602.5394	0.0667
STD5	1.101	0.0334	302.3090	0.0334
STD6	0.530	0.0167	151.3044	0.0167
STD7	0.000	0.0000	0.0000	0.0000

Depletion of the Cysteine Peptide Experiment 1

Cysteine Peptide
Sample	Peak Area at 220 nm	Peptide Conc. [mM]	Peptide Depletion [%]	Mean Peptide Depletion [%]	SD of Peptide Depletion [%]	CV of Peptide Depletion [%]
Positive Control	1320.4471	0.1577	70.95	71.10	0.18	0.25
	1315.4908	0.1571	71.06
	1304.7782	0.1559	71.29
Test Item	4600.2661	0.5437	0.00	0.12	0.20	173.21
	4583.2500	0.5417	0.00
	4529.0562	0.5353	0.35

Depletion of the Cysteine Peptide Experiment 2

Cysteine Peptide
Sample	Peak Area at 220 nm	Peptide Conc. [mM]	Peptide Depletion [%]	Mean Peptide Depletion [%]	SD of Peptide Depletion [%]	CV of Peptide Depletion [%]
Positive Control	4.447	0.1325	74.33	74.15	0.18	0.25
	4.477	0.1334	74.15
	4.510	0.1344	73.96
Test Item	17.353	0.5175	0.00	0.30	0.52	173.21
	17.167	0.5120	0.89
	17.396	0.5188	0.00

Depletion of the Lysine Peptide Experiment 1

Lysine Peptide
Sample	Peak Area at 220 nm	Peptide Conc. [mM]	Peptide Depletion [%]	Mean Peptide Depletion [%]	SD of Peptide Depletion [%]	CV of Peptide Depletion [%]
Positive Control	1690.0214	0.2164	56.37	56.51	0.47	0.83
	1699.1774	0.2175	56.14
	1664.2920	0.2131	57.04
Test Item	3573.1260	0.4572	7.76	13.20	5.57	42.20
	3372.0542	0.4315	12.95
	3141.8584	0.4020	18.89

Depletion of the Lysine Peptide Experiment 2

Lysine Peptide
Sample	Peak Area at 220 nm	Peptide Conc. [mM]	Peptide Depletion [%]	Mean Peptide Depletion [%]	SD of Peptide Depletion [%]	CV of Peptide Depletion [%]
Positive Control	1539.7554	0.1748	64.31	63.12	1.08	1.71
	1602.9802	0.1820	62.84
	1630.8385	0.1852	62.20
Test Item	4162.2075	0.4752	3.52	4.43	0.86	19.40
	4118.6553	0.4702	4.53
	4088.5010	0.4667	5.23

Prediction Model 1

Cysteine 1:10/ Lysine 1:50 Prediction Model ¹

Mean Cystein and Lysine PPD	Reactivity Class	DPRA Prediction (2)
0.00% ≤ PPD ≤ 6.38%	No or minimal Reactivity	Negative
6.38% < PPD ≤ 22.62%	Low Reactivity	Positive
22.62% < PPD ≤ 42.47%	Moderate Reactivity
42.47% < PPD ≤ 100%	High Reactivity

1 The numbers refer to statistically generated threshold values and are not related to the precision of the measurement.

2 DPRA predictions should be considered in the framework of an IATA.

Prediction Model 2

Cysteine 1:10 Prediction Model

Cysteine PPD	Reactivity Class	DPRA Prediction (2)
0.00%≤PPD≤13.89%	No or minimal Reactivity	Negative
13.89% <PPD≤23.09%	Low Reactivity	Positive
23.09% <PPD≤98.24%	Moderate Reactivity
98.24% <PPD≤100%	High Reactivity

Categorization of the Test Item Experiment 1

Prediction Model	Prediction Model 1 (Cysteine Peptide and Lysine Peptide / Ratio: 1:10 and 1:50)			Prediction Model 2 (Cysteine Peptide / Test Item Ratio: 1:10)
Test Substance	Mean Peptide Depletion [%]	Reactivity Category	Prediction	Mean Peptide Depletion [%]	Reactivity Category	Prediction
Test Item	6.66	Low Reactivity	sensitiser	0.12	Minimal Reactivity	no sensitiser
Positive Control	63.81	High Reactivity	sensitiser	71.10	Moderate Reactivity	sensitiser

Categorization of the Test Item Experiment 2

Prediction Model	Prediction Model 1 (Cysteine Peptide and Lysine Peptide / Ratio: 1:10 and 1:50)			Prediction Model 2 (Cysteine Peptide / Test Item Ratio: 1:10)
Test Substance	Mean Peptide Depletion [%]	Reactivity Category	Prediction	Mean Peptide Depletion [%]	Reactivity Category	Prediction
Test Item	2.36	Minimal Reactivity	no sensitiser	0.30	Minimal Reactivity	no sensitiser
Positive Control	68.63	High Reactivity	sensitiser	74.15	Moderate Reactivity	sensitiser

Applicant's summary and conclusion

Interpretation of results:

other: peptide depletion negative

Remarks:

The data generated with this method may be not sufficient to conclude on the absence of skin sensitisation potential of chemicals and should be considered in the context of integrated approach such as IATA.

Conclusions:

In this study under the given conditions the test item showed minimal reactivity towards both peptides.

Executive summary:

A study was conducted according to OECD TG 442C in order to evaluate the reactivity of the test item towards cysteine (Cys-) and lysine (Lys-) containing peptides. The DPRA is an in chemico method which quantifies the remaining concentration of cysteine- or lysine-containing peptide following 24 ± 2 hours incubation with the test item at 25 ± 2.5°C. The test item was dissolved at a concentration of 100 mM in acetonitrile. Relative peptide concentration is measured by high-performance liquid chromatography (HPLC) with gradient elution and UV detection at 220 nm.

The 100 mM stock solution of the test item showed low reactivity towards the synthetic peptides. The mean depletion of both peptides was > 6.38% (6.66%). Based on the prediction model 1 the test item might be considered as sensitiser. According to the evaluation criteria in the guideline, for test items with combined cysteine/lysine peptide depletion between 3% and 10% a second run was performed

In the second experiment 100 mM stock solution of the test item showed minimal reactivity towards the synthetic peptides. The mean depletion of both peptides was ≤ 6.38% (2.36%). Based on the prediction model 1 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 68.63%. The controls confirmed the validity of the study for both, the cysteine and lysine run. As this experiment shows that the test item exhibits minimal reactivity (2.36%), the slightly positive result (6.66%) in the former experiment is not regarded as having a higher impact but a lower one. Therefore, the test item can be considered as non-sensitiser.