L-Histidine Hydrochloride Monohydrate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-10-17 to 2018-01-05

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:

adopted 04 February 2015

Deviations:

no

GLP compliance:

yes

Type of study:

direct peptide reactivity assay (DPRA)

Justification for non-LLNA method:

n.a.

Test material

Test material information

Constituent 1

Reference substance name:

L-Histidine Hydrochloride Monohydrate

EC Number:

611-821-4

Cas Number:

5934-29-2

Molecular formula:

C6-H9-N3-O2.Cl-H.H2-O

IUPAC Name:

L-Histidine Hydrochloride Monohydrate

Test material form:

solid: crystalline

Details on test material:

- Name: L-Histidine Hydrochloride Monohydrate (HHM)
- Appearance: White crystals or crystalline powder
- Batch: P817T011
- CAS no.: 5934-29-2
- Purity/Composition: 100.1%
- Molecular weight: 209.63 g/mol
- Test item storage: room temperature
- Expiry date: 31 October 2020

In chemico test system

Details on the study design:

Preparation of the Test Item:
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. 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) and ACN:MQ (1:1, v/v). Test item stock solutions were prepared freshly for each reactivity assay. For both the cysteine and lysine reactivity assay 33.02 mg of test item was pre-weighed into a clean amber glass vial and dissolved, just before use, in 1575 µL MQ 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. Any residual volumes were discarded.

Test System:
Test system: Synthetic peptides containing cysteine (SPCC) (Ac RFAACAA COOH) or synthetic peptides containing lysine (SPCL) (Ac RFAAKAA COOH). The molecular weight is 750.9 g/mol for SPCC and 775.9 g/mol for SPCL.
Source: JPT Peptide Technologies GmbH, Berlin, Germany.
Batch: 111016HS_MHeW0817
Storage: The peptides were stored in the freezer (≤ 15 °C) for a maximum of 6 months.

Preparation of Solutions for Cysteine Reactivity Assay:
Synthetic Peptide Containing Cysteine (SPCC) Stock solution:
A stock solution of 0.667 mM SPCC (0.501 mg SPCC/mL) was prepared by dissolving 10 mg of SPCC in 19.96 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 RCcysCMQ sample was included to evaluate the effect of the solvent that was used to dissolve the test item on the Percent Peptide Depletion. The RCcysCMQ sample was prepared by mixing 750 µL of the 0.667 mM SPCC stock solution with 200 µL ACN and 50 µL MQ.

Co-elution Control, Test Item and Positive Control Samples:
Co-elution control (CC):
Replicates: 1
Sample Code: CCcys-208859/A
Preparation: 750 µL Phosphate buffer pH 7.5, 200 µL ACN and 50 µL test item solution (100 mM)

Cinnamic aldehyde (PC):
Replicates: 3
Sample code: PCcys-1 to PCcys-3
Preparation: 750 µL Stock solution of 0.667 mM SPCC, 200 µL ACN and 50 µL Cinnamic aldehyde solution (100 mM in ACN)

Test item:
Replicates: 3
Sample code: 208859/A-cys-1 to 208859/A-cys-3
Preparation: 750 µL Stock solution of 0.667 mM SPCC, 200 µL ACN 50 µL test item solution (100 mM)

Preparation of Solutions for Lysine Reactivity Assay:
Synthetic Peptide Containing Lysine (SPCL) Stock solution:
A stock solution of 0.667 mM SPCL (0.518 mg SPCL/mL) was prepared by dissolving 10 mg of SPCL in 19.31 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 RClysCMQ sample was included to evaluate the effect of the solvent that was used to dissolve the test item on the Percent Peptide Depletion. The RClysCMQ sample was prepared by mixing 750 µL of the 0.667 mM SPCL stock solution with 250 µL MQ.

Co-elution Control, Test Item and Positive Control Samples:
Co-elution control (CC):
Replicates: 1
Sample Code: CClys-208859/A
Preparation: 750 µL Ammonium acetate buffer pH 10.2, 250 µL test item solution (100 mM)

Cinnamic aldehyde (PC):
Replicates: 3
Sample code: PClys-1 to PClys-3
Preparation: 750 µL Stock solution of 0.667 mM SPCL, 250 µL Cinnamic aldehyde solution (100 mM in ACN)

Test item:
Replicates: 3
Sample code: 208859/A-lys-1 to 208859/A-lys-3
Preparation: 750 µL Stock solution of 0.667 mM SPCL, 250 µL test item solution (100 mM)

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. Prior to HPLC-PDA analysis the samples were visually inspected for precipitation.

Results and discussion

Positive control results:

The mean Percent SPCC Depletion for the positive control cinnamic aldehyde was 65.7% ± 0.6%. This was within the acceptance range of 60.8% to 100% with a SD that was below the maximum (SD< 14.9%). The mean Percent SPCL Depletion for the positive control cinnamic aldehyde was 52.1% ± 2.2%. This was within the acceptance range of 40.2% to 69.0% with a SD that was below the maximum (SD< 11.6%).

In vitro / in chemico

Results

Results 1

Run / experiment:

other: Mean of calculated peptide depletion values for Lys- and Cys-Peptide

Parameter:

other: Mean peptide depletion (%)

Value:

0.1

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Results 2

Key result

Run / experiment:

other: mean of triplicates

Parameter:

other: Mean peptide depletion for the Lys-peptide (%)

Value:

0.2

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Results 3

Key result

Run / experiment:

other: mean of triplicates

Parameter:

other: Mean peptide depletion for the Cys-peptide (%)

Value:

0

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

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

Table 2: SPCC and SPCL Depletion, DPRA Prediction and Reactivity Classification for the Test Item

Test item	SPCC depletion		SPCL depletion		Mean of SPCC and SPCL depletion	DPRA prediction and     reactivity classification
	Mean	± SD	Mean	± SD		Cysteine 1:10 / Lysine 1:50 prediction model
L-Histidine Hydrochloride Monohydrate (HHM)	0.0%	±0.0%	0.2%	±0.4%	0.1%	Negative: No or minimal reactivity

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The results from an in vitro skin sensitisation assay conducted in accordance with OECD 442C showed, that the test item was tested negative in the Direct Peptide Reactivity Assay.

Executive summary:

In a dermal sensitization study conducted according to OECD guideline 442C, the test item (100.1% purity) dissolved in water was tested using the direct peptide reactivity assay. 100 mM test item was incubated 24.5 h at 25 °C together with cysteine and lysine peptides, respectively and the peptide concentration after the incubation was measured using HPLC-PDA. Three replicates were prepared using 1:10 and 1:50 molar ratio of the test item with the Cys- and Lys-peptide. All acceptance criteria were fulfilled and the test was considered as valid. The Cys-peptide depletion was 0.0%, the Lys-peptide depletion was 0.2% and the resulting mean peptide depletion was 0.1%. Based on the results, the DPRA prediction for the test item L-Histidine Hydrochloride Monohydrate (HHM) was negative with reactivity class no or minimal according to the Cysteine 1:10/Lysine 1:50 prediction model.