L-Histidine Hydrochloride Monohydrate

Endpoint summary

Description of key information

The skin sensitisation potential of the target substance L-Histidine Hydrochloride Monohydrate (HHM) (100.1% purity) was assessed in two in vitro skin sensitisation studies conducted according to OECD 442C and OECD 442D. In both in vitro studies the target substance showed no skin sensitising potential. Based on the results, the test item can be considered as not skin sensitising.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

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

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.

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.

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%).

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

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

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

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

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.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-10-27 to 2018-02-22

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)

Version / remarks:

adopted February 2015

Deviations:

no

GLP compliance:

yes

Type of study:

activation of keratinocytes

Justification for non-LLNA method:

n.a.

Details on the study design:

Preparation of Test Item:
No correction was made for the composition/purity of the test item. A solubility test was performed. The test item was dissolved in Milli-Q water to a final concentration of 200 mM (clear colourless). The 100-fold dilution of the 200 mM Milli-Q water stock in DMEM glutamax formed a clear solution (2000 µM). This concentration was selected as highest concentration for the main assay (highest dose required in the current guideline). In the main experiments the test item was dissolved in Milli-Q water at 200 mM (colourless). From this stock 11 spike solutions in Milli-Q water were prepared (2-fold dilution series). The stock and spike solution were diluted 25-fold with exposure medium. These solutions were diluted 4-fold in the assay resulting in final test concentrations of 2000, 1000, 500, 250, 125, 63, 31, 16, 7.8, 3.9, 2.0 and 0.98 µM (final concentration Milli-Q water of 1%). All concentrations of the test item were tested in triplicate. All formulations formed a clear solution. No precipitation was observed at the start and end of the incubation period in the 96-well plates. Test item concentrations were used within 2.5 hours after preparation. Any residual volumes were discarded.

Preparation of the Positive Control:
The positive control used in the case of KeratinoSensTM is Ethylene dimethacrylate glycol (EDMG, Sigma, Zwijndrecht, The Netherlands), for which a 2-fold dilution series ranging from 0.78 to 25 mM were prepared in DMSO and diluted as described in paragraph 4.7.1, so that the final concentration of the positive control ranges from 7.8 to 250 µM (final concentration DMSO of 1%). All concentrations of the positive control were tested in triplicate.

Preparation of the Solvent Control:
The solvent control was 1% DMSO and 1% Milli-Q water in exposure medium. Eighteen wells were tested per plate.

Blank:
On each plate three blank wells were tested (no cells and no treatment).

Test System:
A transgenic cell line having a stable insertion of the luciferase reporter gene under the control of the ARE-element is used (e.g. the KeratinoSens™ cell line). The KeratinoSens™ cell line was generated by and obtained from Givaudan (Duebendorf, Switserland). Upon receipt, cells are propagated (e.g. 2 to 4 passages) and stored frozen as a homogeneous stock. Cells from this original stock can be propagated up to a maximum passage number from the frozen stock (i.e. 25) and are employed for routine testing using the appropriate maintenance medium.

Environmental Conditions:
All incubations, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 72 – 99 %), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0 °C (actual range 35.9 – 36.6 °C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the temperature and humidity occurred due to opening and closing of the incubator door. Based on laboratory historical data these deviations are considered not to affect the study integrity

Subculturing:
Cells were subcultured upon reaching 80-90% confluency. To maintain the integrity of the response, the cells were grown for more than one passage from the frozen stock, and were not cultured for more than 25 passages from the frozen stock (p+25).

Experimental Design:
Plating of cells:
For testing, cells were 80-90% confluent. One day prior to testing cells were harvested, and distributed into 96-well plates (10000 cells/well) in basic medium. For each repetition, three replicates were used for the luciferase activity measurements, and one parallel replicate used for the MTT cell viability assay. The cells were incubated overnight in the incubator. The passage number used was P+12 in experiment 1 and P+8 in experiment 2.
Treatment of Cells:
The medium was removed and replaced with fresh culture medium (150 μL culture medium containing serum but without Geneticin) to which 50 μL of the 25-fold diluted test chemical and control items were added. Three wells per plate were left empty (no cells and no treatment) to assess background values. The treated plates were then incubated for about 48 hours at 37 ± 1.0 °C in the presence of 5% CO2. Initially, experiments 1 and 2 did not pass all the acceptability criteria and therefore this part of the study was repeated. In total 2 valid experiments were performed.

Luciferase Activity Measurement:
The Steady-Glo Luciferase Assay Buffer (10 mL) and Steady-Glo Luciferase Assay Substrate (lyophilized) from Promega were mixed together. The assay plates were removed from the incubator and the medium is removed. Then 200 µL of the Steady-Glo Luciferase substrate solution (prior to addition 1:1 mixed with exposure medium) was added to each well. The plates were shaken for at least 3 minutes at room temperature. Plates with the cell lysates were placed in the TECAN Infinite® M200 Pro Plate Reader to assess the quantity of luciferase (integration time two seconds).

Cytotoxicity Assessment:
For the KeratinoSensTM cell viability assay, medium was replaced after the 48 hour exposure time with fresh medium containing MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue tetrazolium bromide; CAS No. 298-93-1) and cells were incubated for 3 hours at 37 °C in the presence of 5% CO2. The MTT medium was then removed and cells were lysed overnight by adding 10% SDS solution to each well. After shaking, the absorption was measured at 570 nm with the TECAN Infinite® M200 Pro Plate Reader.

Positive control results:

- Experiment I : The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 2.32 and the EC1.5 67 µM.
- Experiment II: The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 2.73 and the EC1.5 58 µM.

Key result

Run / experiment:

other: Experiment I

Parameter:

other: Maximum Luciferase Activity Induction (Imax)

Value:

1.09

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Run / experiment:

other: Experiment II

Parameter:

other: Maximum Luciferase Activity Induction (Imax)

Value:

1.07

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:

Experiment 1
- No precipitation was observed at the start and end of the incubation period in the 96-well plates.
- L-Histidine Hydrochloride Monohydrate (HHM) showed no toxicity. The viability of the cells was higher than 70% at all test concentrations and therefore no IC30 and IC50 values could be calculated.
- No luminescence activity induction compared to the vehicle control was observed at any of the test concentrations after treatment with L-Histidine Hydrochloride Monohydrate (HHM). The Imax was 1.09 and therefore no EC1.5 could be calculated.
- The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 2.32 and the EC1.5 67 µM.

Experiment 2
- No precipitation was observed at the start and end of the incubation period in the 96-well plates.
- L-Histidine Hydrochloride Monohydrate (HHM) showed no toxicity. The viability of the cells was higher than 70% at all test concentrations and therefore no IC30 and IC50 values could be calculated.
- No luminescence activity induction compared to the vehicle control was observed at any of the test concentrations after treatment with L-Histidine Hydrochloride Monohydrate (HHM). The Imax was 1.07 and therefore no EC1.5 could be calculated.
- The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 2.73 and the EC1.5 58 µM.

Both tests passed the acceptance criteria:
- The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, was above the threshold of 1.5-fold in at least one concentration.
- The EC1.5 of the positive control was between 5 and 125 µM (67 µM and 58 µM in experiment 1 and 2, respectively). A dose response was observed and the induction at 250 µM was higher than 2-fold (2.32-fold and 2.73-fold in experiment 1 and 2, respectively).
- The average coefficient of variation of the luminescence reading for the negative (solvent) control Milli-Q water was below 20% (7.3% and 7.3% in experiment 1 and 2, respectively).
- Finally, the average coefficient of variation of the luminescence reading for the negative (solvent) control DMSO was below 20% (7.7% and 7.7% in experiment 1 and 2, respectively).

Overall it is concluded that the test conditions were adequate and that the test system functioned properly.

Table 1: Overview of EC1.5, Imax, IC30 and IC50 values

	EC1.5(µM)	Imax	IC30(µM)	IC50(µM)
Test item Experiment 1	NA	1.09	NA	NA
Test item Experiment 2	NA	1.07	NA	NA
Pos Control Experiment 1	67	2.32	NA	NA
Pos Control Experiment 2	58	2.73	NA	NA

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, L-Histidine Hydrochloride Monohydrate (HHM) is classified as negative (no activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) under the experimental conditions described in this report.

Executive summary:

In a dermal sensitization study conducted according to OECD 442D with item L-Histidine Hydrochloride Monohydrate (HHM) (100.1% purity) in water, the sensitization potential of the test item was assessed on the basis of the activation of keratinocytes using the genetically modified keratinocyte cell line 'KeratinoSens'. Cells were incubated with the test item for 48 h at different concentrations (0.98, 2.0, 3.9, 7.8, 16, 31, 63, 125, 250, 500, 1000 and 2000 µM) and later checked for luciferase activity. Sensitization was scored by measuring the luciferase activity induction and relative cell viability. For both experiment I and II, the fold induction was less than a 1.5 and the cell viability was greater than 70%. Therefore, in this study, the test item is not considered to be a skin sensitizer under UN GHS guidelines.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

The skin sensitisation potential of the target substance was assessed in two in vitro skin sensitisation studies conducted according to OECD 442C and OECD 442D. In the first study conducted according to OECD 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.

In the second study conducted according to OECD 442D the sensitization potential of the test item was assessed on the basis of the activation of keratinocytes using the genetically modified keratinocyte cell line 'KeratinoSens'. Cells were incubated with the test item for 48 h at different concentrations (0.98, 2.0, 3.9, 7.8, 16, 31, 63, 125, 250, 500, 1000 and 2000 µM) and later checked for luciferase activity. Sensitization was scored by measuring the luciferase activity induction and relative cell viability. For both experiment I and II, the fold induction was less than a 1.5 and the cell viability was greater than 70%.

Based on the assessment of the available data, the test item is not considered as a skin sensitiser.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

In two in vitro skin sensitisation studies conducted according to OECD 442C and OECD 442D, the target substance was tested for skin sensitisation. Based on the results, no classification of the target substance for skin sensitisation is warranted.