L-Histidine Hydrochloride Monohydrate

Endpoint summary

Description of key information

The potential of L-Histidine Hydrochloride Monohydrate (HHM) (100.1% purity) to induce skin irritation (OECD 439) and eye irritation (OECD 492, OECD 437) was tested in suitable in vitro test methods. Based on the results, the target substance can be considered non-irritant to the skin. By assessing the results from both in vitro eye irritation tests in a weight-of-evidence approach, the target substance must be considered as irritating to the eye (Eye Irrit. 2, H319).

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-10-17 to 2018-01-19

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

adopted 28 July 2015

Deviations:

no

GLP compliance:

yes

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

EPISKIN Small ModelTM is a three-dimensional human epidermis model, which consists of adult human-derived epidermal keratinocytes which have been seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. The keratinocytes were cultured for 13 days, which results in a highly differentiated and stratified epidermis model comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum. In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimize the need of in vivo testing. One of the validated in vitro skin irritation tests is the EPISKIN test, which is recommended in international guidelines (e.g. OECD and EC).

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN Small ModelTM (EPISKIN-SMTM), 0.38 cm²
- Source: SkinEthic Laboratories, Lyon, France

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation (if applicable): 37 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
- rinsed with phosphate buffered saline (PBS)

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: MTT concentrate (3 mg/mL in PBS) diluted (10x) in Assay medium (final concentration 0.3 mg/mL)
- Incubation time: 3 hours
- Spectrophotometer: the amount of extracted formazan was determined at 570 nm in duplicate with the TECAN Infinite M200 Pro Plate Reader
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 3

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be irritant to skin in accordance with regulation EC 1272/2008 (UN GHS "Category 2") if the viability after exposure and post-incubation is less or equal to 50%.
- The test substance is considered to be non-irritant to skin in accordance with regulation EC 1272/2008 (UN GHS "No Category") if the viability after exposure and post-incubation is more than 50%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 22.9 - 27.9 mg

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 25 µL PBS

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 25 µL
- Concentration (if solution): 5%

Duration of treatment / exposure:

15 +/- 0.5 minutes at room temperature

Duration of post-treatment incubation (if applicable):

42 hours at 37 °C

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean of triplicates

Value:

110

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

L-Histidine Hydrochloride Monohydrate (HHM) was checked for color interference in aqueous conditions and possible direct MTT reduction by adding the test item to MTT medium. Because no color changes were observed it was concluded that the test item did not interact with the MTT endpoint. The mean tissue viability obtained after 15 ± 0.5 minutes treatment with the test item compared to the negative control tissues. Skin irritation is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with the test item compared to the negative control tissues was 110%. Since the mean relative tissue viability for the test item was above 50% the test item is considered to be non-irritant. The positive control had a mean cell viability after 15 ± 0.5 minutes exposure of 10%. The absolute mean OD570 of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was < 10%, indicating that the test system functioned properly.

Table 1: Mean Tissue Viability

	Mean tissue viability (percentage of control)	Standard deviation (percentage)
Negative control	100	5.7
Test item	110	9.4
Positive control	10	2.3

Interpretation of results:

GHS criteria not met

Conclusions:

In this study under the given conditions the test item showed no irritant effects in an validated in vitro system (EPISKIN). The test item is classified as ‘non-irritant’ in accordance with UN GHS ‘No Category’.

Executive summary:

The potential for the test item to induce skin irritation was tested by using the three-dimensional human skin model EpiSkin-SM (SkinEthic) comprising a reconstructed human epidermis with functional stratum corneum. The test item was applied directly atop the EpiSkin-SM tissue for 15 min, followed by 42 h post incubation period and immediate determination of cytotoxic effects via MTT reduction assay. Irritant potential from the test item was predicted from the relative mean tissue viabilities obtained compared to the corresponding negative control tissues concurrently treated with PBS. The test item showed no irritant effects. The mean relative tissue viability (% negative control) was ≥ 50% (110%) after 15 min treatment and 42 h post incubation. The controls confirmed the validity of the study. The positive control had a mean cell viability after 15 ± 0.5 minutes exposure of 10%.  The absolute mean OD570 of the negative control tissues was within the laboratory historical control data range. In this study under the given conditions the test item showed no irritant effects. The test item is classified as ‘non-irritant’ in accordance with UN GHS ‘No Category’.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-10-17 to 2018-01-19

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Version / remarks:

adopted 28 July 2015

Deviations:

no

GLP compliance:

yes

Species:

human

Details on test animals or tissues and environmental conditions:

- Justification of the test method: The EpiOcular tissue construct is a nonkeratinized epithelium prepared from normal human keratinocytes. It models the cornea epithelium with progressively stratified, but not cornified cells. A cell suspension is seeded into the insert in specialized medium. After an initial period of submerged culture, the medium is removed from the top of the tissue so that the epithelial surface is in direct contact with the air. This allows the test material to be directly applied to the epithelial surface in a fashion similar to how the corneal epithelium would be exposed in vivo. In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimize the need of in vivo testing. One of the validated in vitro eye irritation tests is the EpiOcular test, which is recommended in international guidelines and scientific publications (e.g. OECD).

- Description of the cell system used: The EpiOcular tissue construct is a non-keratinized epithelium (0.6 cm²) prepared from normal human keratinocytes (MatTek). It models the cornea epithelium with progressively stratified, but not cornified cells. These cells are not transformed or transfected with genes to induce an extended life span in culture. The “tissue” is prepared in inserts with a porous membrane through which the nutrients pass to the cells. A cell suspension is seeded into the insert in specialized medium. After an initial period of submerged culture, the medium is removed from the top of the tissue so that the epithelial surface is in direct contact with the air. This allows the test material to be directly applied to the epithelial surface in a fashion similar to how the corneal epithelium would be exposed in vivo.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): at least 50 mg

Duration of treatment / exposure:

6 hours ± 15 minutes

Observation period (in vivo):

n.a.

Duration of post- treatment incubation (in vitro):

Post exposure post-soak plate: 25 ± 2 min at room temperature
Post exposure post-treatment plate: 18 hours ± 15 minutes at 37 °C

Number of animals or in vitro replicates:

2 tissues

Details on study design:

Experimental Design:
Test for the Interference of the Test Item with the MTT Endpoint:
A test item may interfere with the MTT endpoint if it is colored and/or it is able to directly reduce MTT. The cell viability measurement is affected only if the test item is present on the tissues when the MTT viability test is performed.

Test for Color Interference by the Test Item:
L-Histidine Hydrochloride Monohydrate (HHM) was checked for possible color interference before the study was started. Some non-colored test items may change into colored items in aqueous conditions and thus stain the tissues during the exposure. To assess the color interference, approximately 50 mg of the test item or 50 µL sterile Milli-Q water as a negative control was added to 1.0 mL Milli-Q water. The mixture was incubated for at least 1 hour at 37.0 ± 1.0 °C in the dark. After incubation approximately 1 mL of the mixture was centrifuged for 30 seconds at 16000 g. Furthermore, approximately 50 mg of the test item or 50 µL sterile Milli-Q water as a negative control was added to 2.0 mL isopropanol. The mixture was incubated for 2 - 3 hours at room temperature with gentle shaking. After incubation approximately 1 mL of the mixture was centrifuged for 30 seconds at 16000 g. At the end of the exposure time, the absorbance of the solutions was determined spectrophotometrically at 570 nm in duplicate with the TECAN Infinite® M200 Pro Plate Reader. Centrifugation was not considered necessary. If after subtraction of the negative control, the OD for the test item solution is > 0.08, the test item is considered as possibly interacting with the MTT measurement.

Test for Reduction of MTT by the Test Item:
L-Histidine Hydrochloride Monohydrate (HHM) was checked for possible direct MTT reduction before the study was started. To assess the ability of the test item to reduce MTT, approximately 50 mg of the test item was added to 1 mL MTT solution (1 mg/mL MTT in phosphate buffered saline). The mixture was incubated for approximately 3 hours at 37.0 ± 1.0 °C in the dark. A negative control, 50 µL sterile Milli-Q water was tested concurrently. If the MTT solution color turned blue / purple or if a blue / purple precipitate was observed the test item interacts with MTT. Only test items which bind to the tissue after rinsing can interact with MTT in the main assay.

Test System Set Up:
On the day of receipt the tissues were equilibrated (in its 24-well shipping container) to room temperature. Subsequently, tissues were transferred to 6-well plates and incubated for 20 ± 4 hours at 37 °C in 1.0 mL fresh pre-warmed Assay Medium, which was refreshed after approximately 1 hour. Assay Medium was supplied by MatTek Corporation, Ashland, USA.

Environmental conditions:
All incubations, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 68 - 85%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0 °C (actual range 36.3 - 37.1 °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, humidity and CO2 percentage may occur due to opening and closing of the incubator door. Based on laboratory historical data these deviations are considered not to affect the study integrity.

Test Item Preparation:
No correction was made for the purity/composition of the test item. The solid test item (57.2 to 58.2 mg) was applied directly on top of the skin tissue.
L-Histidine Hydrochloride Monohydrate (HHM) was spread to match the size of the tissue. Any residual volumes were discarded.


Application of the Test Item:
The test was performed on a total of 2 tissues per test item together with a negative control and positive control. Before the assay was started the entire tissues were pre-wetted with 20 μL of Ca2+/Mg2+-Free-DPBS. The tissues were incubated at standard culture conditions for 30 ± 2 minutes.
Two tissues were treated with 50 µL Milli-Q water (negative control) and 2 tissues with 50 µL Methyl Acetate (positive control) respectively. At least 50 mg solid was added into the 6-well plates on top of the tissues. After the exposure period with the test item(6 hours ± 15 minutes at 37.0 ± 1.0 °C), the tissues were thoroughly rinsed with Ca2+/Mg2+-free D-PBS (brought to room temperature) to remove residual test item. After rinsing the cell culture inserts were each dried carefully and immediately transferred to and immersed in 5 mL of previously warmed Assay Medium (room temperature) in a pre-labeled 12-well plate for a 25 ± 2 minutes immersion incubation at room temperature (Post-Soak). After the Post-Soak period cell culture inserts were each dried carefully and transferred to the 6-well plate containing 1.0 mL of warm Assay Medium and were incubated for 18 hours ± 15 minutes at 37 °C.

Cell Viability Measurement:
After incubation, cell culture inserts were dried carefully to remove excess medium. The cell culture inserts were transferred into a 24-wells plate prefilled with 0.3 mL MTT-medium (1.0 mg/mL). The tissues were incubated for 180 ± 10 minutes at 37 °C. After incubation with MTT-medium the tissues were placed on blotting paper to dry the tissues and then transferred to a pre-labeled 6-well plate containing 2 mL isopropanol in each well so that no isopropanol is flowing into the insert. Formazan was extracted with 2 mL isopropanol for 2 - 3 hours at room temperature with gentle shaking. The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate with the TECAN Infinite® M200 Pro Plate Reader. Cell viability was calculated for each tissue as a percentage of the mean of the negative control tissues. Eye hazard potential of the test item was classified according to remaining cell viability following exposure of the test item.

Irritation parameter:

other: Relative Tissue Viability (%)

Run / experiment:

Mean of replicates

Value:

3.9

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

The relative mean tissue viability obtained after 6 hours ± 15 minutes treatment with the test item compared to the negative control tissues was 3.9%. Since the mean relative tissue viability for the test item was below 60% it is considered to be potentially irritant or corrosive to the eye. The positive control had a mean cell viability after 6 hours ± 15 minutes exposure of 29%. The absolute mean OD570 of the negative control tissues was within the laboratory historical control data range. The difference between the percentage of viability of two tissues treated identically was less than 13%, indicating that the test system functioned properly.

OTHER EFFECTS:
L-Histidine Hydrochloride Monohydrate (HHM) was checked for possible direct MTT reduction by adding the test item to MTT medium. Because no color changes were observed it was concluded that the test item did not interact with the MTT endpoint. The test item was checked for color interference in aqueous conditions. Addition of the test item to Milli-Q and isopropanol resulted after subtraction of the blank in an OD of -0.0043 and -0.001, respectively. Therefore it was concluded that the test item did not induce color interference.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes

Table 1: Mean Absorption in the EpiOcular™ Test with L-Histidine Hydrochloride Monohydrate (HHM)

	A (OD570)	B (OD570)	Mean (OD570)		SD
Negative control	1.218	1.389	1.304	±	0.120
Test item	0.046	0.056	0.051	±	0.007
Positive control	0.377	0.366	0.372	±	0.008

OD = optical density

SD = Standard deviation

Table 2: Mean Tissue Viability in the EpiOcular™ Test with L-Histidine Hydrochloride Monohydrate (HHM)

	Mean tissue viability (percentage of control)	Difference between two tissues  (percentage)
Negative control	100	13
Test item	3.9	0.79
Positive control	29	0.86

Interpretation of results:

other: UN GHS Category 1 or Category 2

Conclusions:

In this study under the given conditions, the test item showed irritant effects. The test item is classified as “irritant“ in accordance with UN GHS “Category 1” or “Category 2”.

Executive summary:

In the present study the eye irritant potential of L-Histidine Hydrochloride Monohydrate (100.1% purity) was analysed according to OECD 492 using the three-dimensional human corneal epithelium model EpiOcular, consisting of normal, human-derived epidermal keratinocytes mimicking characteristics of the corneal epithelium. Hereby, L-Histidine Hydrochloride Monohydrate was applied directly atop the EpiOcular™ tissue. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment. L-Histidine Hydrochloride Monohydrate showed no non-specific reduction of MTT and no colouring potential. Therefore, no additional controls for correction of results were necessary. L-Histidine Hydrochloride Monohydrate showed irritant effects. The mean relative tissue viability of two replicates (% negative control) was < 60% (3.9%). Therefore, L-Histidine Hydrochloride Monohydrate is considered to be irritating to the eye in accordance with UN GHS “Category 1" or "Category 2”.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018-04-04 to 2018-04-10

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Version / remarks:

adopted 09 October 2017

Deviations:

no

GLP compliance:

yes

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Bovine eyes from young cattle were obtained from the slaughterhouse (Vitelco, -'s Hertogenbosch, The Netherlands)
- Transport conditions of ocular tissue: Eyes were collected and transported in physiological saline in a suitable container under cooled conditions.
- indication of any existing defects or lesions in ocular tissue samples: The eyes were checked for unacceptable defects, such as opacity, scratches, pigmentation and neovascularization by removing them from the physiological saline and holding them in the light. Those exhibiting defects were discarded

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 403.9 to 434.1 mg

Duration of treatment / exposure:

240 +/- 10 minutes at 32 °C

Duration of post- treatment incubation (in vitro):

The optical density at 490 nm was measured upon 90 minutes of incubation with fluorescein after exposure to the test item by using a spectrophotometer.

Number of animals or in vitro replicates:

Three corneas each for the test item, negative control (physiological saline 0.9% NaCl) and positive control (imidazole 20% in physiological saline 0.9% NaCl).

Details on study design:

Preparation of corneas
The eyes were checked for unacceptable defects, such as opacity, scratches, pigmentation and neovascularization by removing them from the physiological saline and holding them in the light. Those exhibiting defects were discarded. The isolated corneas were stored in a petri dish with cMEM (Earle’s Minimum Essential Medium (Life Technologies, Bleiswijk, The Netherlands) containing 1% (v/v) L-glutamine (Life Technologies) and 1% (v/v) Fetal Bovine Serum (Life Technologies)). The isolated corneas were mounted in a corneal holder (one cornea per holder) of BASF (Ludwigshafen, Germany) with the endothelial side against the O-ring of the posterior half of the holder. The anterior half of the holder was positioned on top of the cornea and tightened with screws. The compartments of the corneal holder were filled with cMEM of 32 ± 1 °C. The corneas were incubated for the minimum of 1 hour at 32 ± 1 °C.

Selection of corneas and Opcaity Reading
After the incubation period, the medium was removed from both compartments and replaced with fresh cMEM. Opacity determinations were performed on each of the corneas using an opacitometer (BASF-OP3.0, BASF, Ludwigshafen, Germany). The opacity of each cornea was read against a cMEM filled chamber, and the initial opacity reading thus determined was recorded. Corneas that had an initial opacity reading higher than 7 were not used. Three corneas were selected at random for each treatment group.

Treatment of corneas
The medium from the anterior compartment was removed and 750 µL of the negative control and 20% (w/v) Imidazole solution (positive control) were introduced onto the epithelium of the cornea. L-Histidine Hydrochloride Monohydrate (HHM) was weighed in a bottle and applied directly on the corneas in such a way that the cornea was completely covered (403.9 to 434.1 mg). The holder was slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the solutions over the entire cornea. Corneas were incubated in a horizontal position for 240 ± 10 minutes at 32 ± 1 °C. After the incubation the solutions and the test item were removed and the epithelium was washed at least three times with MEM with phenol red (Earle’s Minimum Essential Medium Life Technologies). Possible pH effects of the test item on the corneas were recorded. Each cornea was inspected visually for dissimilar opacity patterns. The medium in the posterior compartment was removed and both compartments were refilled with fresh cMEM and the opacity determinations were performed.

Opacity measurements
The opacity of a cornea was measured by the diminution of light passing through the cornea. The light was measured as illuminance (I = luminous flux per area, unit: lux) by a light meter.The opacity value (measured with the device OP-KIT) was calculated according to:
Opacity=(Io/I-0.9894)/0.0251
With Io the empirically determined illuminance through a cornea holder but with windows and medium, and I the measured illuminance through a holder with cornea.
The change in opacity for each individual cornea (including the negative control) was calculated by subtracting the initial opacity reading from the final post-treatment reading. The corrected opacity for each treated cornea with the test item or positive control was calculated by subtracting the average change in opacity of the negative control corneas from the change in opacity of each test item or positive control treated cornea. The mean opacity value of each treatment group was calculated by averaging the corrected opacity values of the treated corneas for each treatment group.

Application of Sodium Fluorescein
Following the final opacity measurement, permeability of the cornea to Na-fluorescein (Sigma-Aldrich, Germany) was evaluated. The medium of both compartments (anterior compartment first) was removed. The posterior compartment was refilled with fresh cMEM. The anterior compartment was filled with 1 mL of 5 mg Na-fluorescein/mL cMEM solution (Sigma-Aldrich Chemie GmbH, Germany). The holders were slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the sodium-fluorescein solution over the entire cornea. Corneas were incubated in a horizontal position for 90 ± 5 minutes at 32 ± 1 °C.

Permeability Determinations
After the incubation period, the medium in the posterior compartment of each holder was removed and placed into a sampling tube labelled according to holder number. 360 µL of the medium from each sampling tube was transferred to a 96-well plate. The optical density at 490 nm (OD490) of each sampling tube was measured in triplicate using a microplate reader (TECAN Infinite® M200 Pro Plate Reader). Any OD490 that was 1.500 or higher was diluted to bring the OD490 into the acceptable range (linearity up to OD490 of 1.500 was verified before the start of the experiment). OD490 values of less than 1.500 were used in the permeability calculation. The mean OD490 for each treatment was calculated using cMEM corrected OD490 values. If a dilution has been performed, the OD490 of each reading of the positive control and the test item was corrected for the mean negative control OD490 before the dilution factor was applied to the reading.

Irritation parameter:

in vitro irritation score

Run / experiment:

mean of triplicates

Value:

10

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: no prediction can be made

Other effects / acceptance of results:

The individual in vitro irritancy scores for the negative controls ranged from 0.3 to 1.9. The individual positive control in vitro irritancy scores ranged from 101 to 113. The corneas treated with the positive control were turbid after the 240 minutes of treatment. The corneas treated with L-Histidine Hydrochloride Monohydrate (HHM) showed opacity values ranging from 6.6 to 7.0 and permeability values ranging from -0.030 to 0.687. The corneas were slightly translucent after the 240 minutes of treatment with the test item. A pH effect of the test item was observed on the rinsing medium, the corneas were rinsed until no colour change of the medium was observed. Hence, the in vitro irritancy scores ranged from 6.1 to 17 after 240 minutes of treatment with L-Histidine Hydrochloride Monohydrate (HHM). For detailed results please refer to box "Any other information on results incl. tables".

Table 1: In vitro irritancy scores

Treatment	Final Opacity^2	Final OD490^2	In vitro Irritancy Score^1
Negative control	0.9	0.031	1.3
	0.1	0.012	0.3
	1.5	0.028	1.9
Positive control	101	0.796	113
	85	1.076	101
	91	1.022	107
L-Histidine Hydrochloride Monohydrate (HHM)	6.6	-0.030	6.1
	7.0	0.687	17
	6.9	-0.029	6.4

¹  In vitro irritancy score (IVIS) = opacity value + (15 x OD490 value).

²  Positive control and test item are corrected for the negative control.

Table 2: Summary of opacity, permeability and in vitro scores

Treatment	Mean Opacity	Mean Permeability	Mean In vitro Irritation Score^1, 2
Negative control	0.8	0.024	1.2
Positive control	92	0.965	107
L-Histidine Hydrochloride Monohydrate (HHM)	6.8	0.209	10.0

¹    Calculated using the negative control mean opacity and mean permeability values for the positive control and test item.

²    In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value).

Interpretation of results:

other: no prediction can be made

Conclusions:

In conclusion, based on the mean in vitro irritation score of 10 obtained in the bovine corneal opacity and permeability assay (BCOP, OECD 437), no prediction can be made regarding the classification of the test substance.

Executive summary:

The eye irritation potential of L-Histidine Hydrochloride Monohydrate (HHM) (100.1% purity) was investigated in the bovine corneal opacity and permeability assay (BCOP, OECD 437). A mean in vitro irritation score of 10 was determined. The positive control induced the appropriate responses, indicating the validity of the assay. Based on the results obtained, no prediction can be made regarding the classification of the substance.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The potential of L-Histidine Hydrochloride Monohydrate (HHM) (100.1% purity) to induce skin irritation (OECD 439) and eye irritation (OECD 492, OECD 437) was tested in suitable in vitro test methods. Based on the results, the target substance can be considered non-irritant to the skin. To assess the eye irritant potential of the target substance, the EpiOcular Eye Irritation Test was conducted in accordance to OECD 492. This test is able to correctly identify chemicals that do not require classification or require labelling for eye irritation (Eye Irrit. 2, H319) or serious eye damage (Eye Dam. 1, H318). However, is not possible to distinguish between serious eye irritation and serious eye damage in this test. The test item showed irritant effects in the EpiOcular test. The mean relative tissue viability (% negative control) was ≤ 60% (3.9%). Therefore, the test item can be considered to require classification. Subsequently, the test item was tested in the Bovine Corneal Opacity & Permeability (BCOP) assay. The BCOP allows classification as “not classified” and “Eye Dam 1, H318” based on the In Vitro Irritancy Score (IVIS). Results which are in between both categories are identified as “No prediction can be made”. Based on the results from the BCOP test, no prediction can be made regarding the classification of the target substance. Therefore, the test item was considered not to be an ocular corrosive or severe irritant.

By assessing the results from both in vitro eye irritation tests in a weight-of-evidence approach, it can be concluded that classification as Eye Irrit 2, H319 is warranted as it was shown that the substance cannot be considered as non-irritant, but also not as inducing severe eye damage (Eye Dam 1, H318).