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Administrative data

Description of key information

L-histidine is not irritating to the skin or the eyes.

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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
test system CORROSITEX®
Deviations:
no
Principles of method if other than guideline:
The commercial reconstructed human epidermis (RhE) model named EPISKIN was used. The test item and controls are placed on the model and their ability to penetrate the stratum corneum and impair cell viability is assessed. The exposure time is 15 minutes, followed by a 42 hours recovery period. Colorimetric measurement of MTT reduction (blue formazan salt) is used to assess the cell viability.
GLP compliance:
yes
Species:
other: reconstituted collagen matrix
Details on test animals and environmental conditions:
According to the supplier procedure, tissues were prepared as follows:
– Alive tissues: at arrival, plates were opened under a sterile airflow and each insert, containing the epidermal tissue, was carefully taken out and placed in a 12-well plate in which each well had previously been filled with 2 mL/well SkinEthic Maintenance Medium. Culture plates were placed in the incubator at 37°C, 5% CO2 and saturated humidity for approximately 24 hours.
– Killed tissues: a sufficient number of epidermis units were placed in a 12- well plate in which each well had previously been filled with 2 mL/well sterile water for injection. Tissues were incubated for approximately 48 hours, then transferred into a new plate and stored at -20°C. The day of the experiment, tissues were thawed at room temperature with 2 mL of maintenance medium.

Media:
- Maintenance Medium SkinEthic; batch: 15-MAIN3-034
- Assay Medium SkinEthic; batches: 15-ESSC-034 and 15-ESSC-025
Type of coverage:
other: not applicable
Preparation of test site:
other: not applicable
Vehicle:
unchanged (no vehicle)
Controls:
not required
Amount / concentration applied:
20mg
Duration of treatment / exposure:
15 minutes
Observation period:
42 hours
Number of animals:
Not applicable
Details on study design:
Control items:
- Positive control: 5% (v/v) sodium dodecyl sulphate (SDS) (SIGMA, batch 041M8713V) in sterile water
- Negative control: D-PBS (GIBCO, batch 1605086)

Experimental procedure:
- Direct MTT reduction test: non-specific reduction of MTT is evaluated
- Colouring potential test: chemicals’ colouring potential is assessed for potential interaction with the test system.
- Main assay (3 replicates): alive tissues are treated with the test item, positive and negative controls; an additional control using alive treated tissues without MTT was performed. Additional controls were performed using killed tissues treated with test item and negative control.

Interpretation of the results:
- Result: After appropriate blank subtractions and/or corrections for the background controls, means, standard deviations, coefficients of variation, mean relative viability values (percentage relative to the negative control) were calculated.
- Cut-off values: mean relative viability =< 50%: irritant (GHS Category 2) and mean relative viability > 50%: not irritant (GHS No category)
Irritation / corrosion parameter:
other: cell viability
Run / experiment:
Mean
Value:
106.9
Remarks on result:
other: Remarks: expressed in % relative to the negative control.
Interpretation of results:
GHS criteria not met
Conclusions:
L-HISTIDINE should be considered as not irritating to the skin.
Executive summary:

In the current study the skin irritation potential of the test item L-HISTIDINE was assessed in an in vitro membrane barrier assay, using a commercial reconstructed human epidermis (RhE) model named EPISKINTM . The study was according to OECD 439 and GLP.

In a first step, the test item was assayed for the ability to reduce MTT. At the end of the incubation period, a yellow/grey solution, without precipitate, was observed, indicating that the test item could directly interact with MTT. Thus, additional controls were added in the Main Assay for the evaluation of MTT non specific reduction (NSMTT).

In a second step, the test item was assayed for the ability to colour water. A colourless suspension, with plenty white precipitate, was observed, which indicates that the test item does not have a colouring ability. For this reason, no additional controls were added in the Main Assay for the evaluation of non specific colouring potential (NSC).

Using alive tissues, the negative control gave the expected baseline value and variability, in agreement with guideline indications. According to the method, the mean negative control value is considered the baseline value of the experiment and thus represents 100% of cell viability.

The positive control showed cell death with an acceptable relative cell viability of 6.2 % in function of the negative control. Variability between replicates was acceptable (SD of % viability = 4.3). Based on the criteria the study was accepted as valid.

The test item did not induce any relevant reduction of MTT staning in any replicate. The mean cell viability was 106.9% in function of the negative control. The variability between the replicates was 18.8, which is slightly higher than the stated in the Study Acceptability Criteria, however, the study was considered valid since the Optical Density value of each test item replicate sample was comparable to the negative control values.

The classification criteria in the CLP legislation are not directly applicable to an in vitro test. Therefore, as described in OECD 439 the following cut-off time values are used.

mean relative viability =< 50%: irritant (GHS Category 2)

mean relative viability > 50%: not irritant (GHS No category)

Based on these results L-Histidine should be considered as not irritant to the skin.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
17-11-2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
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:
July 2013
Deviations:
no
Qualifier:
according to
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
Version / remarks:
December 2010
Deviations:
no
GLP compliance:
yes
Species:
cattle
Strain:
other: Bos primigenius Taurus
Details on test animals or tissues and environmental conditions:
Fresh bovine eyes were obtained from the slaughterhouse Müller Fleisch GmbH, Enzstr. 2-4, 75217 Birkenfeld, Germany.
Bovine eyes were obtained on the day of the test.
Age of cattle: 12-60 months.
Transport to test facility in Hank's balanced salts solution supplemented with 0.01% streptomycin and 0.01% penicillin.
After dissection the corneas are incubated in medium at 32 +/- 1°C for 1h.
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
Replicate 1: 506.8 mg
Replicate 2: 503.4 mg
Replicate 3: 508.2 mg
Duration of treatment / exposure:
4 hours exposure time at 32 +/-1°C
Duration of post- treatment incubation (in vitro):
90 minutes at 32 +/- 1°C (for premeability measurement)
Number of animals or in vitro replicates:
3 test item replicates, 3 negative control replicates and 3 positive control replicates.
Details on study design:
SELECTION AND PREPARATION OF CORNEAS
Only corneas free from damage were used.
Corneas were excised with a scalpel and cut from the globe with a 2-3 mm ring of sclera around the outside.
Each cornea was transferred to a cornea holder in which pre-warmed (32+/-1°C) cMEM without phenol was filled.
The holders were then incubated for 1 hour in the incubation chamber at 32+/-1°C.

QUALITY CHECK OF THE ISOLATED CORNEAS
Quality check after initial incubation: the medium was changed and the baseline opacity for each cornea was recorded.
None of the cornea showed tissue damage, therefore all corneas were used.

TREATMENT METHOD:
Open chamber method.
The solid was applied directly on the cornea, in such a manner that as much as possible of the cornea was covered with test item.

REMOVAL OF TEST SUBSTANCE, POST-EXPOSURE INCUBATION AND MEASURING OPACITY AND PERMEABILITY
Thorough rinsins with cMEM with phenol red, followed by rinsing with cMEM without phenol red.
Subsequently, both chambers were filled with cMEM without phenol red and the final opacity value of each cornea was recorded.
The cMEM without phenol red was then removed from the front chamber and 1 ML sodium fluorescein solution (5 mg/mL) was added to the front chamber.
The chambers were then closed again and incubated for 90 minutes at 32+/-1°C.
After incubation, the content of the posterior chamber was thoroughly mixed. Then the permeability of the cornea was measured as optical density of the liquid at 492 nm.

SCORING SYSTEM:

1) Opacity = [(I0/I)-b]a

with:
a = 0.0251 (opacitometer-specific, empirically determined)
b = 0.9894 (opacitometer-specific, empirically determined)
I0 = empirically determined illuminance through a cornea holder with windows and medium (expressed in LUX) (here: I0 = 1078.3)
I = measured illuminance (expressed in LUX)

2) Permeability
The corrected OD492 value of each cornea treated with test item and positive control was calculated by substracting the mean negative control conrea value from the original permeability value for each cornea.
The mean OD492 value for each treatment group (test item, positive control and negative control) was determined by averaging the final OD492 values of the treated corneas for one treatment group.

3) In Vitro Irritancy Score (IVIS)

For the negative control:
IVIS = opacity difference + (15 x corrected OD492 value)

For the positice control and the test item treatment group:
IVIS = (opacity difference - mean opacity difference of the negative control) + (15 x (OD492 - mean OD492 of the negative control))

DECISION CRITERIA:

IVIS <= 3: no category
IVIS >3 and <= 55: no prediction can be made
IVIS > 55: eye damage Category 1
Irritation parameter:
in vitro irritation score
Run / experiment:
Mean of 3 replicates
Value:
0.32
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
in vitro irritation score
Run / experiment:
Replicate 1
Value:
-0.46
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
in vitro irritation score
Run / experiment:
Replicate 2
Value:
0.27
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
in vitro irritation score
Run / experiment:
Replicate 3
Value:
1.15
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Criterion: IVIS <= 3
- Found: IVIS = -0.04

- Acceptance criteria met for positive control: yes
- Criterion: IVIS = 73.16-164.28
- Found: IVIS = 118.15

Opacity Values:

 

Treatment group

Replicate

Before exposure

After exposure

Negative control

1

1026

1019

2

986

1030

3

1009

1016

Test item

1

1021

1037

2

1026

1021

3

1031

2008

Positive control

1

1008

334

2

995

441

3

997

350

 

Optical density at 492 nm of Blank:

 

Measurement

cMEM without phenol red

1

0.033

2

0.031

3

0.039

Mean

0.034

 

Permeability Values:

 

Repl.

Measurem. #

Measured value

Measured value- blank

Mean of replicate

Mean of 3 replicates

Corrected

Neg. contr.

1

1

2

3

0.046

0.042

0.046

0.0117

0.0077

0.0117

0.0103

0.0388

--

2

1

2

3

0.044

0.042

0.042

0.0097

0.0077

0.0077

0.0083

--

3

1

2

3

0.137

0.134

0.125

0.1027

0.0997

0.0907

0.0977

--

Test item

1

1

2

3

0.043

0.045

0.045

0.0087

0.0107

0.0107

0.0100

--

-0.0288

2

1

2

3

0.035

0.035

0.037

0.0007

0.0007

0.0027

0.0013

-0.0374

3

1

2

3

0.042

0.042

0.050

0.0077

0.0077

0.0157

0.0103

-0.0284

Pos. contr.

1

1

2

3

0.724

0.720

0.714

0.6897

00.6857

0.6797

0.6850

--

3.3862

2

1

2

3

0.576

0.576

0.576

0.5417

0.5417

0.5417

0.5417

2.6696

3

1

2

3

0.605

0.601

0.594

0.5707

0.5667

0.5597

0.5657

2.7896

Interpretation of results:
GHS criteria not met
Conclusions:
Based on the outcome of the BCOP assessment, L-histidine was found to be not irritant or corrosive to the eyes.
Executive summary:

The potential of L-HISTIDINE to cause eye irritation or eye dammage was assessed with the in vitro BCOP assay. The study was performed according to OECD 437 and GLP.

The positive and negative control results met the criteria, indicating that the test system was valid.

The IVIS score for the test item was calculated to be 0.32. As this is below the threshold value of 3, L-histidine was found to not induce effects to the eye.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

In a first study the skin corrosion potential of the test item L-HISTIDINE was assessed with the in vitro membrane barrier assay CORROSITEX® . The study was according to OECD 435 and GLP.

The positive and negative control results met the criteria, indicating that the test system was valid.

The test item did not not have the ability to penetrate the biobarrier.

In the second study the skin irritation potential of the test item L-HISTIDINE was assessed in the in vitro membrane barrier assay EPISKINTM. The study was according to OECD 439 and GLP.

Both the negative and positive controls gave results in agreement with guideline indications and based on the criteria the study was accepted as valid. The test item did not induce any reduction in cell viability.

The potential of L-HISTIDINE to cause eye irritation or eye dammage was assessed with the in vitro BCOP assay. The study was performed according to OECD 437 and GLP.

The positive and negative control results met the criteria, indicating that the test system was valid.

The test item was found to not induce effects to the eye.

As the vitro studies are sufficient to conclude upon the classification and labelling for L-HISTIDINE with regard to both irritation to the skin and to the eyse, in vivo skin irritation/corrosion and in vivo eye dammage tests were not performed. This is in line with the REACh regulation as no additional animal testing was conducted when deemed unnecessary.

Justification for classification or non-classification

The classification criteria in the CLP legislation are not directly applicable to the in vitro tests. Therefore, the cut off values described in the relevant OECD guidelines are used for the classification of the substance.

Skin irritation:

For the first study the following cut-off time values are:

Category GHS 1A: 0-3 minutes

Category GHS 1B: >3-30 minutes

Category GHS 1C: >30-60 minutes

Non-corrosive >60 minutes.

L-Histidine did not penetrate the barrier for 65 minutes, therefore it should be considered as non corrosive to the skin.

For the second study the following cut-off time values are:

mean relative viability =< 50%: irritant (GHS Category 2)

mean relative viability > 50%: not irritant (GHS No category)

The mean relative viability of L-Histidine was 106.9%, therefore, it should be considered as not irritant to the skin.

Eye irritation:

The BCOP study allows to conlude on the classification of a substance if the IVIS is found to be <= 3 (no classification), or >= 55 (eye damage Cat. 1).

For L-HISTIDINE the IVIS score was found to be 0.32, thus allowing to conclude that the substance is not corrosive or irritant to the eyes.