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Toxicological information

Skin irritation / corrosion

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Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2017

Materials and methods

Test guideline
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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Identity: L-Histidine
- Batch n°: P816P001
- Expiry date: 03 January 2016
- Storage conditions: room temperature protected from light
- RTC number: 14549

Test animals

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

Test system

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)

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
other: cell viability
Run / experiment:
Mean
Value:
106.9
Remarks on result:
other: Remarks: expressed in % relative to the negative control.

Applicant's summary and conclusion

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.