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Description of key information

L-Phenylalanine is neither irritating or corrosive to skin nor irritating to eyes as well as to the respiratory system.

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:
2014-05-08 to 2014-06-30
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Reference:
Composition 0
Qualifier:
according to
Guideline:
other: OECD Guideline 439 In vitro Skin Irritation: Reconstructed Human Epidermis Test method, adopted July 26, 2013
Deviations:
no
Qualifier:
according to
Guideline:
other: Council Regulation (EC) No. 761/2009, method B.46. In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test, adopted August 24, 2009
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test material information:
Composition 1
Species:
other: three-dimensional reconstructed human epidermis model EpiDermTM
Strain:
other: Not applicable
Details on test animals and environmental conditions:
The following Reconstructed Human Epidermis Model was used:
EpiDermTM (EPI-212-SIT, Lot no. 19642) MatTek In Vitro Life Science Laboratories, Bratislava, Slovak Republic.
Type of coverage:
other: The test substance was applied to the skin model to uniformly cover the skin surface.
Preparation of test site:
other: Not applicable as three-dimensional reconstructed human epidermis model EpiDermTM.
Vehicle:
unchanged (no vehicle)
Remarks:
But the epidermis surface was moistened with Dulbecco's phosphate buffered saline (D-PBS) before application to ensure good contact with the skin.
Controls:
other: Concurrent negative (Dulbecco's phospahe buffered sline) and positive control (SDS) each in triplicate, to demonstrate that viability (NC), barrier function and resulting issue sensitivity (PC) of the tissues are acceptance range.
Amount / concentration applied:
25 mg on the surface area of 0.63 cm².
Duration of treatment / exposure:
See "Details on study design"
Observation period:
See "Details on study design"
Number of animals:
3 samples were applied to the skin model
Details on study design:
1.1 General model conditions
Normal human keratinocytes were used to construct the epithelium. Multiple layers of viable epithelial cells (basal layer, stratum spinosum, stratum granulosum) were present under a functional stratum corneum. Stratum corneum was multilayered containing the essential lipid profile to produce a functional barrier with robustness to resist rapid penetration of the cytotoxic marker substance sodium dodecyl sulphate (SDS). The barrier function is assessed either by determination of the concentration at which a marker substance reduces the viability of the tissues by 50% (IC50) after a fixed exposure time, or by determination of the exposure time required to reduce cell viability by 50% (ET50) upon application of the marker substance at a specified, fixed concentration. The containment properties of the model prevented the passage of material around the stratum corneum to the viable
tissue, which would lead to poor modelling of skin exposure. The skin model was free of contamination by bacteria, viruses, mycoplasma, or fungi.

1.2 Functional model conditions
The preferred assay for determining the magnitude of viability was the MTT. The optical density (OD) of the extraction solvent alone were sufficiently small, i.e. OD < 0.1. The tissue treated with NC should exhibit stability in culture (provide similar viability measurements) for the duration of the test exposure period.

2. Quality controls (QC) of the model
The EpiDerm™ System was manufactured according to defined quality assurance procedures. All biological components of the epidermis and the culture medium were tested by manufacturer for viral, bacterial, fungal and mycoplasma contamination. The supplier determines the ET50 value following exposure to Triton-100 (1%) for each EpiDerm lot. The ET50 must fall within a range established based on a historical database of results.
The certificate of the EpiDermTM model has been attached to the study report.

3. Test for interference of chemicals with MTT assay
To identify the potential of the test item to interfere with MTT assay, the following check was performed:
25 mg test item was mixed with 300 μL sterile deionised water and incubated in the dark at 37°C, 5% CO2 and 95% humidity for 60 minutes. No discolorations were noted. Hence, no possible interacting with the MTT measurement had to be considered and no additional test had to be performed.

4. Administration of the test, negative and positive reference items
25 mg of test item were applied to the phosphate buffered saline moistened skin model with a surface area of 0.63 cm2 to uniformly cover the skin surface. A minimum of 25 mg or 25 μL substance applied per cm2 is required by the guidelines. Three replicate tissues were employed. At the end of the exposure period, the test item was carefully washed from the skin surface with Dulbecco's phosphate buffered saline (D-PBS).
The whole exposure period for the used EpiDermTM skin model was 60 minutes.
The incubation conditions were 37°C, 5% CO2 and 95% humidity for the first 35 minutes followed by 25 minutes at room temperature under a sterile hood. Concurrent negative and positive controls were used, each in triplicate, to demonstrate that viability (NC), barrier function and resulting issue sensitivity (PC) of the tissues are within a defined historical acceptance range. The positive control item was 5% aqueous sodium dodecyl sulphate (SDS) and the negative control was D-PBS. 30 μL of negative and positive controls were used.

5. Cell viability measurements
The MTT conversion assay is a quantitative validated method which is used to measure cell viability. It is compatible with use in a three-dimensional tissue construct. The most important element of the test procedure was that viability measurements were not performed immediately after the exposure to the test item, but after a posttreatment incubation period of the rinsed tissues in fresh assay medium of 42 hours.
This period allows both for recovery from weakly irritant effects and for appearance of clear cytotoxic effects. Each skin sample was placed in an MTT assay solution of 1 mg/mL (37°C incubation temperature, 5% CO2, 95% humidity) for 3 hours. The precipitated blue formazan product was extracted using the extraction solution (isopropanol), and the concentration of the formazan was measured by determining the optical density (OD) at a wavelength of 570 nm in a spectrophotometer. The measurements were made for each of the three tissues in duplicate.

6. Assay acceptability criteria
Assay acceptance criterion 1: Negative control
The absolute OD of the negative control (NC) tissues (treated with sterile PBS buffer) in the MTT test is an indicator of tissue viability obtained in the testing laboratory after shipping and storing procedures and under specific conditions of use.
The assay meets the acceptance criterion if the mean OD570 of the NC tissues is ≥ 1.0 and ≤ 2.5.
Assay acceptance criterion 2: Positive control
A 5% SDS (in H2O) solution was used as positive control (PC) and tested concurrently with the test chemicals. Concurrent means here the PC has to be
tested in each assay, but not more than one PC is required per testing day.
The assay meets the acceptance criterion if the mean viability of PC tissues expressed as % of the negative control tissues is ≤ 20%.
Assay acceptance criterion 3: Standard deviation
Since in each test skin irritancy potential is predicted from the mean viability determined on 3 single tissues, the variability of tissue replicates should be acceptably low.
The assay meets the acceptance criterion if the standard deviation (SD) calculated from individual % tissue viabilities of the 3 identically treated replicates is < 18%.

7. Interpretation of results
The OD values obtained for each test sample were used to calculate mean percentage viability relative to the negative control, which was arbitrarily set at 100%. The cut-off mean percentage cell viability value that distinguishes irritant from non-classified test substances is given below:
The test item has to be considered to be irritant to skin in accordance with UN GHS category 2, if the tissue viability after exposure and post-treatment incubation is below or equal 50%.
The test item has to be non-irritant if the tissue viability after exposure and posttreatment incubation is higher than 50%.
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
mean of 3 experiments
Value:
103.1
Vehicle controls valid:
not specified
Negative controls valid:
yes
Positive controls valid:
yes

The EpiDermTM model was employed.

Three tissues were used for each treatment and concurrent control groups. The optical density (OD) was determined by using the MTT (3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide, Thiazolyl blue) reduction assay and expressed as relative percentage of viability of the negative control-treated tissues.

L-Phenylalanine was applied topically as solid test item to the model skin surface, which was moistened with phosphate buffered saline. Dulbecco’s phosphate buffered saline (D-PBS) was used as the negative control. 5% aqueous sodium dodecyl sulphate (SDS) was used as the positive reference item.

The mea n viability of cells exposed to L-Phenylalanine was 103.1% of the negative controls and, hence, was well above the cut-off percentage cell viability value that distinguishes irritant from non-irritant test items of > 50%. L-Phenylalanine was considered to be non-cytotoxic and predicted to be nonirritant to skin. The mean optical density (OD) of the negative control of 3 tissues was 2.160 and was well within the acceptable range of ≥ 1.0 to ≤ 2.5. The viability of cells treated with the positive reference item, 5% SDS, was 3.1% of the negative control and fulfilled the acceptance criteria of < 20%. The standard deviation of all triplicates determined was below the limit of acceptance of 18%. Hence, all acceptance criteria were fulfilled. Conclusion: Under the present test conditions, L-Phenylalanine tested at an exposure time of 60 minutes and a 42-hour post-treatment incubation period, was non-cytotoxic and, hence, was considered to be non-irritant to skin in an experiment employing an artificial three-dimensional model of human skin.

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: OECD GHS
Conclusions:
In a GLP gudeline study according to OECD 439 (three-dimensional reconstructed human epidermis model) L-phenylalanine did not show to be irritatting 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:
2014-05-08 to 2014-06-30
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study. Test procedure in accordance with generally accepted scientific standards and described in sufficient detail.
Reference:
Composition 0
Principles of method if other than guideline:
The study was carried out according to:
ICCVAM-Recommended Test Method Protocol: Hen’s Egg Test – Chorioallantoic Membrane (HET-CAM) Test Method, published 2010.
GLP compliance:
yes (incl. certificate)
Test material information:
Composition 1
Species:
other: CAM (chorioallantoic membrane) of fertile chicken eggs.
Strain:
other: CAM of chicken eggs
Details on test animals or tissues and environmental conditions:
Biological materials
Test system: Fertilized white Leghorn chicken eggs
- Source: Charles River Laboratories, Avian Products and Services, Germany GmbH, Sulzfeld, Germany
- Weight: 53 - 59 g
- Old: 6 days (at study initiation, before incubation)

Vehicle:
unchanged (no vehicle)
Controls:
other: Negative control item: 3 eggs treated with physiological saline. Positive control items: 3 eggs treated with NaOH (0.1 N), 3 eggs treated with SDS (1%).
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 300 mg/egg (used undiluted, grounded in a mortar to a fine dust)
Observation period (in vivo):
Starting 20 seconds after the application the reactions on the CAM were observed over a period of 300 seconds.
Number of animals or in vitro replicates:
3 eggs treated with the test item.
Details on study design:
1. Principle of the test
This method is increasingly used for the detection of eye mucosa membrane irritants. Eye irritation caused by external contact with chemical substances is characterized by corneal damage and/or conjunctival injury and/or iris defects.
The CAM of fertile eggs incubated for 9 days is a vital vascular membrane with a blood vessel complex. Effects which might produce irritancy in the conjunctiva are assessed by exposing the CAM to liquid or solid test items.

2. CAM preparation
a. Fresh, clean, fertile chicken eggs were selected. The eggs were candled and any eggs that were nonviable or defective were discarded. Excessively misshapen eggs or eggs with cracked or thin shells were not used. Shaking, unnecessary tilting, knocking, and all other mechanical irritation of the eggs were avoided when preparing.
b. The eggs were placed in an incubator with a rotating tray. The eggs were incubated at 38.3 ± 0.2°C and 58 ± 2% relative humidity in a Grumbach Compact S 84 incubator. The eggs were rotated five times per day until the day 8.
c. The eggs were candled on incubation day 8 and any nonviable or defective eggs were removed. Eggs were returned to the incubator (without hand rotation) with the large end of the eggs upwards for an additional day.
d. Eggs were removed from the incubator on day 9 for use in the assay. The eggs were again candled and any nonviable or defective eggs were discarded.
e. The air cell of the egg was marked. The section marked as the air cell was cut and then pared off. Care was taken when removing the eggshell to ensure that the inner membrane was not injured.
f. The inner membrane was moistened with 0.9% NaCl. A disposable pipette was used to apply the solution. The egg was placed into the incubator for a maximum of 30 minutes.
g. The egg was removed from the incubator, prior to its use in the assay, and the 0.9% NaCl solution was decanted. The inner membrane was carefully removed with forceps.

3. Observations
The chorioallantoic membrane (CAM) was carefully rinsed with 0.9% NaCl solution immediately before evaluation. Starting 20 seconds after the application the reactions on the CAM were observed over a period of 300 seconds. The time for the appearance of each of the noted endpoints was monitored and recorded, in seconds.
The following endpoints were observed:
- haemorrhage (bleeding from the vessels)
- vascular lysis (blood vessel disintegration)
- coagulation (intra- and extra-vascular protein denaturation)
Whereby,
Haemorrhage time = observed start (in seconds) of haemorrhage reactions on CAM
Lysis time = observed start (in seconds) of vessel lysis on CAM
Coagulation time = observed start (in seconds) of coagulation formation on CAM.
Irritation parameter:
in vitro irritation score
Run / experiment:
mean - haemorrhage
Value:
0
Vehicle controls valid:
not specified
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: All 3 individual values were 0 at each time interval of 0.5, 2 and 5 min. Max. score 7 after 0.5 min, 5 after 2 min, 3 after 5 min.
Irritation parameter:
in vitro irritation score
Run / experiment:
mean - lysis
Value:
0
Vehicle controls valid:
not specified
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: All 3 individual values were 0 at each time interval of 0.5, 2 and 5 min. Max. score 5 after 0.5 min, 3 after 2 min, 1 after 5 min.
Irritation parameter:
in vitro irritation score
Run / experiment:
mean - coagulation
Value:
0
Vehicle controls valid:
not specified
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: All 3 individual values were 0 at each time interval of 0.5, 2 and 5 min. Max. score 9 after 0.5 min, 7 after 2 min, 5 after 5 min.
Irritation parameter:
in vitro irritation score
Run / experiment:
cumulative irritation acore
Value:
0
Vehicle controls valid:
not specified
Negative controls valid:
yes
Positive controls valid:
yes
Remarks:
Max. Score: 21

Evaluation

The current ICCVAM recommended protocol for which HET-CAM is recommended as a screening test to identify non-labelled ocular irritants uses an analysis method (i.e., the Irritation Score IS(A)) which is based on development of each of the three

HET-CAM endpoints at fixed time intervals of 0.5, 2, and 5 minutes (Table 1; Lüpke 1985).

The numerical time-dependent scores for lysis, haemorrhage, and coagulation (Table 2) are summed to give a single numerical value indicating the irritation potential of the test substance on a scale with a maximum value of 21.

Table 1: Scoring scheme for irritation testing

 

Effect

Score

0.5 min

2 min

5 min

Lysis

5

3

1

Haemorrhage

7

5

3

Coagulation

9

7

5

 

Table 2: Classification of cumulative scores

Cumulative Score

Irritation Assessment

0 - 0.9

Practically none

1 - 4.9

Slight

5 - 8.9

Moderate

9 - 21

Strong

 

Criteria for an acceptable test

The HET-CAM assay is considered acceptable if the negative and positive controls each induce a response that falls within the classification of nonirritating and severely irritating, respectively. Historical control studies indicate that using 0.9% NaCl, as a negative control, the IS value was 0.0 (35 studies in the time period between 2010 and 2012). Historical control studies demonstrate that using 1% SDS and 0.1 N NaOH as positive controls results in IS values of approximately 8 to 12 or 17 to 19, respectively.

References

Lüpke, N. P., 1985: Hen's egg chorioallantoic membrane test for irritation potential Fd. Chem. Toxic. Vol. 23, No. 2, pp. 287 - 291

ICCVAM. 2010: ICCVAM-Recommended Test Method Protocol: Hen’s Egg Test - Chorioallantoic Membrane (HET-CAM) Test Method. NIH Publication No: 10-7553, published 2010.

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: OECD GHS
Conclusions:
L-Phenylalanine did not show any eye irritancy potential in an in vitro GLP study using fertile chicken eggs (HET-CAM test).
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Respiratory irritation

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

Additional information

Skin

The dermal irritant potential of L-phenylalanine was assessed in a GLP gudeline study according to OECD 439 (three-dimensional reconstructed human epidermis model) by application of the substance to the test system. L-Phenylalanine did not show to be irritating to the skin and is not classified as irritating or corrosive to human skin.

Eye

The eye irritant potential of L-phenylalanine was assessed in an in vitro GLP study using fertile chicken eggs (HET-CAM test).

The substance did not show any eye irritation. L-Phenylalanine is classified as not irritating to human eye.

Respiratory System

There are no data available which indicate that L-phenylalanine is irritant to the respiratory system.

 

Justification for selection of skin irritation / corrosion endpoint:

Key study

Justification for selection of eye irritation endpoint:

Key study

Justification for classification or non-classification

L-Phenylalanine does not show any irritating or corrosive properties towards both the skin and the eyes. Thus classification is not required.