N-L-alanyl-L-tyrosine

Endpoint summary

Description of key information

L-Alanyl-L-Tyrosine (L-Ala-L-Tyr) showed inconclusive results in the KeratinoSens™ assay and negative results in the U-Sens™ assay.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30.03.2020 - 29.05.2020

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:

2018

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EURL ECVAM DB-ALM Protocol n° 155: KeratinoSens™

Version / remarks:

2018

Deviations:

no

GLP compliance:

yes

Type of study:

activation of keratinocytes

Details on the study design:

PREPARATION OF TEST ITEM SOLUTIONS
- The test item was suspended in DMSO at 50 mM (white homogenous suspension). The stock solution was treated with ultrasonic waves to obtain a homogeneous suspension. From this stock 11 spike solutions in DMSO were prepared (2-fold dilution series). The stock and spike solutions were diluted 25-fold with exposure medium. These solutions were diluted 4-fold with exposure medium in the assay resulting in final test concentrations of 500, 250, 500, 125, 63, 31, 16, 7.8, 3.9, 2.0, 0.98, 0.49 and 0.24 µM (final concentration DMSO of 1%). At concentrations of 1.6 mM and higher the test item formed a suspension in DMSO whereas at 0.78 mM and lower it was fully soluble.
- All concentrations of the test item were tested in triplicate.
- 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.

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, Switzerland).
- All incubations, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 50 – 93 %), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 35.5 – 37.0°C).
- For testing, cells were 80-90% confluent. One day prior to testing cells were harvested, and distributed into 96-well plates (10,000 cells/well) in basic medium. One plate was used for the luciferase activity measurements, and one parallel replicate was used for the MTT cell viability assay. The cells were incubated overnight in the incubator.
- 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 covered with foil and then incubated for about 48 hours ± 1 h at 37±1.0°C in the presence of 5% CO2.

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 5 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; Sigma) and cells were incubated for 3 - 4 hours at 37°C ± 1.0°C in the presence of 5% CO2. The MTT medium was then removed and cells were lysed overnight by adding 10% SDS solution (Sigma) to each well. After shaking, the absorption was measured at 570 nm with the TECAN Infinite® M200 Pro Plate Reader.

Positive control results:

Experiment 1: The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 3.62 and the EC1.5 was 42 µM.
Experiment 2: The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 2.84 and the EC1.5 was 58 µM.

Run / experiment:

other: 1

Parameter:

other: maximum luciferase activity induction (Imax)

Value:

1.19

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Run / experiment:

other: 2

Parameter:

other: maximum luciferase activity induction (Imax)

Value:

1.11

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Other effects / acceptance of results:

Both tests passed the acceptance criteria:
• The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, was statistically significant above the threshold of 1.5-fold in at least one concentration.
• The EC1.5 of the positive control was within two standard deviations of the historical mean (42 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 (3.62-fold and 2.48-fold in experiment 1 and 2, respectively).
• Finally, the average coefficient of variation of the luminescence reading for the vehicle (negative) control DMSO was below 20% (8.6% and 5.9% in experiment 1 and 2, respectively).
Overall it is concluded that the test conditions were adequate and that the test system functioned properly.

The test item showed no toxicity (no IC30 and IC50 value) and no biologically relevant induction of the luciferase activity (no EC1.5 value) was measured at any of the test concentrations in both experiments. The maximum luciferase activity induction (Imax) was 1.19-fold and 1.11-fold in experiment 1 and 2 respectively. the test item is classified as negative in the KeratinoSensTM assay since negative results (<1.5-fold induction) were observed at test concentrations up to 2000 µM.

Overview EC_1.5, I_max, IC₃₀and IC₅₀Values

	EC1.5(µM)	Imax	IC30(µM)	IC50(µM)
Test item Experiment 1	NA	1.19	NA	NA
Test item Experiment 2	NA	1.11	NA	NA
Pos Control Experiment 1	42	3.62	NA	NA
Pos Control Experiment 2	58	2.48	NA	NA

NA = Not applicable

Interpretation of results:

other: inconclusive

Conclusions:

L-Alanyl-L-Tyrosine (L-Ala-L-Tyr) is classified as inconclusive (based on the absence
of a biologically relevant activation of the antioxidant/electrophile responsive element
(ARE)-dependent pathway in keratinocytes at test concentrations < 1000 μM) under the experimental conditions described.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17.04.2020 - 14.05.2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 442E – Annex II "In Vitro Skin Sensitisation: U937 Cell Line Activation Test (U-SENS™)"

Version / remarks:

2018

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EURL ECVAM DB-ALM Protocol n° 183: U937 Cell Line Activation Test for Skin Sensitization (U-SENS™)

Version / remarks:

2017

Deviations:

no

GLP compliance:

yes

Type of study:

activation of dendritic cells

Details on the study design:

TEST ITEM PREPARATION
- In the main experiments the test item was dissolved (clear solution) in complete medium at 0.4 mg/mL. The stock was diluted to a final test concentrations of 200, 100, 50, 20, 10 and 1 µg/mL in the first experiment and 100, 140, 180 and 200 μg/mL in the second experiment in the 96-well plate.
- No precipitation was observed at the end of the incubation period in the 96-well plates.
- Test item concentrations were used within 4 hours after preparation.

TEST SYSTEM
- U937 human monocytes (Inducible CD86-expressing cells; ATCC (American Type Culture Collection, Virginia, USA)
- Stock and treatment cultures were performed in RPMI-1640 medium supplemented with 10% (v/v) heat-inactivated (56°C; 30 min) foetal calf serum (FCS), L-glutamine (2 mM), penicillin/streptomycin (50 U/mL and 50 μg/mL respectively).
- All incubations were carried out in a humid atmosphere of 80 - 100% (actual range 55 - 91%) containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 36.3 - 36.8°C).
- Cultures were initiated in 96-well plates using 100 µL/well of a cell suspension adjusted at 5.0 x 10E5 viable cells/mL. Cell viability was > 90%. All assays were performed using two replicate culture-wells for the test item. One replicate was dedicated to the nonspecific IgG1 binding and the other one to the CD86 binding. Three replicates of untreated control (RPMI), vehicle control (in case of DMSO as vehicle), negative (LA) and positive (TNBS) controls were tested.
- Two valid experiments were conducted per test item.

TREATMENT OF CELLS
- Cells are treated for 45 ± 3 hours with the selected doses or controls (100 µL). The test item was in the first experiment evaluated up to 200 µg/mL using six doses: 1.0, 10, 20, 50, 100 and 200 µg/mL.
- In the second experiment cells were treated with four selected doses of test item. At least 2 concentrations were common with the previous experiment. The concentrations selected in the second experiment were 100, 140, 180 and 200 µg/mL.
- In all experiments, an untreated control (RPMI), vehicle control (in case of DMSO as vehicle) and the positive (TNBS) and negative control (LA) items were included. The final volume in the wells was 200 µL.

CELL ANTIBODIES STAINING FOR IgG1 AND CD86
Cultures were transferred into V-shaped 96-well plates. The cells were separated from the exposure medium by centrifugation (5 min, 200 g). The supernatant was discarded and cells were rinsed once with 100 µL/well Phosphate Buffered Saline (PBS) containing 5% FCS. After a second centrifugation step (5 min, 200 g) 100 µL/well of staining buffer (PBS containing 5% FCS) was applied to the cells.
FITC-conjugated antibodies was used for both IgG1 and CD86 staining:
- Mouse IgG1 of unknown specificity, for isotypic control
- Human CD86 specific mouse IgG1
The cells were transferred into new V-shaped 96-well plates (keeping the same plate template) containing 5 µL/well of the appropriate antibody (1:1 diluted in PBS) and placed refrigerated in the dark for 30 minutes. After this staining period, the cells were rinsed twice with a mixture of PBS/FCS and once in PBS alone and re-suspended in 90 µL of PBS.

FLOW CYTOMETRY METHOD
Just before acquisition, 5 µL of a 0.5 µg/mL propidium iodide (PI) solution was added to each well. The size (FSC) was set linear and the granularity (SSC) parameter was set to logarithmic scale and a R1 region was defined in which approximately 10,000 events were acquired for each culture. The acquisition parameters remained unchanged for the acquisition of all the wells. For the acquisition the BD FACSCanto™ flow cytometer was used and for further analysis BD FACSDiva™ software was used.
All analysis parameters were set on the RPMI wells for IgG1 and remained unchanged, for the analysis of all the other wells.

Positive control results:

Experiment 1: The positive control (TNBS) showed a S.I. ≥ 633% in all wells and was non-cytotoxic at all concentrations (cell viability ≥ 70%). The negative control (Lactic acid) showed a S.I. ≤ 97% in all wells and was non-cytotoxic at all concentrations (cell viability ≥ 70%).
Experiment 2: The positive control (TNBS) showed a S.I. ≥ 1091% in all wells and was non-cytotoxic at all concentrations (cell viability ≥ 70%). The negative control (LA) showed a S.I. ≤ 18% in all wells and was non-cytotoxic at all concentrations (cell viability ≥ 70%).

Run / experiment:

other: 1

Parameter:

other: % Viability (Mean)

Value:

100

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Run / experiment:

other: 2

Parameter:

other: % Viability (Mean)

Value:

100

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Run / experiment:

other: 1

Parameter:

other: CD86-IgG1 S.I.

Value:

88

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Run / experiment:

other: 2

Parameter:

other: CD86-IgG1 S.I.

Value:

145

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Run / experiment:

other: 1

Parameter:

other: Colour Interference S.I.

Value:

101

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Run / experiment:

other: 2

Parameter:

other: Colour Interference S.I.

Value:

96

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Other effects / acceptance of results:

Two independent experiments were performed. The cell viability before incubation with the test item was > 90% (99% and 91% in experiment 1 and 2, respectively). The cells were in these experiments incubated with the test item in a concentration range of 1.0 – 200 µg/mL. The increase of CD86 cell surface marker expression was assessed by measuring the amount fluorescent cell staining of the CD86 cell surface marker compared to the vehicle control. In addition, the viability was assessed with propidium iodide.

Experiment 1:
- No precipitation was observed at the end of the incubation period in the 96-well plates.
- The test item showed no toxicity, the viability of the cells was higher than 70% at all test concentrations and therefore no CV70 values could be calculated and is considered to be higher than 200 µg/mL.
- No increase in expression levels of CD86 compared to the vehicle control was observed at any of the test concentrations after treatment with the test item. No EC150 could be calculated and is considered to be higher than 200 µg/mL.
- The test item showed no colour interference.
- The positive control (TNBS) showed a S.I. ≥ 633% in all wells and was non-cytotoxic at all concentrations (cell viability ≥ 70%). The negative control (LA) showed a S.I. ≤ 97% in all wells and was non-cytotoxic at all concentrations (cell viability ≥ 70%).

Experiment 2:
- No precipitation was observed at the end of the incubation period in the 96-well plates.
- The test item showed no toxicity, the viability of the cells was higher than 70% at all test concentrations and therefore no CV70 values could be calculated and is considered to be higher than 200 µg/mL.
- No increase in expression levels of CD86 compared to the vehicle control was observed at almost all of the test concentrations after treatment with the test item. A fluctuation in increase in the expression of CD86 was observed after treatment with the test item, at only the intermediate dose level of 180 µg/mL (S.I. 164). However, this is considered to be biologically not relevant.
- The test item showed no colour interference.
- The positive control (TNBS) showed a S.I. ≥ 1091% in all wells and was non-cytotoxic at all concentrations (cell viability ≥ 70%). The negative control (LA) showed a S.I. ≤ 18% in all wells and was non-cytotoxic at all concentrations (cell viability ≥ 70%).

Both tests passed the acceptance criteria:
- At the end of the incubation treatment period, the mean viability of the triplicate untreated U937 cells was above the threshold of 90% (100% in experiment 1 and 100% in experiment 2).
- The CD86 basal expression of untreated U937 cells is within the range of ≥ 2% and ≤ 25% in both experiments.
- At least two out of three IgG1 values of untreated U937 cells fell within the range of ≥ 0.6% and < 1.5% in both experiments.
- No drift in CD86 expression was observed in the untreated controls and negative controls.

In both experiments the positive and negative control were considered valid and the positive control fell within the historical control data or above. Overall it is concluded that the test conditions were adequate and that the test system functioned properly.

Interpretation of results:

GHS criteria not met

Conclusions:

The test item showed no toxicity (No CV70 value) and no biologically relevant induction of the CD86 activity (No EC150 value) was measured at any of the test concentrations in both experiments. The test item is classified as negative in the U-Sens™ assay since negative results (< 150% increase) were observed at all test concentrations with a cell viability of >70% compared to the vehicle control. In conclusion, L-Alanyl-L-Tyrosine (L-Ala-L-Tyr) is classified as negative (no increase in the expression levels of CD86 cell surface marker in the U937 cell line) under the experimental conditions described.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

Expert statement for skin sensitising properties of N-Alanyl-L-tyrosine:

Based on Annex XI, 1.1(2) of Regulation (EC) No 1907/2006 (REACH) a skin sensitization study is scientifically not justified as explained in detail below: 

Background:

Skin sensitization is a process which can be basically divided into two parts. First, there must be an exposure to a sensitizer which induces specific molecular signaling cascades and second, the induction of these specific pathways must subsequently lead to elicitation of an allergic hypersensitivity reaction.

In order to induce the so-called Adverse Outcome Pathway (AOP) the substance suspected to act as a sensitizer must be able to penetrate the stratum corneum and be absorbed by keratinocytes. Within the keratinocytes the substance should either react directly with certain proteins or first bind to protein-complexes to form a hapten-protein complex capable of initiating an immunological response. Nevertheless, a covalent binding to proteins is a prerequisite of the sensitizing process. It is well known that most of the identified skin sensitizers are electrophiles that form covalent bonds with nucleophilic centers of proteins mainly consistent of distinct amino acid residues like lysine, tyrosine or cysteine. By binding proteins, transcription factors or other regulatory proteins are activated. These activations in turn initiate the expression of genes involved in the latter allergic reaction via intracellular signaling cascades. The subsequent elicitation of sensitization is dependent upon the induction of specific T lymphocyte responses (Kimber et al. 2000, 2002). 

In order to evaluate if a substance is able to cause skin sensitization a stepwise testing approach is recommended (ECHA amendment Annex VII adopted October 11th 2016). In order to reduce the use of animals for risk assessment, the information needed for the classification or risk assessment shall be obtained through non-animal methods as a first step. In vivo studies shall only be conducted if in vitro/in chemico test methods are not applicable. 

There are validated and regulatory accepted methods for non-animal testing of skin sensitization, including an in chemico test system named Direct Protein Reactivity Assay (DPRA test) and two in vitro test systems using the ARE-Nrf2 Luciferase test method

(KeratinoSens™ assay) or the activation of dendritic cells (DC), i.e. the human cell line activation test, which is considered to be the third key event in the AOP of skin sensitization as depicted in figure 1. DC activation can be caused through activated keratinocytes or directly by skin proteins which were changed by electrophilic chemicals.

 

DPRA:

Given that for a chemical to function as a contact sensitizer (or allergen), it must be capable of penetrating into the viable epidermis, react with protein, induce local trauma, and be recognized by the immune system (Gerberick et al., 2007), the DPRA represents one of the initial steps in sensitization - protein binding -. 

Although penetration of dipeptides into the skin is likely due to their molecular weight which is far below 500 Da, only a low toxicity is expected for the dipeptide because it is already an integral part of the intracellular intermediary metabolism and an indispensable part of mitosis. It is well known that dipeptides are hydrolyzed thereby forming amino acids which are readily metabolized once they have entered the cell (Amino Acid Metabolism: Bender D., 2012). Furthermore, although amino acids are formally substrates of protein synthesis, i.e. covalently bound to each other, it is highly unlikely that they are capable of reacting directly with proteins (or other amino acids) without energetic input. However, once incorporated into larger proteins many of the amino acid residues exhibit nucleophilic character.

This nucleophilic reactivity of certain amino acids is the underlying mechanism of the DPRA. The majority of chemical allergens (or their metabolites) have electrophilic properties and are able to react with various nucleophiles to form covalent bonds. In proteins, the side chains of many amino acids contain electron-rich groups, nucleophiles, capable of reacting with electrophilic allergens (Gerberick et al., 2004). Lysine and cysteine are those most often cited, but other amino acids containing nucleophilic heteroatoms, such as histidine, methionine, arginine and tyrosine can also react with electrophiles (Ahlfors et al., 2003, Gerberick et al., The Report and Recommendations of ECVAM Workshop 64a, 2008). It is highly unlikely that the assay is capable of detecting a sensitizing property of an added amino acid (in case of tyrosine) already included in the method and known to react nucleophilic. Some amino acids, e.g. non-charged molecules like asparagine or alanine are not expected to react as electrophiles, therefore these molecules will most probably not be detected by this method. Even if amino acids belonging to the group of acidic amino acids like glutamate or aspartate are tested with this method there would be a negative result because under acidic conditions as present in keratinocytes these amino acids would be present in their ionized form and therefore not capable of reacting as an electrophile.

 

KeratinoSens™ assay

The underlying mechanism of the KeratinoSens™ assay is the interaction of electrophile substances with certain inhibitor proteins, resulting in an activation of signaling cascades which in turn promote elicitation of sensitization. One of these signaling pathways is the activation of the transcription factor Nrf-2. It is well known that Nrf-2 is constitutively expressed in various cells. In its inactive form it is bound to its inhibitor KeapI, which serves as a sensor for electrophiles. KeapI consists of several predisposed cysteine residues which can bind to electrophiles thereby changing KeapI´s conformation and enable its proteasomal degradation. The result of KeapI degradation is the activation and delocalization of the transcription factor Nrf-2 into the nucleus where it subsequently induces the expression of genes involved in an inflammatory response. As mentioned above most dipeptides or amino acids are not assumed to react electrophilic enough to modify the respective cysteine residues responsible for KeapI´s degradation. Although the pH in this test system is more physiological than compared with to the acidic milieu within the DPRA test, most of the amino acids will not change their reactivity towards proteins.

Thus, both assays are based on the same underlying mechanism, interaction of an electrophile substance with a nucleophilic centre of a target protein and amino acids of neutral or hydrophobic origin, i.e. methionine, alanine or asparagine are not reactive enough to form covalent bonds without energetic expenditure. 

Furthermore, since the KeratinoSens assay is an in vitro system the addition of amino acids in form of serum proteins is an indispensable prerequisite for cellular growth and optimal performance of the test system. Therefore, increasing the concentration of amino acids is considered to enhance growth and not to mediate adverse effects. Several Vendors recommend even to supplement the growth media with certain amino acids to ensure optimal culture conditions (ATCC(American Type Culture Collection; www.lgcstandards-atcc.org)).

 

h-CLAT / U-SENS^TM

The third test system which was adopted on July 29^th, 2016 by the OECD is the human Cell Line Activation Test (h-CLAT). Also in the OECD TG 442E the method U937 cell line activation Test (U-SENS™) is described. The underlying mechanism of these tests is the activation of DC which constitutes the third key step within the AOP of skin sensitization. As a result of their activation, the expression pattern of DCs changes and the expression of certain surface markers, chemokines and cytokines is augmented. Nevertheless, this event is supposed to happen only if the specific cellular signaling cascades are activated and this activation in turn is dependent of the previously described reaction of an electrophilic substance with proteins. As outlined above, for amino acids and also dipeptides formation of a covalent bond is an energy-consuming process thus, N-Alanyl-L-tyrosine is not assumed to be able to activate denditric cells.

 

Since the dipeptide to be registered falls out of the applicability domain of the validated and accepted in vitro/in chemico test methods, the next step would be in vivo testing of the substance. However, also in vivo testing of N-Acetyl-L-tyrosine is not justified according to Annex XI. A detailed explanation is given in the following section:

As already explained in the first section of this justification, the capability of electrophilic chemicals to react with nucleophilic centres of proteins is only one key step in skin sensitization. After modification of cellular proteins like KeapI and expression of inflammatory response genes and/ or forming hapten-protein complex another important event is the recognition of the allergen by the immune system. It was shown that the inducing or mature allergen needs to bind at least two antibody molecules to be recognized by the immune system. The requirement for two IgE binding sites, i.e. two epitopes, in turn requires a peptide with a minimum of 15 amino acid residues in order to bind these antibodies (Huby et al., 2000). Although amino acids are not likely bound to proteins without energetic support we consider that even if they are bound to proteins they would not turn into an allergen because this reaction most probably will not generate a molecule that is recognized as “foreign” or “not-self” which in turn is a basic requirement for an allergen (Gerberick et al., The Report and Recommendations of ECVAM Workshop 64a, 2008).

Further evidence is provided by negative in vivo test results obtained with the related dipeptide L-Alanyl-L-glutamine. L-Alanyl-L-glutamine was tested in Guinea Pig Maximization Test. Under the conditions used in the study the test substance showed no sensitizing potential (93-0103-DNT).

Recapitulatory, a reaction of the dipeptide N-Alanyl-L-tyrosine is not expected because it is 1) not electrophilic enough to react with the used oligopeptide in case of the DPRA test, the KeratinoSens^TM assay or the h-CLAT/ U-SENS™ and 2) it is too small to be recognized by the immune system even under the assumption that it might has been bound to a protein would be recognized as “own-protein”.

Therefore, neither in vitro nor in vivo testing of skin sensitizing properties of either L-Alanine, L-Tyrosine or N-Alanyl-L-tyrosine is scientifically justified.

Nevertheless, two in vitro assays covering the second and third key steps of the AOP, the KeratinoSens™ assay and the U-SENS™ assay, were performed for the sake of data completeness and to provide further evidence that no skin sensitizing potential has to be expected from a dipeptide like N-Alanyl-L-tyrosine. The KeratinoSens™ assay was concluded inconclusive as the negative results were obtained with concentration <1000 μM without reaching cytotoxicity at the maximal tested concentration. The U-SENS™ assay however, was clearly negative. The two in vitro assays therefore confirmed the absence of a skin sensitizing potential of the dipeptide N-Alanyl-L-tyrosine.

 

 

References:

Guidance: Information Requirements and Chemical Safety Assessment, Chapter R 7.a, section R.7.3 Skin sensitisation, ECHA, 2017-07.

Bender, D. Amino Acid Metabolism, 3^rd ed., John Wiley & Sons Ltd. 2012

Gerberick et al. (2008). Chemical Reactivity Measurement and the Predictive Identification of Skin Sensitisers. The Report and Recommendations of ECVAM Workshop 64a. ATLA 36, 215-242.

Gerberick  et  al.  (2007). Quantification  of  chemical  peptide  reactivity  for  screening  contact allergens: A classification tree model approach. Toxicological Sciences 97:417-427.

Gerberick  et  al.  (2004).  Development  of  a  peptide  reactivity  assay  for  screening  contact  allergens. Toxicological Sciences 81:332-343.

Ahlfors, S. R., Sterner, O., and Hansson, C. (2003). Reactivity of contact allergenic haptens to amino acid residues in a model carrier peptide, and characterization of formed petide-hapten adducts. Skin Pharmacol. Appl. Skin Physiol. 16, 59–68.

Huby, R. D. J., Dearman, R.J., and Kimber, I. (2000). Why are some Proteins allergens? Toxicological Sciences 55, 235-246.

93-0103-DNT, Study report, L-Alanyl-L-Glutamine: Testing the cutaneous sensitizing properties in the guinea pig (Maximization Test), ASTA Medica AG, Institute of Toxicology, 1994.

2020-5034-DGT, Study report, Evaluation of in vitro Skin Sensitization Potential of L-Alanyl-L-Tyrosine (L-Ala-L-Tyr) with the KeratinoSens^TM Assay, Charles River Laboratories Den Bosch BV, 2020

2020-5036-DGT, Study report, Evaluation of in vitro Skin Sensitization Potential of L-Alanyl-L-Tyrosine (L-Ala-L-Tyr) with the U937 Cell Line Activation Test (U-SENS™) Assay, Charles River Laboratories Den Bosch BV, 2020

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

L-Alanyl-L-Tyrosine (L-Ala-L-Tyr) showed inconclusive results in the KeratinoSens™ assay and negative results in the U-Sens™ assay. Therefore, no classification and labelling according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2017) (including all amendments) and Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendment) is needed.