Registration Dossier

Administrative data

Description of key information

The Skin Sensitization endpoint has been assessed using a weight of evidence approach, as indicated in Annex VII, 8.3.1 of the REACH Regulation.

- In Chemico Determination of the Skin Sensitization Potential of N-acetylglycyl beta-alanine unsing the Direct Peptide Reactivity Assay (DPRA). The objective of this study was to determine the reactivity of N-acetylglycyl beta-alanine towards model synthetic peptides containing either cysteine (SPCC) or lysine (SPCL).All acceptability criteria were met, therefore this DPRA is considered to be valid.

N-acetylglycyl beta-alanine was negative in the DPRA and was classified in the “no or minimal reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model.

-Evaluation of in vitro Skin Sensitization Potential of N-acetylglycyl beta-alanine with the KeratinoSensTM Assay

The objective of this study was to evaluate the ability of N-acetylglycyl beta-alanine to activate the antioxidant/electrophile

responsive element (ARE)-dependent pathway in the KeratinoSensTM assay.

The result of the study shows the test item is classified as negative (no activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) under the experimental conditions described in this report.

- Q(SAR) prediction using DEREK NEXUS. DEREK NEXUS version 5.0.2 did not yield any alerts for skin sensitization for the test item.

N-acetylglycyl beta-alanine is predicted to be not sensitizing to the skin.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation, other
Remarks:
in silico model
Type of information:
(Q)SAR
Adequacy of study:
weight of evidence
Study period:
February 2017
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification
Justification for type of information:
1. SOFTWARE
DEREK NEXUS

2. MODEL (incl. version number)
DEREK NEXUS version 5.0.2
DEREK NEXUS is a knowledge-based system that contains 80 alerts for skin sensitization based on the presence of molecular substructures. LHASA (see Appendix I) has inserted validation comments for the skin sensitization alerts.
The level of likelihood of a structure being sensitizing to skin is expressed in terms of:
Certain There is proof that the proposition is true.
Probable There is at least one strong argument that the proposition is true and there are no arguments against it.
Plausible The weight of evidence supports the proposition.
Equivocal There is an equal weight of evidence for and against the proposition.

The default of DEREK NEXUS for the level of likelihood, mentioning all alerts which are evaluated as being equivocal or greater was used in this assessment.

If a substance is predicted to be a skin sensitizer, its potency will be predicted by calculating an EC3 value based on experimental data from its closest structurally-related substances (at
least 3 closest substances should be present).The EC3 is the estimated concentration needed to produce a stimulation index of 3.

3. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL
Smiles: CC(NCC(NCCC(O)=O)=O)=O
Average Mol Mass Exact Mol Mass Log Kp Log P
188.18 188.0797 -4.97 -1.55

4. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL
[Explain how the model fulfils the OECD principles for (Q)SAR model validation. Consider attaching the QMRF or providing a link]

Endpoint (OECD Principle 1):
Endpoint: Skin sensitisation.
Dependent variable: Data from several toxicity assays (e.g. LLNA, GPMT, HRIPT) ad mechanistic studies (e.g. DPRA) are synthesised to arrive at an expert conclusion of whether compounds within the model training set is likely to be a skin sensitizer.

Algorithm (OECD Principle 2)
a. Model or submodel name: DEREK NEXUS – skin sensitization.
b. Model version: DEREK NEXUS 5.0.2.
c. Reference to QMRF: The QMRF DEREK NEXUS 5.0.2 – skin sensitization was prepared by the provider LHASA Ltd (UK).
d. Predicted value (model result): Assessment with DEREK NEXUS did not yield any skin sensitization alerts.
e. Predicted value (comments): Not relevant; if the structure does not does not contain one of the currently 80 skin sensitisation alerts, the structure is predicted to be
negative.
f. Input for prediction: See section 1.5.
g. Descriptor values: Not relevant.

Applicability domain (OECD principle 3):
a. Domains:
i. descriptor domain: The scopes of the structure-activity relationships describing the skin sensitisation endpoint are defined by the developer to be the applicability domain for the model. Therefore, if a chemical activates an alert describing a structure-activity for skin sensitisation it can be considered to be within the applicability domain. The applicability of potency predictions may be judged, and modified, by the user based on the displayed data for nearest neighbours. If a compound does not activate an alert or reasoning rule in Derek, a result of ‘nothing to report’ is presented to the user. This can be interpreted as a negative prediction or that the query compound is outside the domain of the model. Which of these is more appropriate may dependon the endpoint of interest. For the endpoint of skin sensitisation, which features multiple alerts believed to cover most of the mechanisms and chemical classes responsible for activity, ‘nothing to report’ may be extrapolated to a negative prediction.
Method used to assess the applicability domain: The applicability domain of each alert is defined by the alert developer on the basis of the training set data and expert judgement on the chemical and biological factors which affect the mechanism of action for each alert. For potency predictions, at least three nearest neighbours are required within alerting space to make a prediction.
ii. structural fragment domain: for skin sensitisation, which features multiple alerts believed to cover most of the mechanisms and chemical classes responsible for activity, ‘no alerts fired’ may be extrapolated to a negative prediction.
iii. mechanism domain: as the prediction is ‘no alerts fired’ none of the mechanisms for skin sensitisation is predicted to be applicable to this structure.
iv. metabolic domain: not relevant.
b. Structural analogues: Not applicable, because DEREK Nexus is alert-based and alerts are based on positive results mostly, only analogues are presented if a result is positive.
c. Considerations on structural analogues: Not applicable.

The uncertainty of the prediction (OECD principle 4):
DEREK NEXUS predictive performance against a combined human dataset had an accuracy of 77%.
https://www.lhasalimited.org/Public/Library/2014/Derek%20Nexus%20predicts%20human%20skin%20sensitisation%20accurately.pdf

The chemical and biological mechanisms according to the model underpinning the predicted result (OECD principle 5).
Not relevant; DEREK NEXUS did not yield any alerts for skin sensitisation.


5. APPLICABILITY DOMAIN
[Explain how the substance falls within the applicability domain of the model]
- Descriptor domain: The scopes of the structure-activity relationships describing the skin sensitisation endpoint are defined by the developer to be the applicability domain for the model. Therefore, if a chemical activates an alert describing a structure-activity for skin sensitisation it can be considered to be within the applicability domain. The applicability of potency predictions may be judged, and modified, by the user based on the displayed data for nearest neighbours. If a compound does not activate an alert or reasoning rule in Derek, a result of ‘nothing to report’ is presented to the user. This can be interpreted as a negative prediction or that the query compound is outside the domain of the model. Which of these is more appropriate may dependon the endpoint of interest. For the endpoint of skin sensitisation, which features multiple alerts believed to cover most of the mechanisms and chemical classes responsible for activity, ‘nothing to report’ may be extrapolated to a negative prediction.
- Structural and mechanistic domains: for skin sensitisation, which features multiple alerts believed to cover most of the mechanisms and chemical classes responsible for activity, ‘no alerts fired’ may be extrapolated to a negative prediction. As the prediction is ‘no alerts fired’ none of the mechanisms for skin sensitisation is predicted to be applicable to this structure.
- Similarity with analogues in the training set: Not applicable, because DEREK Nexus is alert-based and alerts are based on positive results mostly, only analogues are presented if a result is positive.

6. ADEQUACY OF THE RESULT
[Explain how the prediction fits the purpose of classification and labelling and/or risk assessment]
Regulatory purpose: The present prediction may be used for preparing the REACH Registration Dossier on the substance for submission to ECHA, as required by Regulation (EC) 1907/2006 and related amendments.
Approach for regulatory interpretation of the model result: This result can be directly used within a weight-of-evidence approach to complete the endpoint skin sensitization.
Outcome: Substance should not be classified according to DEREK NEXUS; however, this (Q)SAR prediction cannot be used as stand-alone for classification purposes or for covering the endpoint skin sensitization for registration under REACH.
Conclusion: The result is adequate to be used in a weight-of-evidence approach together with in chemico/in vitro studies to complete the endpoint skin sensitization.
Principles of method if other than guideline:
- Software tool(s) used including version: DEREK NEXUS version 5.0.2
- Model(s) used: In silico model Derek Nexus
- Model description: see field 'Justification for non-standard information'
- Justification of QSAR prediction: see field 'Justification for type of information'
Remarks on result:
no indication of skin sensitisation
Interpretation of results:
other: The result is adequate to be used in a weight-of-evidence approach together with in chemico/in vitro studies to complete the endpoint skin sensitization.
Conclusions:
DEREK NEXUS version 5.0.2 did not yield any alerts for skin sensitization for the test item.
N-acetylglycyl beta-alanine is predicted to be not sensitizing to the skin.
Executive summary:

The objective of this study was to obtain a prediction on the potential for skin sensitization of N-acetylglycyl beta-alanine with the in silico model DEREK NEXUS. In this assessment version 5.0.2 of DEREK NEXUS was used.

DEREK NEXUS is a knowledge-based system that contains 80 alerts for skin sensitization based on the presence of molecular substructures. LHASA (see Appendix I) has inserted validation comments for the skin sensitization alerts.

The level of likelihood of a structure being sensitizing to skin is expressed in terms of:

Certain There is proof that the proposition is true.

Probable There is at least one strong argument that the proposition is true and there are no arguments against it.

Plausible The weight of evidence supports the proposition.

Equivocal There is an equal weight of evidence for and against the proposition.

The default of DEREK NEXUS for the level of likelihood, mentioning all alerts which are evaluated as being equivocal or greater was used in this assessment.

If a substance is predicted to be a skin sensitizer, its potency will be predicted by calculating an EC3 value based on experimental data from its closest structurally-related substances (at least 3 closest substances should be present). The EC3 is the estimated concentration needed to produce a stimulation index of 3.

DEREK NEXUS version 5.0.2 did not yield any alerts for skin sensitization for the test item.

N-acetylglycyl beta-alanine is predicted to be not sensitizing to the skin.

Endpoint:
skin sensitisation: in chemico
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
April-May 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
Version / remarks:
4 February 2015
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
direct peptide binding assay
Details on study design:
The objective of the study was to determine the reactivity of N-acetylglycil beta-alanine towards model synthetic peptides containing either cysteine (SPCC) or lysine (SPCL). After incubation of GenoWhite with either SPCC or SPCL, the relative peptide concentration was determined by High-Performance Liquid Chromatography (HPLC) with gradient elution and photodiode array (PDA) detection at 220 nm and 258 nm. SPCC and SPCL Percent Depletion Values were calculated and used in a prediction model which allows assigning the test chemical to one of four reactivity classes used to support the discrimination between sensitizers and non-sensitizers.

Milli-Q water (MQ) was found to be an appropriate solvent to dissolve GenoWhite was therefore used in this Direct Peptide Reactivity Assay (DPRA) study.
Positive control results:
The results of the positive control cinnamic aldehyde are presented in the following table:

SPCC Peak Area, Concentration and Depletion of the Cinnamic Aldehyde Positive Control Samples

Sample code Peak area at 220 nm (μAU) Concentration (mM) SPCC Depletion
PCcys-1 1083065 0.126 74.5%
PCcys-2 1103315 0.129 73.9%
PCcys-3 1037781 0.120 75.7%
Mean 74.7%
SD 0.9%
SD = Standard Deviation.

The Percent SPCC Depletion was calculated versus the mean SPCC peak area of Reference Controls C. The mean Percent SPCC Depletion for the positive control cinnamic aldehyde was 74.7% ± 0.9%. This was within the acceptance range of 60.8% to 100% with a SD that was below the maximum (SD <14.9%).
Key result
Parameter:
other: Mean SPCC depletion
Run / experiment:
Mean
Value:
4.5
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Parameter:
other: Mean SPCL depletion
Run / experiment:
Mean
Value:
0
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Parameter:
other: Mean of SPCC and SPCL depletion
Run / experiment:
mean
Value:
2.3
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
Acceptability of the Cysteine Reactivity Assay

The correlation coefficient (r2) of the SPCC standard calibration curve was 0.994. Since the r2 was >0.99, the SPCC standard calibration curve was accepted.
The mean peptide concentration of Reference Controls A was 0.511±0.005 mM, the mean peptide concentration of Reference Controls C was
0.494±0.012 mM and the mean peptide concentration of Reference Controls Cwater was 0.454±0.007 mM. The mean Reference Control samples A, and C and Cwater were all within the acceptance criteria of 0.50±0.05 mM. This confirms the suitability of the HPLC system and indicates that the solvent (MQ) used to dissolve N-acetylglycyl beta-alanine did not impact the Percent SPCC Depletion.

The Coefficient of Variation (CV) of the peptide areas for the nine Reference Controls B and C was 2.2%. This was within the acceptance criteria (CV <15.0%) and confirms the stability of the HPLC run over time.

The mean area ratio (A220/A258) of the Reference Control samples was 18.40. The mean A220/A258 ratio ± 10% range was 16.56-20.24. Each sample showing an A220/A258 ratio within this range gives an indication that co-elution has not occurred.

The Percent SPCC Depletion was calculated versus the mean SPCC peak area of Reference Controls C. The mean Percent SPCC Depletion for the positive control cinnamic aldehyde was 74.7% ± 0.9%. This was within the acceptance range of 60.8% to 100% with a SD that was below the maximum (SD <14.9%).


Results Cysteine Reactivity Assay for N-acetylglycyl beta-alanine

Preparation of a 100 mM the test item stock solution in MQ showed that the test item was dissolved completely. Upon preparation and after approximately 24 hours of incubation, both the co-elution control (CC) as well as the test item samples were visually inspected. No
precipitate was observed in any of the samples.
In the CC sample no peak was observed at the retention time of SPCC. This demonstrated that there was no co-elution of the test
item with SPCC. For the 207182/B-cys samples, the mean SPCC A220/A258 area ratio was 18.61. Since this was within the 16.56-20.24 range, this again indicated that there was no coelution of the test item with SPCC.
The Percent SPCC Depletion was calculated versus the mean SPCC peak area of Reference Controls Cwater. The mean Percent SPCC Depletion for N-acetylglycyl beta-alanine was 4.5% ± 4.5%.


Acceptability of the Lysine Reactivity Assay

The correlation coefficient (r2) of the SPCL standard calibration curve was 0.993. Since the r2 was >0.99, the SPCL standard calibration curve was accepted.
The mean peptide concentration of Reference Controls A was 0.488±0.015 mM, the mean peptide concentration of Reference Controls C was
0.500±0.006 mM and the mean peptide concentration of Reference Controls Cwater was 0.475±0.011 mM. The mean Reference Control samples A, and C and Cwater were all within the acceptance criteria of 0.50±0.05 mM. This confirms the suitability of the HPLC system and indicates that the solvent (MQ) used to dissolve N-acetylglycyl beta-alanine did not impact the Percent SPCL Depletion.

The CV of the peptide areas for the nine Reference Controls B and C was 3.1%. This was within the acceptance criteria (CV <15.0%) and confirms the stability of the HPLC run over time.

The mean area ratio (A220/A258) of the Reference Control samples was 13.91. The mean A220/A258 ratio ± 10% range was 12.52-15.30. Each sample showing an A220/A258 ratio within this range gives an indication that co-elution has not occurred.

The Percent SPCL Depletion was calculated versus the mean SPCL peak area of Reference Controls C. The mean Percent SPCL Depletion for the positive control cinnamic aldehyde was 60.9% ± 3.1%. This was within the acceptance range of 40.2% to 69.0% with a SD that was below the maximum (SD <11.6%).


Results Lysine Reactivity Assay for N-acetylglycyl beta-alanine
Preparation of a 100 mM N-acetylglycyl beta-alanine
stock solution in MQ showed that the test item was dissolved completely. Upon preparation and after approximately 24 hours of incubation, both the CC as well as the test item samples were visually inspected. No precipitate was observed
in any of the samples.

In the CC sample no peak was observed at the retention time of SPCL. This demonstrated that there was no co-elution of the test item with SPCL. For the 207182/B-lys samples, the mean SPCL A220/A258 area ratio was 13.56. Since this was within the 12.52-15.30 range, this again indicated that there was no coelution of the test item with SPCL.

The Percent SPCL Depletion was calculated versus the mean SPCL peak area of Reference Controls Cwater. The mean Percent SPCL Depletion for
N-acetylglycyl beta-alanine was 0.0% ± 0.0% .
Interpretation of results:
GHS criteria not met
Conclusions:
All acceptability criteria were met, therefore this DPRA is considered to be valid.
N-acetylglycyl beta-alanine was negative in the DPRA and was classified in the “no or minimal reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model.
Executive summary:

The objective of this study was to determine the reactivity of N-acetylglycyl beta-alanine towards model synthetic peptides containing either cysteine (SPCC) or lysine (SPCL). After incubation of N-acetylglycyl beta-alanine with either SPCC or SPCL, the relative peptide concentration was determined by High-Performance Liquid Chromatography (HPLC) with gradient elution and photodiode array (PDA) detection at 220 nm and 258 nm. SPCC and SPCL Percent Depletion Values were calculated and used in a prediction model which allows assigning the test chemical to one of four reactivity classes used to support the discrimination between sensitizers and non-sensitizers.

The study procedures described in this report were based on the most recent OECD guidelines.

Milli-Q water (MQ) was found to be an appropriate solvent to dissolve the test item and was therefore used in this Direct Peptide Reactivity Assay (DPRA) study. An overview of the obtained assay validation parameters is presented in the table below.

Acceptability of the Direct Peptide Reactivity Assay (DPRA)

Cysteine reactivity assay                                   Lysine reactivity assay

Acceptability criteria       Results forSPCC       Acceptability criteria       Results for SPCL

Correlation coefficient (r2)

standard calibration curve              >0.99                              0.994                     >0.99                            0.993

Mean peptide concentration

RC-A samples (mM)                     0.50 ± 0.05              0.511 ± 0.005              0.50 ± 0.05              0.488 ± 0.015

Mean peptide concentration

RC-C samples (mM)                     0.50 ± 0.05              0.494 ± 0.012              0.50 ± 0.05              0.500 ± 0.006

Mean peptide concentration

RC-Cwater samples (mM)             0.50 ± 0.05              0.454 ± 0.007              0.50 ± 0.05              0.475 ± 0.011

CV (%) for RC samples

B and C                                                 <15.0                        2.2                            <15.0                            3.1

Mean peptide depletion

cinnamic aldehyde (%)                      60.8-100                     74.7                            40.2-69.0                     60.9

SD of peptide depletion

cinnamic aldehyde (%)                     <14.9                            0.9                               <11.6                            3.1

SD of peptide depletion for

N-actylglycyl beta-alanine (%)         <14.9                            4.5                                <11.6                             0.0

RC = Reference Control; CV = Coefficient of Variation; SD = Standard Deviation; NA = Not Applicable.

The validation parameters, i.e. calibration curve, mean concentration of Reference Control (RC) samples A, C and Cwater, the CV for RC samples B and C, the mean percent peptide depletion values for the positive control with its standard deviation value and the standard deviation value of the peptide depletion for N-acetylgycyl beta-alanine were all within the acceptability criteria for the DPRA.

No co-elution of the test item with SPCC or SPCL was observed.

An overview of the individual results of the cysteine and lysine reactivity assays as well as the mean of the SPCC and SPCL depletion are presented in the tables below.

SPCC and SPCL Depletion and Reactivity Classification for N-acetylglycyl beta-alanine

SPCC depletion              SPCL depletion              Mean of SPCC and SPCL depletion                     Reactivity class

Mean       ± SD           Mean              ± SD                                                                             Cysteine 1:10 / Lysine 1:50 prediction model

4.5%       ±4.5%          0.0%              ±0.0%                             2.3%                                    No or minimal reactivity

SD = Standard Deviation.

In the cysteine reactivity assay N-acetylglycyl beta-alanine showed 4.5% SPCC depletion while in the lysine reactivity assay N-acetylglycyl beta-alanine showed 0.0% SPCL depletion. The mean of the SPCC and SPCL depletion was 2.3% and as a result N-acetylglycyl beta-alanine was classified in the “no or minimal reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model.

In conclusion, since all acceptability criteria were met this DPRA is considered to be valid. N-acetylglycyl beta-alanine was negative in the DPRA and was classified in the “no or minimal reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model.

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
April-June 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
Version / remarks:
February 2015
Deviations:
no
GLP compliance:
yes
Type of study:
activation of keratinocytes
Details on study design:
Vehicle
The vehicle of the test item, i.e. Milli-Q water (Millipore Corp., Bedford, MA., USA).

Negative (solvent) Controls
The negative (solvent) control of the test item was 1% Milli-Q water in exposure medium.
The negative (solvent) control of the positive control is 1% DMSO in exposure medium.
Eighteen wells were tested per plate.

Positive Control
Identification Ethylene dimethacrylate glycol
CAS Number 97-90-5
Molecular formula
Molecular weight 198.22
Appearance Clear colorless liquid
Batch SHBF2397V
Purity 98%
Storage conditions In refrigerator (2-8°C)
Stable under storage conditions until 06 May 2019

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). Upon receipt, cells are propagated (e.g. 2 to 4 passages) and stored frozen as a homogeneous stock. Cells from this original stock can be propagated up to a maximum passage number (i.e. 25) and are employed for routine testing using the appropriate maintenance medium.

Cell Culture
Basic medium: Dulbecco’s minimal supplemented with 9.1% (v/v) heat-inactivated (56°C; 30 min) fetal calf serum.
Maintenance medium: Dulbecco’s minimal supplemented with 9.1% (v/v) heat-inactivated (56°C; 30 min) fetal calf serum and geneticin (500 μg/ml).
Exposure medium: Dulbecco’s minimal supplemented with 1% (v/v) heat-inactivated (56°C; 30 min) fetal calf serum.

Environmental conditions
All incubations, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 59 - 86%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 36.5 - 37.5°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the humidity occurred due to opening and closing of the incubator door. Based on laboratory historical data these deviations are considered not to affect the study integrity.

Subculturing
Cells were subcultured upon reaching 80-90% confluency. To maintain the integrity of the response, the cells were grown for more than one passage from the frozen stock, and were not cultured for more than 25 passages.

Experimental Design
Plating of Cells: 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. For each repetition, three replicates were used for the luciferase activity measurements, and one parallel replicate used for the MTT cell viability assay. The cells were incubated overnight in the incubator. The passage number used was 21 in experiment 1 and 16 in experiment 2.
Treatment of Cells: 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 then incubated for about 48 hours at 37±1.0oC in the presence of 5% CO2. Two experiments were performed. Initially the second experiment did not pass the acceptance criteria and was rejected. The second experiment was repeated.
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 3 minutes at room temperature. Plates with the cell lysates were placed in the luminometer 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) and cells were incubated for 3 hours at 37°C in the presence of 5% CO2. The MTT medium was then removed and cells were lysed overnight by adding 10% SDS solution to each well. After shaking, the absorption was measured with the TECAN Infinite® M200 Pro Plate Reader.

ACCEPTABILITY CRITERIA
The KeratinoSensTM test is considered acceptable if it meets the following criteria:
a) The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, should be above the threshold of 1.5 in at least one of the tested concentrations (from 7.81 to 250 μM).
b) The EC1.5 should be between 5 and 125 μM. Moreover, the induction for Ethylene dimethacrylate glycol at 250 μM should be higher than 2-fold. If the latter criterion is not fulfilled, the dose-response of Ethylene dimethacrylate glycol should be carefully checked, and tests may be accepted only if there is a clear dose-response with increasing luciferase activity induction at increasing concentrations for the positive control.
c) Finally, the average coefficient of variation of the luminescence reading for the negative (solvent) control DMSO should be below 20% in each repetition which consists of 18 wells tested. If the variability is higher, results should be discarded.
Positive control results:
The EC1.5 of the positive control was between 5 and 125 μM (16.3 μM and 31.6 μM in experiment 1 and 2, respectively).
A dose response was observed and the induction at 250 μM was higher than 2-fold (8.05-fold and 3.51-fold in experiment 1 and 2, respectively).
Key result
Parameter:
other: IC50
Run / experiment:
1
Value:
0
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Remarks:
no IC50 for the test item
Key result
Parameter:
other: IC30
Run / experiment:
1
Value:
0
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Remarks:
no IC30 for the test item
Key result
Parameter:
other: EC1.5
Run / experiment:
1
Value:
0
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Remarks:
no EC1.5 for the test item
Key result
Parameter:
other: IC30
Run / experiment:
2
Value:
0
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Remarks:
no IC30 for the test item
Key result
Parameter:
other: IC50
Run / experiment:
2
Value:
0
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Remarks:
no IC50 for the test item
Key result
Parameter:
other: EC1.5
Run / experiment:
2
Value:
0
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Remarks:
no EC1.5 for the test item
Key result
Parameter:
other: Imax
Run / experiment:
1
Value:
1.39
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Parameter:
other: Imax
Run / experiment:
2
Value:
1.32
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:
Results
N-acetylglycyl beta-alanine was evaluated for the ability to activate the antioxidant/electrophile responsive element (ARE)-dependent pathway. An overview of the viability and luciferase activity induction is summarized in Table 1. The results of the positive control are summarized in Table 2. An overview of EC1.5, Imax, IC30 and IC50 values is given in Table 3. The individual raw data are presented in Appendix 4 and Appendix 5. The historical control data are presented in Appendix 6.
Two independent experiments were performed. The cells were in these experiments incubated with the test item in a concentration range of 0.98 – 2000 μM (2-fold dilution steps) for 48 hours. The activation of the ARE-dependent pathway was assessed by measuring the luminescence induction compared to the vehicle control. In addition, the viability was assessed with an MTT assay.
Experiment 1
 N-acetylglycyl beta-alanine showed no toxicity. The viability of the cells was higher than 70% at all test concentrations and therefore no IC30 and IC50 values could be calculated.
 No luminesce activity induction compared to the vehicle control was observed at any of the test concentrations after treatment with N-acetylglycyl beta-alanine. The Imax was 1.39 and therefore no EC1.5 could be calculated.
 The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 8.05 and the EC1.5 16.3 μM.
Experiment 2
 N-acetylglycyl beta-alanine showed no toxicity. The viability of the cells was higher than 70% at all test concentrations and therefore no IC30 and IC50 values could be calculated.
 No luminesce activity induction compared to the vehicle control was observed at any of the test concentrations after treatment with the test item. The Imax was 1.32 and therefore no EC1.5 could be calculated.
 The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 3.51 and the EC1.5 31.6 μM.

Both tests passed the acceptance criteria:
 The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, was above the threshold of 1.5-fold in at least one concentration.
 The EC1.5 of the positive control was between 5 and 125 μM (16.3 μM and 31.6 μM in experiment 1 and 2, respectively). A dose response was observed and the induction at 250 μM was higher than 2-fold (8.05-fold and 3.51-fold in experiment 1 and 2, respectively).
 Finally, the average coefficient of variation of the luminescence reading for the negative (solvent) control DMSO was below 20% (8.8% and 8.2% in experiment 1 and 2, respectively). The average coefficient of variation of the luminescence reading for the negative (solvent) control Milli-Q water was also below 20% (14.6% and 2.6% in experiment 1 and 2, respectively).

Overall it is concluded that the test conditions were adequate and that the test system functioned properly.

Discussion
N-acetylglycyl beta-alanine 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 1.39-fold and 1.32-fold in experiment 1 and 2 respectively. N-acetylglycyl beta-alanine is classified as negative in the KeratinoSensTM assay since negative results (<1.5-fold induction) were observed at test concentrations of ≥1000 μM with a cell viability of >70% compared to the vehicle control.
Interpretation of results:
GHS criteria not met
Conclusions:
The current study was valid and N-acetylglycyl beta-alanine is classified as negative (no activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) under the experimental conditions described in this report.
Executive summary:

The objective of this study was to evaluate the ability of N-acetylglycyl beta-alanine to activate the antioxidant/electrophile responsive element (ARE)-dependent pathway in the KeratinoSensTM assay.

The study procedures described in this report were based on the most recent OECD guideline.

Batch PT034901 of N-acetylglycyl beta-alanine was a white powder. N-acetylclycyl beta-alanine was dissolved in Milli-Q water. From this stock 11 spike solutions in Milli-Q water were prepared. The stock and spike solutions were diluted 100-fold in the assay resulting in test concentrations of 0.98 – 2000 μM (2-fold dilution series). Two independent experiments were performed.

Both experiments passed the acceptance criteria:

- The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, was above the threshold of 1.5-fold in at least one concentration.

- The EC1.5 of the positive control was between 5 and 125 μM (16.3 μM and 31.6 μM in experiment 1 and 2, respectively). A dose response was observed and the induction at 250 μM was higher than 2-fold (8.05-fold and 3.51-fold in experiment 1 and 2, respectively).

- Finally, the average coefficient of variation of the luminescence reading for the negative (solvent) control DMSO was below 20% (8.8% and 8.2% in experiment 1 and 2, respectively). The average coefficient of variation of the luminescence reading for the negative (solvent) control Milli-Q water was also below 20% (14.6% and 2.6% in experiment 1 and 2, respectively).

Overall it is concluded that the test conditions were adequate and that the test system functioned properly.

N-acetylglycyl beta.alannine 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 1.39-fold and 1.32-fold in experiment 1 and 2 respectively. N-acetylglycyl beta-alanine is classified as negative in the KeratinoSensTM assay since negative results (<1.5-fold induction) were observed at test concentrations of ≥1000 μM with a cell viability of >70% compared to the vehicle control.

In conclusion, the current study was valid and N-acetylglycyl beta-alanine

+ is classified as negative (no activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) under the experimental conditions described in this report.

Endpoint:
skin sensitisation, other
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2012
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Reason / purpose:
reference to other study
Qualifier:
no guideline available
Principles of method if other than guideline:
Assess on 10 volunteers the irritant potential of the studied test item after its unique application, maintained for 48 hours in contact with the skin, with the help of a semi-occlusive patch.
GLP compliance:
no
Remarks:
semi-occlusive patch test in human, no guideline available an no GLP compliance - see Justification for non-LLNA method.
Type of study:
patch test
Justification for non-LLNA method:
The substance is a cosmetic active ingredient. The patch test was available, as it is a common study with cosmetics.
The information obtained in this study is used for the Skin sensitisation endpoint, in a weight of evidence approach, taking also into account several additinal in vitro and in chemico studies.
Species:
other: Human
Details on test animals and environmental conditions:
11 volunteers of the female or male sex from 18 to 65 years of age, with a normal skin, without any dermatological lesion on the experimental area, should be included in the study.
Key result
Reading:
other: Mean irritation index
Hours after challenge:
0.5
Group:
test group
Dose level:
160 mg diluted at 5%
No. with + reactions:
0
Total no. in group:
11
Remarks on result:
no indication of skin sensitisation

The individual scores of product for each of the 11 volunteers is 0.00. The mean irritation index of the test item is 0.

Interpretation of results:
study cannot be used for classification
Conclusions:
No irritating or sensitising effects observed.
Executive summary:

The patch test objective is to assess on 11 volunteers (female or male sex from 18 to 65 years of age, with a normal skin, without any

dermatological lesion on the experimental area) the irritant potential of the studied test item after its unique application, maintained for 48 hours in contact with the skin, with the help of a semi-occlusive patch.

The mean irritation index of the tst item is 0.

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

Justification for classification or non-classification

Based on the results of the available studies assessing skin sensitization, N-acetylglycyl β-alanine is considered to be non-sensitizing. The classification criteria stated in EU Regulation 1272/2008 are not met.