Registration Dossier

Administrative data

Description of key information

The substance is only used as active ingredient for cosmetic products. The acute toxicity endpoint (oral route) has been assessed using a weight of evidence approach, avoiding the use of animals for in vivo tests.

-In vitro acute toxicity test:: 3T3 NRU cytotoxicity assay oaccording to the OECD guidance document nº 129, the ECVAM OB-ALM nº139 protocol and the EURL ECVAM Recommendation, April 2013.

Under the retained experimental conditions, the mean LD50 of the test item is equal to 3229 mg/kg, which is higher than 2000 mg/kg. The test item  N-acetylglycyl beta-alanine may be not classified for acute toxicity according to the CLP classification.

-In vitro 3T3 NRU PhototoxicityTest according to the OECD guideline Nº432. The substance was found to be not phototoxic.

-Predictive Acute Toxicity Assessment with the MultiCASE (CASE Ultra version 1.6.0.3).

-Read-across approach from analogues (breakdown products)

Key value for chemical safety assessment

Acute toxicity: via oral route

Link to relevant study records

Referenceopen allclose all

Endpoint:
acute toxicity: oral
Type of information:
(Q)SAR
Adequacy of study:
weight of evidence
Study period:
April 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:
See document in "Attached justification"
Principles of method if other than guideline:
-Predictions of the acute oral toxicity of acetylglycyl beta-alanine were obtained with the MultiCASE programme. Case Ultra Version 1.6.0.3 was used.

CASE (Computer Automated Structure Evaluation) Ultra, a MultiCASE program, is a statistics-based computer program that organizes data and predicts various toxicological endpoints on the basis of structural fragments. The program automatically identifies molecular substructures that have a high probability of being relevant or responsible for an observed toxicological effect from a learning set containing both active and inactive molecules of diverse structures. CASE Ultra assumes that the presence of these molecular fragments is indicative of the toxicological endpoint and is a reasonable basis to assess the activity of new
molecules. Compounds are submitted to the database of interest and an expert prediction of the potential activity of the molecule is obtained.

- Model description: see field 'Attached justification'
- Justification of QSAR prediction: see field 'Attached justification'
Key result
Dose descriptor:
LD50
Effect level:
> 2 000 - < 5 000 mg/kg bw
Based on:
test mat.
Remarks on result:
other: Predition for Acute Tox. 5 category was positive

No positive alerts were identified in the test chemical for Acute Tox. 1, Acute Tox. 2 and Acute Tox. 3 categories, thus the prediction for these categories was negative.

One positive alert indicated in Figure 1 was obtained for Acute Tox. 4 category. The calculated probability of being positive for this category for this compound was45.1%. As the calculated probability falls within the model's gray zone (40-60%) around the model’s current classification threshold (50.0%), the result of activity prediction was inconclusive in the test using the model LD50CAT4 (GHS Category 4). Finally, one positive alert shown in Figure 2 has been obtained for Acute Tox 5 category. The calculated probability of being positive for this compound for this category was 65.1%. As the calculated probability is higher than the model's current classification threshold (50.0%) and not within the gray zone (40-60%), the compound was predicted to be positive in the test using the model LD50CAT5 (GHS Category 5).

As the prediction for Acute Tox. 4 category was inconclusive and based on a normal group present in amino acids, and the prediction for Acute Tox. 5 category was positive, classification of the substance into Acute Tox. 5 category is considered to be justified (LD50 2000-5000 mg/kg bw).

Interpretation of results:
study cannot be used for classification
Remarks:
Acute toxic prediction adequate to be used in a weight-of-evidence approach together with literature data or data on analogues to complete the endpoint acute oral toxicity.
Conclusions:
No positive alerts were identified in the test chemical for Acute Tox. 1, Acute Tox. 2 and Acute Tox. 3 categories, thus the prediction for these categories was negative.
One positive alert indicated in Figure 1 was obtained for Acute Tox. 4 category. The calculated probability of being positive for this category for this compound was45.1%. As the calculated probability falls within the model's gray zone (40-60%) around the model’s current classification threshold (50.0%), the result of activity prediction was inconclusive in the test using the model LD50CAT4 (GHS Category 4). Finally, one positive alert shown in Figure 2 has been obtained for Acute Tox 5 category. The calculated probability of being positive for this compound for this category was 65.1%. As the calculated probability is higher than the model's current classification threshold (50.0%) and not within the gray zone (40-60%), the compound was predicted to be positive in the test using the model LD50CAT5 (GHS Category 5).


As the prediction for Acute Tox. 4 category was inconclusive and based on a normal group present in amino acids, and the prediction for Acute Tox. 5 category was positive, classification of the substance into Acute Tox. 5 category is considered to be justified (LD50 2000-5000 mg/kg bw).
Executive summary:

Predictions of the acute oral toxicity of acetylglycyl beta-alanine were obtained with the MultiCASE programme. Case Ultra Version 1.6.0.3 was used.

CASE (Computer Automated Structure Evaluation) Ultra, a MultiCASE program, is a statistics-based computer program that organizes data and predicts various toxicological endpoints on the basis of structural fragments. The program automatically identifies molecular substructures that have a high probability of being relevant or responsible for an observed toxicological effect from a learning set containing both active and inactive molecules of diverse structures. CASE Ultra assumes that the presence of these molecular fragments is indicative of the toxicological endpoint and is a reasonable basis to assess the activity of new

molecules. Compounds are submitted to the database of interest and an expert prediction of the potential activity of the molecule is obtained.

Acute oral toxicity of the substance was predicted for the five GHS Acute toxicity categories (Acute Tox. 1: LD50 0-5 mg/kg bw; Acute Tox. 2: LD50 5-50 mg/kg bw; Acute Tox. 3: LD50 50-300 mg/kg bw; Acute Tox. 4: LD50 300-2000 mg/kg bw and Acute Tox. 5: LD50 2000-5000 mg/kg bw).

Endpoint:
acute toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
January to February 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
other: OECD guideline Nº 432
Version / remarks:
April 13th, 2004
GLP compliance:
yes
Test type:
other: In vitro acute toxicity test (3T3 NRU citotoxicity assay)
Limit test:
no
Species:
other: in vitro
Details on study design:
In vitro acute toxicity test oaccording to OECD guidance document number 129, July 20th 2010,
the ECVAM OB-ALM nº139 protocol and the EURL ECVAM Recommendation, April 2013

This assay aims to evaluate the cytotoxicity by neutral red release method on fibroblast Balblc 3T3
line in order to identify substances not requiring classification for acute oral toxicity.
After 48 hours contact, cells are coloured using a vital dye: the neutral red. This colorant diffuses
through the plasma membrane and concentrates in Iysosomes where it electrostatically binds to the
anionic lysosomal matrix. The concentration of neutral red dye desorbed from the cultured cells is
directly proportional to the number of living cells. After dissolution of the neutral red to incorporate
during 20- 45 minutes, the absorbances are measured. The results are expressed in viability percentage
compared with the negative control.

Reference items
Negative control: dilution medium
Positive control: SDS solution from 6.75 to 100 J.1g1ml (CAS Number: IS1-21-3)

Test system
Cells: mouse embryo fibroblasts Balb/c 3T3 clone 31 (ATCC - CCLI63), maintained according to the current working instruction IL 09.
Cells were cultured in DMEM medium with 4.S gil glucose, 4 mM L-glutamine or stabilised L-glutamine, SO IVlml penicillin, SO J.1g1ml streptomycin, 10% non-heat-inactivated new born calf serum.
Cells were maintained in a controlled humid atmosphere (37°C, S% CO2).
Cells are exempt of mycoplasma. Assessment of mycoplasma was performed according to the current working instruction IL 07.
Range finder test: cells were used at passage 97.
Main test N° 1: cells were used at passage 71.
Main test N°2: cells were used at passage 73.

A range finding test with 7 test item dilutions from 160 mg/ml to 2.10E-5 mg/ml was peformed at first place, and no IC50 was found.

Main test
Test item: Two independent studies were realized, with a range of concentrations deternined by the range finder test.
As the viability was greater than 50% in the preliminary test (no IC50), the concentration of the stock solution was increased up to 500 mg/ml.
Reference items:
Negative control: at least 12 wells of dilution medium were tested.
* CSb: control ± solvent without cells (20 wells)
* CS 1 and CS2: control ± solvent with cells ( 12· wells)
* Cxb: test or reference item at the dilution x without cells
* CSx: test or reference item at the dilution x with cells
Positive control: for each series, SDS was tested at 8 concentrations. The concentration varies from 6.75 to 100 micrograms/ml with a dilution factor = l.47.
For each concentration tested in the range finder test or in the main test, for the test item or reference items, 6 replicate were run.

Test protocol
• Cell seeding (day 1)
Cells were treated with trypsin and counted according to the working instruction IL 09. Seeding was the same for the range finder test and the main test. One 96-well culture plate was seeded for the test item and one for the reference item with 100 J.ll of cells at 3.1 04 cells/ml (i.e. 3.103 cells per well) in culture medium. The peripheral wells were used as blanks and were filled with PBS or cellfree culture medium. The plates were placed in an incubator for 24 hours ± 2 hours (37°C, 5% CO2).
• Contact between test item and cells (day 2)
The culture medium was removed.
50 J.l1 culture medium maintained at room temperature and 50 J.ll of test item or reference item dilutions were added. The plates were incubated 48 hours ± 30 minutes (37°C, 5% CO2).
• Neutral Red Uptake test (day 4)
After at least 46 hours, cellular viability was assessed by observation with a phase contrast microscope in order to exclude experimental errors. Observations were recorded in the study note book.
The culture medium was removed and each well was rinsed gently once with 250 III PBS at room temperature before being treated with 250 J.ll of the staining solution. The plates were then incubated for 3 hours ± 10 minutes (37°C, 5% CO2).
After incubation, the staining solution was removed and the cells were washed with 250 f.ll PBS.
The rinsing solution was removed and 100 J.1l of desorption solution were added. The plates were shaken until complete dissolution of crystals, i.e. during 20 to 45 minutes at room temperature, protected from light.
• Plate reading
After dissolution, the plates were left for at least 5 minutes at room temperature. Absorbances were measured at 540 nm ± 10 nm, using the blank CSb as blank. Absorbances were measured within 60 minutes after adding desorption solution.

Key result
Dose descriptor:
LD50
Effect level:
ca. 3 266 other: mg/kg
Based on:
test mat.
Remarks on result:
other: LC50 is 10 mg/ml, leading to a LD50 = 3266 mg/kg
Key result
Dose descriptor:
LD50
Effect level:
ca. 3 192 other: mg/kg
Based on:
test mat.
Remarks on result:
other: LC50 is 10 mg/ml, leading to a LD50 = 3192 mg/kg

Range finding test

The IC50 has been estimated to 5 mg/ml.

Main test

The IC50 for test 1 is 10 mg/ml, leading to a LD50 egal to 3266 mg/kg.

The IC50 for test 2 is 10 mg/ml, leading to a LD50 egal to 3192 mg/kg.

Results calculation

The cells viability was calculated for each dilution and each condition according to the fonnula:

% viability = ( Mean Abs (test item) / Mean Abs (CS1 +CS2) ) x100

Note: The mean absorbance for the blanks CSb was subtracted before the cell viability calculation.

A curve of percentage cell viability against test item concentrations (Viability =f(log[Concentration]) is drawn and the test item concentration resulting in 50% cell viability (lC50) is detennined graphically using a validated Excel matrix locked by a password, R2 of the positive control response dose curve is calculated from a polynomial trendline of order 6 on Excel.

Furthennore, as recommended by DECO guidance document, a Hill function analysis of the replicate cell viability data for each concentration using statistic software (GraphPad PRISM@) is used to calculate the IC50 and the R2 of the positive control response dose curve.

Interpretation

The test has been evaluated in Acute Tox, an integrated in the EU FP6 project on the optimization and prevalidation of an in vitro test for predicting human acute oral toxicity.

The NICEA TMIICCV AM Peer review panel report (2006) has confirmed that the test do not allow to predict each of the GHS acute oral toxicity categories. However the test may be used to detennine the starting doses for acute oral in vivo toxicity. ICCVAM Test method evaluation report (2006) therefore proposed a regression model, based on rat acute oral toxicity data, in order to predict LD50 value from the ICso value obtained with the 3T3 NRU method.

The regression models are as follow:

Millimole regression model: log LD50 (mmol/kg) = 0.439 log IC50 (mM) + 0.621

Weight regression model: log LD50 (mg/kg) = 0.372 log IC50 (microgram/ml) + 2.024

EURL ECV AM Recommendation from April 2013 has confinned that the 3T3 NRU Cytotoxicity Assay can be used in an integrated strategy to support identification of non-classified substances when using a threshold of> 2000 mg/kg. This threshold was used for non-classification by EU CLP.

Test validation

To validate the test, the following validity criteria were controlled:

-Cell

In the case of cryogenically-preserved stock, cells should be cultured at least twice before using then.

It is recommended not to exceed 18 divisions.

-Cytotoxicity

For each plate, at least one calculated cytotoxicty value> 0% and :s 50% viability and at least one calculated cytotoxicty value> 50% and < 100% viability should be present.

Exception: if a test has only one point between 0 and 100% and the smallest practical dilution factor was used and all other test acceptance criteria are met, then the test is acceptable.

- Reference item validity

Solvent control: for each plate, the mean corrected absorbance on the left (CS 1) and the mean corrected absorbance on the right (CS2) do not differ by more than 15% from the mean absorbance of all.

- Positive control

The dose-response curve should have an R2 >= 0.85 for the Hill model fit and the IC50 value should be

within ± 2 standards deviations of the historical mean.

Follow-up of positive control: Updated on 23/02/2017 - N = 27

Mean IC50 value ± 2 SD       23.3 migrogram/ml =< IC50 >= 43.1 microgram/ml

Interpretation of results:
other: study suggests GHS criteria for classification are not met, but it alone cannot be used for classification
Conclusions:
Under the retained experimental conditions, the mean LD50 of the test item is equal to 3229 mg/kg, which is higher than 2000 mg/kg. The test item (INCI : ACETYL GLYCYL beta-ALANINE) may be not classified for acute toxicity according to the CLP classification.
Executive summary:

The acute toxicity of the test item has been evaluated by the in vitro study of cytotoxicity by the neutral red release, acording to OECD guidance document No 129, July 20th 2010, the ECVAM OB-ALM nº139 protocol and the EURL ECVAM Recommendation, April 2013.

Endpoint:
acute toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
January 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
other: OECD guideline Nº 432
Version / remarks:
April 13th, 2004
Deviations:
no
GLP compliance:
yes
Test type:
other: In vitro 3T3 NRU Phototoxicity Test
Details on study design:
Test system:
Cells: mouse embryo fibroblasts from the Balblc 3T3 clone 31 (ATCC - CCL 163), maintained according to the current working instruction IL 09.
Cells are exempt of mycoplasma. Assessment of mycoplasma was performed according to the current working instruction IL 07.
Cells were used at passage 95.
Radiation sensitivity of cells was determined according to the current working instruction IL 12. The last control was done on 1110112017. Cells under 5 J/cm2 UV A showed 11.62% mortality.

Reference items:
Positive control: chlorpromazine solution (CAS number: 69-09-0)
Negative control: reference and test item diluents (buffered saline solution ± 1 % solvent)

Media and reagents:
- Complete DMEM 4.5 gil glucose: DMEM 4.5 gil glucose, 4 mM L-glutamine or stabilised L-glutamine, 10% heat inactived new born calf serum, 50 IU/ml penicillin, 50 J.lglml streptomycin - stored at
5°C ± 3°C
- Cell washing solution: Dulbecco's PBS Ca2+ and Mg2+ free - stored at room temperature
- Trypsin (2.5 gil) - EDTA (0.38 gil) - stored at -20°C ± 5°C
- Diluents for test and reference items: buffered saline solution (for example Hanks) and, when necessary, 1% final maximum of solvent (for example DMSO or ethanol) - stored at room temperature
- Staining solution: 50 J.lglml neutral red solution in not completed culture medium prepared according to the current operating method ML 07 - extemporaneously prepared and used within the day
- Desorption solution: 1 part glacial acetic acid, 50 parts ethanol, 49 parts water - stored at room temperature

FL REAC Oland FL REAC 06 forms ensure the traceability of media and reagents used in the study.

Series definition:
The choice of the test item solvent was determined with information included in the technical data sheet provided by the Sponsor and was shown in the Specific Study Plan.
Dilutions were performed in Hank's salt solution.
The homogeneity and stability of dilutions after UV A exposition were checked and recorded in the study notebook.
The test item was tested at 8 concentrations (cf. results table) on at least four culture wells by assessed concentration, with and without UVA). In agreement with the OECD Guideline n0432 recommendations,
the maximal concentration tested is 1000 microg/ml, followed by 7 dilutions in geometric progression of2.
Negative control: 6 wells, at least, of buffered saline solution were tested with and without UVA.
Positive control: due to the high phototoxic effect of chlorpromazine, 2 dilution ranges were prepared for the reference item in order to correctly frame the ICso• Each dilution was tested at least 4 times with and
without UV A.
For the positive control with irradiation, the maximum concentration tested was 10 microg/ml followed by 7 dilutions in geometric progression of 2.
For the positive control without irradiation, the maximum concentration tested was 80 microg/ml followed by 7 dilutions in geometric progression of 2.
The test and reference items were replicated in two plates, one will be irradiated and the other plate will be protected from UV A.

Test protocol:
Cells were treated with trypsin and counted according to the current working instruction IL 09.
Two 96 wells culture plates were seeded with 100 microl of cells at 2·10E+5 cellslml (Le. 2·10E+4 cells per well) in complete culture medium.
The column I was used as blanks and was filled with PBS.
The plates were incubated 24 hours ± 1 hour (37°C, 5% CO2).
The dilutions were prepared just before being added to the cell cultures. The pH of the highest concentration was measured and is between 6.5 and 7.8.
The culture medium was removed and each well gently rinsed with 150 microL PBS maintained at room temperature before being treated with 100 microL of the test or reference items.
The plates were incubated in darkness for 1 hour ± 5minutes (37°C, 5% CO2).
The irradiation was performed by a BIO SUN solar irradiator (Vilber Lourmat RMX3W).
The BIO SUN is a complete microprocessor controlled UV irradiation based on a programmable microprocessor, the system constantly monitor the UV light emission. The irradiation stops automatically
when the energy received matches the programmed energy. BIO SUN was assessed by a supplier approved by the Quality Assurance according to a planning defined in FG MAT 03.
The spectral irradiance of the device was measured in the wavelength range 250-700 nm with a calibrated spectroradiometer. An integration sphere served as entrance optics to measure direct and indirect radiation.
lrradiance was measured in the center of the irradiation area at a distance of 35 mm from the UV lamp.
The irradiance in different wavelength bands was calculated from the irradiance spectra. It is available in the certificate of calibration established by the supplier. The irradiance measured in range UVA from 320 to 400 nm is around 5 mW/cm2.
The dose applied onto the cells is 5 J/cm2. The dose to be programmed onto the BIO SUN has to take into account the UVA absorption by the plate cover and is defined in the working instruction IL MAT 04.The
exposure time is about 25 minutes.
Manufacturer guaranties a maximum tray temperature during irradiation of 30°C for a room at 20°C. The room temperature is recorded in the study notebook.
One of the two plates was irradiated with its cover at room temperature, and the other plate was protected from the UV A and kept in the dark during this period.
After irradiation, the treatment medium was removed by aspiration and the cells were rinsed with 150 microL PBS.
Then, 100 microL of complete culture medium was gently added and the plates were incubated for 18 to 22 hours (37°C, 5% CO2).
Cellular viability (growth, morphology) was assessed the following day by observation with a phase contrast microscope.
Observations were recorded in the study note book.
The culture medium was removed and each well was gently rinsed once with 150 fll PBS at room temperature before being treated with 100 microL of the staining solution. The plates were then incubated for 3 hours ± 30 minutes (37°C, 5% CO2).
The staining solution was removed and the cells were rinsed with 150 microL PBS. The rinsing solution was eliminate and 150 microL of desorption solution was added. The plates were shaken until complete solubilisation.
Absorbances were measured at 540 nm.
The blank absorbances mean (desorption solution) were removed from the test and reference items absorbances when the raw data are processed.
Key result
Dose descriptor:
other: MPE
Remarks:
mean photo effect
Effect level:
ca. 0.048 other: adimensional
Based on:
test mat.
Key result
Dose descriptor:
LD50
Remarks:
with UVA
Based on:
test mat.
Remarks on result:
not determinable due to absence of adverse toxic effects
Remarks:
Cell viability with UVA is always higher than 50%
Key result
Dose descriptor:
LD50
Remarks:
without UVA
Based on:
test mat.
Remarks on result:
not determinable due to absence of adverse toxic effects
Remarks:
Cell viability without UVA is always higher than 50%

Results are expressed using a calculation of a photo-irritation-factor (PIF) and/or mean photo effect (MPE).

PIF determination:

The PIF was determined as follow:

The cell death rate was calculated for each dilution and each condition (with and without UVA) according to the formula:

% death rate = ( (Mean Abs negative control - Mean Abs test item) / Mean Abs negative control) ) x 100

A curve of percentage cell mortality against test item concentration was drawn and the test item concentration resulting in 50 % cell mortality (IC50) is determined graphically. The photo-irritation-factor

(PIF) was then calculated using the following formula:

PIF = IC50(-UVA)/ IC50(+UVA)

The calculation of PIF was also performed using the COLIPA "Phototox" version 2.0 software provided by the OECD.

The differences observed between the two methods of detennination of the IC50 come from the calculating methods used: a simple graphic detennination on the one hand and a computation software

based on a sophisticated mathematical model on the other hand.

MPE determination:

The mean photo effect (MPE) is based on comparison of the complete concentration response curves. It is defined as the weighted average across a representative set of photo-effect values.

The photo effect (PEc) at any concentration (C) is defined as the product of the response effect (REc) and the dose effect (DEc) i.e. PEc = REc x DEc.

Wi is a weighting factor given by the COLIPA "Phototox" version 2.0 software provided by the OECD.

The response effect (REc) is the difference between the responses observed in the absence and presence of light, i.e. REc = Rc (-UVA) - Rc (+UVA). The dose effect is given by the fonnula:

DEc = (C/C*-1) / (C/C*+l)

where C* represents the equivalence concentration, i.e. the concentration at which the +UV A response equals the -UV A response at concentration C. If C* cannot be detennined because the response values of the +UVA curve are systematically higher or lower than Rc (-UVA), the dose effect is set to 1.

PE calculation at the concentration 0.4 RE0.4 = (66% x 11%)/100% = 0.55, DE0.4 = (0.4/0.16 - 1)/(0.4/0.16 + 1) = 0.43, PE0.4 = 0.55 x 0.43 = 0.24. MPE is obtained by averaging over the values for the photo effect at various concentrations.

The calculation of MPE was perfonned using the COLIPA "Phototox" version 2.0 software provided by the OECD.

Interpretation:

The results were interpreted using the following table:

PIF  < 2 or MPE  < 0.1               :       Not phototoxic

2   PIF  5 or 0.1  MPE  0.15  :       Probably phototoxic

PIF > 5 or MPE > 0.15                 :       Phototoxic

The result validation is performed by the Study Director in agreement with the working instruction IL 04.

Test validation:

Sensitivity of cells to radiation (current working instruction IL 12)

The sensitivity of the cells to UVA is checked after approximately every 10 passages by evaluating their viability after exposure to increasing doses of radiation.

Cells are cultured at the density used in the test. They are irradiated the following day at doses of 2, 5 and 9 J/cm2 and cell viability is determined one day later using the NRU test. The cells fulfil the quality

criteria if their viability after UV A irradiation at 5 J/cm2 is equal to or greater than 80% of control cells kept in the dark. The viability at the higher UV A dose of 9 J/cm2 must be equal to or greater than 50% of control cells kept in the dark.

Radiation sensitivity, checking the current trial

The viability of the cells must be equal to or greater than 80% compared to non-irradiated cells.

Reference item validity

In order to validate the current test, the validity criteria must be checked:

Solvent control: buffered saline solution ± 1 % solvent                            Measured absorbance 0.4

Positive control: chlorpromazine solution                                                IC50 (- UVA) = 7 to 90 µg/ml

IC50 (+ UV A) = 0.1 to 2 µg/ml

Photo-irritation-factor (PIF) 6

The result validation is performed by the Study Director in agreement with the working instruction IL 04.

RESULTS

Test validation:

The negative control shows an absorbance higher or equal than 0.4

The chlorpromazine, positive control, shows an IC50 between 0.1 and 2 µg/ml when irradiated and between 7 and 90 µg/ml whennon irradiated (PIF6)

These results allow to validate the test.

Results:

Graphical determination:

The test item concentration giving 50% cell death with UV A can not be assessed. Mortality never reached 50%.

The test item concentration giving 50% cell death without UV A can not be assessed. Mortality never reached 50%.

COLIPA software « Phototox » version 2:

The test item concentration giving 50% cell viability with UV A can not be assessed. Viability is always higher than 50%.

The test item concentration giving 50% cell viability without UV A can not be assessed. Viability is always higher than 50%.

The MPE is 0.048.

Conclusion

Under the retained experimental conditions, and under the adopted scale, the test item can be assigned as "not phototoxic".

Interpretation of results:
other: not phototoxic
Remarks:
This result is usted in a weight of evidence approach for the assessment of Acute Toxicity
Conclusions:
Acetyl glycyl beta-alanine is not phototoxic.
Executive summary:

The phototoxic potential of acetyl glycyl beta-alanine was evaluated in an in vitro 3T3 NRU phototoxicity test according to the OECD guideline Nº 432.

Results using graphical determination: The test item concentration giving 50% cell death with and without UVA cannot be assessed. Mortality never reached 50%

Results using COLIPA software "Phototox" version 2: The test item concentration giving 50% cell viability with and without UVA cannot be assessed. Viability is alwas higher than 50%.

The MPE is 0.048.

In conclusion, the test substance is found to be not phototoxic.

Endpoint:
acute toxicity: oral
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Study period:
April 2017
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Justification for type of information:
See attached justification.
Reason / purpose:
read-across source
Reason / purpose:
read-across source
Reason / purpose:
read-across source
Key result
Dose descriptor:
LD50
Effect level:
> 2 000 mg/kg bw
Based on:
test mat.
Interpretation of results:
other: CLP criteria for classification is not met
Conclusions:
No analogues with data on acute oral toxicity could be found to complete the acute oral toxicity endpoint for N-acetylglycil-β-alanine.
However, all metabolites are naturally occurring substances and have acute oral toxicity data. All metabolites have an LD50 in rat above 2000 mgkg bw. Based on these data, N-acetylglycyl-β-alanine is considered tohave an LD50>2000 mg/kg bw.
Executive summary:

To cover the endpoint of acute oral toxicity for N-acetylglycyl-β-alanine, a search for analogues with data on acute oral toxicity was performed. Following a search in several databases no analogues with data on acute oral toxicity could be found to complete the acute oral toxicity endpoint for N-acetylglycyl-β-alanine.

However, all metabolites are naturally occurring substances and have acute oral toxicity data. All metabolites have an LD50 in rat above 2000 mg/kg bw. Based on these data, N-acetylglycyl-β-alanine is considered to have an LD50 >2000 mg/kg bw.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed

Additional information

Justification for classification or non-classification

Based on the results of the available studies assessing acute toxicity of N-acetylglycyl β-alanine, the classification criteria stated in EU Regulation 1272/2008 are not met.