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Acute Toxicity: oral

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acute toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
January to February 2017
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report Date:

Materials and methods

Test guideline
according to
other: OECD guideline Nº 432
Version / remarks:
April 13th, 2004
GLP compliance:
Test type:
other: In vitro acute toxicity test (3T3 NRU citotoxicity assay)
Limit test:

Test material

Test material form:
solid: particulate/powder

Test animals

other: in vitro

Administration / exposure

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.

Results and discussion

Effect levelsopen allclose all
Key result
Dose descriptor:
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:
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

Any other information on results incl. tables

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.


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:


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.


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

Applicant's summary and conclusion

Interpretation of results:
other: study suggests GHS criteria for classification are not met, but it alone cannot be used for classification
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.