Registration Dossier

Toxicological information

Acute Toxicity: oral

Currently viewing:

Administrative data

Endpoint:
acute toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
From June 20th to 30th, 2017
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: OECD GD 129 (3T3 NRU ) and the EURL ECVAM’s DataBase for Alternative Methods (DB-ALM) Protocol n. 139 for the assessment of the acute oral toxicity.
GLP compliance:
no
Test type:
other: in vitro test
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder

Test animals

Details on test animals and environmental conditions:
IN VITRO TEST

TEST SYSTEM:
- Cell line: BALB/c 3T3 cells, clone 31, an immortalized mouse fibroblast cell line.
- Initial n. of cell: 2-3 x10^3 cell/well in a 96-well plate.
- Source: American Type Culture Collection (ATCC, CCL-163)
- Growth conditions: 37 °C, 90 % humidity and 5 % CO2/air.
- In vitro test: application of test item dosing solution on Balb T3 monolayer.

Administration / exposure

Route of administration:
other: In vitro test: application of test item dosing solution on Balb T3 monolayer
Vehicle:
DMSO
Details on oral exposure:
VEHICLE
- Justification for use and choice of vehicle (if other than water): test item is a surfactant not completely soluble in water and tends to form micelles so it has been tested in DMSO. DMSO was chosen based on the results of preliminary solubility test.
- Concentration in vehicle: 200 x dosing solution in DMSO.
Doses:
7 concentrations for test item: 680.3, 462.8, 314.8, 214.2, 145.684, 99.1 and 67.4 µg/ml.
8 concentrations for positive control Sodium Dodecyl Sulfate: 100, 82.5, 68.1, 56.2, 46.4, 38.3, 31.6, 26.1 µg/ml.
Details on study design:
MEDIA and CULTURE CONDITIONS:
Cells have been maintained under standard culture conditions (37 °C, 90 % humidity and 5 % CO2/air in Routine Culture Medium (RCM: Dulbecco’s modified Eagle’s medium (DMEM) containing 10 % new born calf serum and 4 mM Glutamine). At confluence of > 50 % and < 80 %, cells have been washed with 5 - 15ml of Dulbecco’s Phosphate Buffered Salin (DPBS) without Ca2+ and Mg2+, detached using Trypsin-EDTA (1-2ml/plate) and seeded at density ranging from 4200-16800 cells/cm^2

PREPARATION OF THE CULTURE:
The day before application, cells have been washed, detached as described above and seeded into 96-well tissue culture microtiter plates at a density of 2.0 – 3.0 x 10^3cells/well (100 μl/well) and cultured in RCM for 24h. Using a multichannel pipette, 100 μl RCM (medium) have been dispensed only into the peripheral wells of a 96-well tissue culture microtiter plate. In the remaining wells, 100 μl of a cell suspension of 2.0 – 3.0 x 10^4 cells/ml (= 2.0 – 3.0 x 103 cells/well) have been dispended. The seeding density should be noted to ensure that the cells in the control wells are not overgrown after three days (i.e., 24 h incubation and 48 h exposure to test substances). One plate per each substance tested has been prepared.

PREPARATION OF TEST ITEM AND POSITIVE CONTROL:
- Test item: 200x dosing solution has been prepared in DMSO and 2x dosing solution has been prepared in Chemical Dilution Medium (CDM= Dulbecco’s modified Eagle’s medium (DMEM) without serum containing 4 mM Glutamine
200 IU/ml Penicillin 200 μg/ml Streptomycin). DMSO was chosen based on the test item is a surfactant not completely soluble in water and based on the results of a preliminary solubility test.
- Positive control: SDS: 2x dosing solution has been prepared in CDM.
The test substance and control dosing solutions have been dispensed into a “dummy” plate.
The day of treatment (after 24h incubations of the cells) the RCM has been removed by careful inversion of the plate (i.e., “dump”) and replaced with 100 μl of fresh prewarmed RCM to all wells, including the blanks. At the time of treatment initiation, a single delivery multi-channel pipettor has been used to transfer 100 μl of the 2x dosing solutions, from the res
ervoirs (dummy) plate to the appropriate wells on the treatment plate.
POSITIVE AND NEGATIVE CONTROLS:
- Negative controls: DMSO 0.5% for test item and Medium for positive control.
- Positive control: Sodium Dodecyl Sulfate.

PROCEDURE
# PHASE I: Range Finder Experiment : the Range Finder Experiment is the initial cytotoxicity test performed to determine the starting doses for the main test. The test item has been tested at 8 concentrations in the range from 1000 µg/ml to 0.0001 µg/ml (six replicates have been used for each concentration). Sodium Dodecyl Sulfate (SDS) at 8 concentrations from 100 µg/ml to 26.1 µg/ml (six replicates have been used for each concentration) has been used as positive control. DMSO 0.5 % has been used as negative control.
At the end of the exposure time the Cytotoxicity has been evaluated by the Neutral Red Uptake (NRU) assay in order to determine the starting dose for the Main Test.
# PHASE II: The main test of the cytotoxicity assay is performed to extrapolate the concentration at which is observed the 50 % of reduction of the cellular viability (IC50). The concentration closest to the range finder test IC50 value serves as the midpoint of the concentrations tested in the main test. Compared to the range finder test, the main test uses a smaller dilution factor for the concentrations tested. For each test chemical and control substance, two independent repetitions of the Main Test (experiments 1 and 2) each consisting of testing the dilution series are needed to derive a prediction.
After 48h of test item exposure on Balb B3 4 step procedure was followed:
- Plate preparation for NRU assay : cells have been rinsed carefully with 250 μl/well pre-warmed Dulbecco’s Phosphate Buffered Saline (DPBS).
- NRU assay : 250 μl of 25 μg/ml NR dye in DMEM medium with 5% NCS, 4 mM/l Glutamine, 100 IU/ml Penicillin and 100 μg/ml Streptomycin have been added to all wells (including the blanks) and incubated at 37 ºC, 90 % humidity and 5.0 % CO2/air for 3.0 hours.
- READ OUT : After incubation, the NR solution have been removed and cells have been carefully rinsed with 250 μl/well pre-warmed D-PBS. After medium removal, 100 μl NR desorb solution (freshly prepared) has been added to all wells (including blanks) to extract the dye by shaking the plate (while protecting the plate from light) on a microtiter plate shaker for 20 – 45 minutes. The light absorption (optical density [OD]) has been read within 60 minutes of adding NR desorb solution to each well at 540 nm ±10 nm (OD540) using the blanks as a reference by TECAN INFINITE M-200 further processed by Software Magellan 6.5 and reported in an Excel file.
- The data have saved in an appropriate electronic file format for subsequent analysis.

DATA ANALYSIS
After subtraction of the blank OD540 value, the mean NRU of the six replicate values for each test substance concentration has been compared with the mean NRU of all VC values. Cell viability has been calculated for each concentration as percent of the mean VC OD540. The Hill function analysis is performed using a statistical software) to calculate the IC50.

DETERMINATION OF LD50 VALUES:
Log LD50 (mmol/kg) = 0.439 log IC50 (mM) + 0.621 : for substances of known molecular weight.
Log LD50 (mg/kg) = 0.372 log IC50 (μg/mL) + 2.024 : for mixtures, substances whose structures or molecular weights are unknown and substances with high impurity ratio.

EVALUATION CRITERIA:
LD50 > 2000 mg/kg bw: no classification according to CLP Regulation (EC n.1272/2008).

ACCEPTANCE CRITERIA:
1 -The SDS fitted dose-response curve should have an R2 (coefficient of determination) ≥ 0.85 for the Hill model fit. The SDS IC50 must be in the range > 52.11 µg/ml and < 85.5 µg/ml based of the historical data. It must meet criteria 2 and 3.
2 - The mean corrected absorbance of the left (VC1) and the mean corrected absorbance of the right (VC2) columns of VCs do not differ by more than 15 % from the mean corrected absorbance of all VCs.
3 - At least one calculated cytotoxicity value at 0 % < and ≤ 50.0 % viability and at least one calculated cytotoxicity value at 50.0 % < and < 100 % viability must be present.
Statistics:
The Hill function analysis is performed using a statistical software to calculate the IC50.

Results and discussion

Preliminary study:
The preliminary test was performed to evaluate the citotoxicity of the test item (IC50) and to define the concentrations for main test. In the preliminary test 8 concentrations of test item were used (1000, 100, 10, 1, 0.1, 0.01, 0.001 and 0.0001 µg/ml). Based on the result of preliminary test the main test was performed starting from 680 µg/ml.
Effect levels
Key result
Dose descriptor:
LD50
Effect level:
ca. 1 025 mg/kg bw
Remarks on result:
other: was calculated based on IC 50 about 450 μg/ml.

Any other information on results incl. tables

The 7 concentrations of test item used in main test was chosen based on the result of preliminary test IC50= 680 µg/ml. IC50 of the test item should be between 462.8 µg/ml and 314.8 µg/ml. However, the statistical software gave an ambiguous value that cannot be used to calculate the LD50.

The statistical software (GraphPadPRISM®http://www.graphpad.com/Prism.htm) gave the following IC50 for Sodium Dodecyl sulfate (SDS): 74.1 µg/ml. This value is within the historical range defined in the testing facility (52.11 µg/ml ≤ C50 ≤ 85.5 µg/ml).

DOSE RANGE FINDING

conc. test item(µg/ml)  1000 100 10 1 0.1 0.01 0.001 0.0001
Mean OD 0.146 0.552 0.616 0.624 0.633 0.626 0.629 0.635
SD 0.007 0.028 0.039 0.034 0.014 0.016 0.015 0.026

conc. test item(µg/ml)  1000 100 10 1 0.1 0.01 0.001 0.0001
% of cell viability 22.6% 85.6% 95.4% 96.7% 98.1% 97.1% 97.5% 98.4%
SD 1.10% 4.40% 6.00% 5.30% 2.20% 2.50% 2.30% 4.00%
CV 4.66% 5.09% 6.26% 5.52% 2.28% 2.56% 2.32% 4.11%

MAIN TEST

conc. test item(µg/ml)  680.3 462.8 314.8 214.2 145,684 99.1 67.4
Mean OD 0.219 0.373 0.457 0.548 0.71 0.754 0.77
SD 0.008 0.012 0.023 0.01 0.019 0.019 0.008

conc. test item(µg/ml)  680.3 462.8 314.8 214.2 145,684 99.1 67.4
% of cell viability 28.50% 48.60% 59.60% 71.40% 92.40% 98.20% 100.30%
SD 1.00% 1.60% 2.90% 1.30% 2.50% 2.50% 1.00%
CV 3.67% 3.29% 4.92% 1.79% 2.65% 2.54% 1.02%

Applicant's summary and conclusion

Interpretation of results:
other: H302 (Acute oral tox cat.4) according to the CLP Regulation (EC n.1272/2008)
Conclusions:
IC50 of the test item should be between 462.8 µg/ml and 314.8 µg/ml. However, the statistical software gave an ambiguous value that cannot be used to calculate the LD50. LD50 was calculated based on IC 50 about 450 μg/ml equivalent to LD50 = 1025 mg/kg
Executive summary:

A feasibility study on the test item has been conducted to verify if the chemical should be consistent with the applicability domain of the OECD GD 129 (3T3 NRU ) and the EURL ECVAM’s DataBase for Alternative Methods (DB-ALM) Protocol n. 139 for the assessment of the acute oral toxicity . The results of the Balb NRU assay confirmed the cytotoxic effect of SDS on BALB 3T3 with a IC50 of 74.1 µg/ml in agreement with the historical data defined in the testing facility (52.11 µg/ml ≤ IC50 ≤ 85.5 µg/ml).

 The results of the NRU assay for the test item were not conclusive: the statistical software (GraphPad PRISM®) gave an ambiguous IC50 value that cannot be used to calculate the LD50. 

 The test item features seem to fall down in the described main troubleshooting described in the OECD 129: 

 I. low solubility in culture medium and in DMSO

 II. unstable dispersion in aqueous environment

 III. the NR could interfere with the test item structure

 IV. possible sedimentation on the plastic plate during the assay 

 All these parameters together determine multiple interferences with optical density data acquisition resulting in ambiguous and not acceptable IC50 value results. For that reason the test item could not be classified according to the applicability domain and prediction model criteria of the OECD 129. However, the data of this feasibility study (IC 50 about 450 μg/ml equivalent to log LD50 (mg/kg) = 0.372 log IC50 (μg/ml) + 2.024 = 0.372*2.65 + 2.024 = 3.011 i.e. LD50= 1025 mg/kg) can be used in the weight of evidence approach to assesso the acute oral toxicity of the test item.