Registration Dossier

Administrative data

Endpoint:
acute toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
in vitro Balb/c 3T3 was performed instead of the in-vivo test since the substance is used in cosmetics

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
other: ICCVAM -Recommended Test Method Protocol BALB/c 3T3 NRU Cytotoxicity Test Method
Version / remarks:
This test method is used to evaluate the cytotoxicity of test substances using the BALB/c 3T3 Neutral Red Uptake (NRU) in vitro cytotoxicity test. The data generated from the in vitro cytotoxicity assays are used to predict the starting doses for rodent acute oral systemic toxicity assays.
Qualifier:
according to
Guideline:
other: ENV/JM/MONO(2010)20-series of testing and Assessment n.129- Guidance document on using cytotoxicity tests to estimate starting doses for acute oral systemic test
Principles of method if other than guideline:
The NRU cytotoxicity assay procedure is based on the ability of VIAble cells to INCORPORATE AND BIND neutral red (NR), a supravital dye. NR is a weak cationic dye that readily diffuses through the plasma membrane and concentrates in lysosomes where it electrostatically binds to the anionic lysosomal matrix. Toxicants can alter the cell surface or the lysosomal membrane to cause lysosomal fragility and other adverse changes that gradually become irreversible. Thus, cell death and/or inhibition of cell growth decreases the amount of neutral red retained by the culture. Healthy proliferating mammalian cells, when properly maintained in culture, continuously divide and multiply over time. A toxic chemical, regardless of site or mechanism of action, will interfere with this process and result in a reduction of the growth rate as reflected by cell number. Cytotoxicity is expressed as a concentration dependent reduction of the uptake of NR after chemical exposure, thus providing a sensitive, integrated signal of both cell integrity and growth inhibition.The concentration of NR dye desorbed from the cultured cells is directly proportional to the number of living cells
GLP compliance:
yes
Test type:
other: in vitro cytotoxicity test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: viscous solid
Details on test material:
Substance obtained by dehydration of an aqueous solution

Administration / exposure

Route of administration:
other:
Vehicle:
other: chemical dilution medium
Doses:
main test: 100.0, 10.0, 1.00, 0.100, 0.0100, 0.000100 ug/ml
Details on study design:
Balb/c 3T3 cells:Balb/c 3T3 Clone A31 (ATCC No.: CCL 163) was obtained from American Type Culture Collection Rockville,Maryland. The cells are checked at regular intervals for the absence of mycoplasmal contamination and for the generation time. Permanent stocks of the Balb/c 3T3 cells are stored in liquid nitrogen, and subcultures are prepared from the frozen stocks for experimental use. Cells are grown in DMEM medium supplemented with 10% Newborn Calf Serum and 5% Foetal Calf Serum (DMEM Complete). All incubations are at 37°C in a 5% carbon dioxide atmosphere (100% nominal relative humidity).Preparation of test culturesOn the day before the experiment, sufficient numbers of wells of 96 microwell plates were inoculated with 3 x 103 freshly trypsinized Balb/c 3T3 cells from a common pool with theexception of blank controls. The cells were incubated at 37°C in a 5% carbon dioxide atmosphere (100% nominal relative humidity) for 24±2 hours prior to treatment.Formulation procedure:Solutions of the test item, as received, were prepared immediately before use in Chemical Dilution Medium on a weight/volume basis without correction for the displacement due to the volume of the test item. Formulates at the concentrations of 200 mg/mL (preliminary experiment) and 2 mg/mL (Main Assay) were prepared by warming at 37°C for 35 and 20 minutes, respectively. Concentrations of solutions were expressed in terms of test item as received.Control items:The solvent used in this study was Chemical Dilution Medium.Negative control: Solvent/vehicle control was prepared as 50ul of DMEM Complete Medium and 50 ul of Chemical Dilution MediumPositive control: dilution of a sterile commercial 10% w/v sodium dodecyl sulfate solution in Chemical Dilution Medium at the concentration of 200ug/mol. Further dilutions spaced by a factor of 1.47 were prepared in Chemical Dilution Medium. Each solution of positive control was diluted in 1:1 culture medium to achieve the final concentrationsBlank control: DMEM complete mediumTreatment of cells cultures and check with Neutral Red UptakeTwo experiments were performed. The preliminary dose-range finder experiment included blank, negative control and eight dose levels of the test item, spaced at approximately log intervals. The Main Assay included blank, negative control, eight dose levels of the positive control and eight dose levels of the test item. Six wells of a 96 microwell plate were preparedfor each experimental point.At the treatment time, the growth medium was removed from the wells and replaced with 50µL of DMEM Complete Medium and 50 µL of treatment solution. Cultures were incubatedat 37°C in CO2 atmosphere (100% relative nominal humidity) for 48±2 hours (differently indicated in the Study Protocol as approximately 48 hours). At this time, cultures wereexamined microscopically in order to evaluate changes in cell morphology due to cytotoxic effects.Culture medium was discarded. Cells were washed with PBS and stained with a 0.4% neutral red solution for 3 hours. Cells were washed rapidly with an aqueous 1% formaldehyde-1% calcium chloride solution. After washing, cells were destained with an aqueous solution of 50% ethanol-1% acetic acid. Plates were read by a spectrophotometer at 530 nm.

Results and discussion

Preliminary study:
the test item was assayed at a maximum dose level of 100000 µg/mL and at a wide range of lower dose levels: 10000, 1000, 100, 10.0, 1.00, 0.100 and 0.010 µg/mL. Dose related reduction of the cell layer was noted at all concentrations tested; changes in cell morphology were observed at the four highest dose levels. Mild to moderate reduction in neutral red uptake was noted at all concentrations tested. Since no concentration gave a cytotoxicity value >50% and <100% viability, the IC50 value was not calculated. After making the appropriate blank correction, mean values of absorbance, standard deviation and percentages over the solvent control values were calculated for each dilution of test item.By the end of treatment, opaque treatment mixtures with precipitate were observed at the two highest dose levels
Effect levels
Dose descriptor:
other: IC50
Effect level:
ca. 9.81 other: ug/ml

Any other information on results incl. tables

Dose related reduction of cell layer and change in cell morphology were noted at the three highest dose levels. Severe reduction in neutral red uptake (4 and 5% over the concurrent negative control) was noted at the two highest concentrations tested, mild toxicity was noted at the next lower dose level of 10.0, while no reduction in neutral red uptake was observed over the remaining dose levels.

The IC50 value was 9.81 µg/mL. After making the appropriate blank correction, mean values of absorbance, standard deviation and percentages over the solvent control values were calculated for each dilution of test item and positive control. By the end of treatment, no precipitation of the test item was observed at any concentration tested.

Data from the in vitro tests can be used for estimating the starting dose for acute oral systemic toxicity tests. The in vivo starting dose is an estimated LD50 value calculated by inserting the in vitro IC50 value into a regression formula derived from 282 substances for which there are both historical rat oral LD50 values and in vitro IC50 values from the RC (ICCVAM, 2006a).

Use the IC50 value in mM in the following regression formula to estimate the log LD50 in mmol/kg:

log LD50 (mmol/kg) = 0.439 log IC50 (mM) + 0.621 (ICCVAM, 2006a).

9.81 ug/ml = 9.81 mg/l

MW (olive oil anfoacetate) = 488 g/mol

=0.439 x log (9.81 mg/l / 488 g/mol) + 0.621

=0.439 x log(0.02mM) + 0.621 = 0.439 x (-1.7) + 0.621 = -0.12

LD50 mmol/kg = 0.75

LD50 mg/kg = 0.75 x 488 =370.2

Applicant's summary and conclusion

Conclusions:
The substance was tested for cytotoxicity following Balb/c 3T3 guideline and OECD 129. Under the experimental conditions the IC50 is equal to 9.81 ug/ml
Executive summary:

The potential in vitro cytotoxicity of the substance has been evaluated on Balb/c 3T3 cells. Cell cultures were treated with different concentrations of the test item. At the end of treatment time, measurement of neutral red uptake was performed to assess cytotoxicity and cell layers were examined in order to evaluate changes in cell morphology. Test item solutions were prepared using Chemical Dilution Medium. A preliminary range-finder experiment was undertaken in order to select appropriate dose levels for the Main Assay. The test item was found to be soluble at 200 mg/mL in Chemical Dilution Medium. Based on this result, in the preliminary solubility trial the test item was assayed at a maximum dose level of 100000 µg/mL and at a wide range of lower dose levels: 10000, 1000, 100, 10.0, 1.00, 0.100 and 0.010 µg/mL. Dose related reduction of the cell layer was noted at all concentrations tested; changes in cell morphology were observed at the four highest dose levels. Mild to moderate reduction in neutral red uptake was noted at all concentrations tested. By the end of treatment, opaque treatment mixtures with precipitate were observed at the two highest dose levels. Since cytotoxicity should be evaluated at soluble dose levels, the Main Assay was performed using the following concentrations: 1000, 100, 10.0, 1.00, 0.100, 0.0100, 0.00100 and 0.000100 µg/mL. Dose related reduction of cell layer and change in cell morphology were noted at the three highest dose levels. Severe reduction in neutral red uptake (4 and 5% over the concurrent negative control) was noted at the two highest concentrations tested, mild toxicity was noted at the next lower dose level of 10.0, while no reduction in neutral red uptake was observed over the remaining dose levels. By the end of treatment, no precipitation of the test item was observed at any concentration. The IC50 value was 9.81 µg/mL. Negative and positive control treatments were included in theMain Assay. Negative control cultures gave acceptable optical density values (0.183 ≤ OD≤ 0.769). Dose related toxicity was observed after treatment with the positive controlSodium Lauryl Sulfate with a calculated IC50 value of 48.0 µg/mL, indicating the correct functioning of the assay system.