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EC number: 943-154-2
CAS number: -
Balb/c 3T3 in vitro test, IC50 = 9.81 ug/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.
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
The potential in vitro cytotoxicity of the analogue 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.
Since the substance is used in cosmetics was tested for acute toxicity
by testing in-vitro its cytotoxicity following the method set for Balb/c
3T3 (Ricci, 2016): Under the experimental conditions the IC50 is equal
to 9.81 ug/ml.
Based on the considerations presented in the attached document for the
analogue substance and the read across consideraation, the substance is
not classified for acute oral toxicity following Regulation 1272/2008
Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.
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