Ozonized olive oil

Acute Toxicity: oral

Administrative data

Endpoint:

acute toxicity: oral

Remarks:

Test performed in 2010 for purpose of medical device

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From July 05, 2010 to July 07, 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: UNI EN ISO 10993-5

Principles of method if other than guideline:

Aim of the test is to evaluate the cytotoxicity of finished products or raw materials aimed to be used on the skin or on the mucosae following the UNI EN ISO 10993-5 rule concerning the biological evaluation of medical devices.
The cytotoxicity assay performed in this study was designed to evaluate the cytotoxic potential of the tested product towards the fibroblasts.
The MTT assay is simple, accurate and yields reproducible results. This method has been developed originally by Mossman. The key component is (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) or MTT. This product is of yellowish colour in solution. Mitochondrial dehydrogenases of viable cells cleave the tetrazolium ring, leading to the formation of purple crystals which are insoluble in aqueous solutions. The crystals are re-dissolved in isopropanol and the resulting purple solution is measured spectrophotometrically.
An increase or decrease in cell number results in a concomitant change in the amount of formazan formed, indicating the degree of cytotoxicity caused by the test material.
After 24h exposure of the cells to the test material, the culture medium is removed and the cells incubated for 2 h in 150 μl/well of 1mg/ml MTT solution at 37°C. The solution is the removed and replaced with 200μl/well of isopropanol, with further 20’ incubation at room temperature under medium speed shaking.
The absorbance at 550 nm is measured with a microplate reader (Tecan modello Sunrise remote), with background clearing at 690 nm. The absorbance values are corrected by subtracting the reading of the blanks, with the diluent only.

GLP compliance:

not specified

Test material

Test material information

Constituent 1

Reference substance name:

Ozonized Tetramers and Pentamers

IUPAC Name:

Ozonized Tetramers and Pentamers

impurity 1

Reference substance name:

Unknown impurities

IUPAC Name:

Unknown impurities

Constituent 2

Reference substance name:

Free Ozonized Acids

IUPAC Name:

Free Ozonized Acids

Constituent 3

Reference substance name:

Ozonized Trimers

IUPAC Name:

Ozonized Trimers

Constituent 4

Reference substance name:

Ozonized Dimers

IUPAC Name:

Ozonized Dimers

Constituent 5

Reference substance name:

Ozonized Triglycerides

IUPAC Name:

Ozonized Triglycerides

Test material form:

other: oiy gel

Test animals

Species:

other: murine fibroblasts (Balb/c 3T3 cells, clone A31-1-1)

Strain:

Balb/c

Details on test animals or test system and environmental conditions:

In vitro test system employed consists of:
Murine fibroblasts (Balb/c 3T3 cells, clone A31-1-1)
Source: TIMS (JCRB-Japanese Collection of Research Bioresources)

This clone is derived from mouse embryonic fibroblasts obtained from Balb-c mouse embryo cultures (Aaronson and Todaro, 1968).
This subclone was derived by Dr. Takeo Kakunaga, National Cancer Institute, Bethesda, MD.
The employed cell line is representative of the tissue supposed to come in contact with the product.

Administration / exposure

Route of administration:

other: Fresh medium supplemented with 6 scalar dilutions of the tested product ranging from 5 to 0.15mg/ml

Vehicle:

other: The medium used was composed as following (pH 7.2-7.4):

Remarks:

Minimum essential medium (MEM) (Lonza) Pennicillin-Streptomicyn 0,1mg/ml (Euroclone) Kanamicyn 0,1 mg/ml (SIGMA) Glutammine 4mM (Euroclone) Non essential amoniacids (SIGMA) Fetal Bovin serum (FBS) (Euroclone)

Doses:

6 scalar dilutions of the tested product ranging from 5 to 0.15mg/ml:
-0,.15625mg/ml
-0.3125mg/ml
-0.625mg/ml
-1.25mg/ml
-2.50mg/ml
-5,00mg/ml

No. of animals per sex per dose:

Cells are seeded in 96 wells plates (10000cells/well), treated with fresh medium supplemented with 6 scalar dilutions of the tested product in three replica for each test dilution.

Control animals:

yes

Remarks:

Murine fibroblasts (Balb/c 3T3 cells, clone A31-1-1)

Details on study design:

Cells are seeded in 96 wells plates (10000cells/well) and allowed to grow for 24 h at 37°C and 0.5% CO 2.
The second day fresh medium is added, supplemented with 6 scalar dilutions of the tested product ranging from 5 to 0.15mg/ml.
The sample has been previously dissolved in isopropanol at 37°C at 250mg/ml concentration and than diluted in the culture medium.
The test is carried out in three replica for each test dilution. At the end of the incubation period, the cells are tested for their viability with the citotoxicity (MTT) assay. Cells treated with a known irritating surfactant (Sodium Lauryl Sulfate – SLS) in concentration ranging from 0.5mg/ml to 0.03mg/ml are used as positive control. Untreated cells are used as negative control.The MTT assay is able to evaluate the toxic impact of the tested compound on the cells viability.

Results and discussion

Preliminary study:

Cytotoxicity by MTT test: cell survival assay using cultured on human fibroblasts in monolayer cultures to assess biocompatibility with skin.
If the sample is not sterile a preliminary sterility test has made for the determination of eventually bacterial contamination of the sample that can lead to a false assessment of cytotoxicity.
In this case the microbiological evaluation has been not made because the sample has not water.

Effect levels

Key result

Dose descriptor:

LD50

Remarks:

The value from test is IC50. LD50 was calcuated from IC50 using the following expression: log LD50 [mg/kg]=0.372 log IC50[µg/ml] + 2.024 LD50=10E(log LD50)

Effect level:

1 050 mg/kg bw

Based on:

test mat.

Remarks on result:

other: Data calculated using:

Remarks:

Follow-up study on the predictive capacity of the 3T3 Neutral Red Uptake cytotoxicity assay to correctly identify substances not classified for acute oral toxicity under the EU CLP system (LD50 > 2 000 mg/kg) Final Study Report Prepared by the European Centre for the Validation of Alternative Methods (ECVAM) Authors: Pilar Prieto, Agnieszka Kinsner-Ovaskainen, Anita Tuomainen

Other findings:

The results are expressed in terms of viability:
% of cell viability = [OD(550 nm - 690 nm) test product / OD(550 nm - 690 nm) negative control] x 100
Reduction of cell viability by more than 30% is considered a cytotoxic effect.
In that case is it possible calculated IC50 value (Inhibiting Concentration 50) is the concentration of test compound which inhibit viability cell of 50%.

Applicant's summary and conclusion

Interpretation of results:

Category 4 based on GHS criteria

Conclusions:

The product did show cytotoxic effects at 5; 2.5; 1.25 and 0.625mg/ml concentrations
and an IC50 of 0.48mg/ml on fibroblasts.
The value of IC50=0,38mg/ml correspond to LD50=1050mg/kg

Executive summary:

The MTT assay is simple, accurate and yields reproducible results. This method has been developed originally by Mossman. The key component is (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) or MTT. This product is of yellowish colour in solution. Mitochondrial dehydrogenases of viable cells cleavethe tetrazolium ring, leading to the formation of purple crystals which are insoluble in aqueous solutions. The crystals are re-dissolved with the MTT Solubilization Solution and the resulting purple solution is measured spectrophotometrically. An increase or decrease in cell number results in a concomitant change in the amount of formazan formed, indicating the degree of cytotoxicity caused by the test material. After 24h exposure of the cells to the test material, the culture medium is removed and the cells incubated for 2 h in 150 μl/well of 1mg/ml MTT solution at 37°C. The solution is the removed and replaced with 200 μl/well of isopropanol, with further 20’ incubation at room temperature under medium speed shaking.

The absorbance at 550 nm is measured with a microplate reader (Tecan modello Sunrise remote), with background clearing at 690 nm. The absorbance values are corrected by subtracting the reading of the blanks, with the diluent only.

The results are expressed in terms of viability:

% of cell viability = [OD(550 nm - 690 nm) test product / OD(550 nm - 690 nm) negative control] x 100

Reduction of cell viability by more than 30% is considered a cytotoxic effect.

In that case is it possible calculated IC50 value (Inhibiting Concentration 50) is the concentration of test compound which inhibit viability cell of 50%.

The product did show cytotoxic effects at 5; 2.5; 1.25 and 0.625mg/ml concentrations and an IC50 of 0.48mg/ml on fibroblasts, corresponding to LC50=1050mg/kg.

This value of LC50 correspond to GHS Oral Acute Tox Cat 4 H302.