Ozonized olive oil

Endpoint summary

Description of key information

NRU Test

 

On the test item "NOVOX 5ml" a toxicological study was carried out aimed to evaluate any cytotoxic effect.

The following test was performed:

cytotoxicity direct contact according to ISO 10993-5:2009.

For the cytotoxicity test by direct contact, a subconfluent Balb/c 3T3 cell culture in exponential phase of growth was used.

A qualitative evaluation was performed observing cell culture by means of an inverted microscope, while a quantitative evaluation was performed using the Neutral Red Uptake method (NRU).

The NRU is a method that allows to measure cell viability using their capacity to incorporate and to bind a cellular viability dye, the Neutral Red.

The test item was applied to the monolayer of Balb/c 3T3 cells and was incubated at 37°C ±1°C in CO₂ atmosphere for 24 hours.

Qualitative evaluation

After 24 hours of incubation the cells were observed under the microscope (qualitative evaluation) to evaluate the biological reaction.

After 24 hours of contact, in the wells treated with test sample, a zone beyond specimen further than 1,0cm shown malforrned or degenerated cells (reactivity grade 4).

Quantitative eva/uation

After the qualitative evaluation cells were treated for 3 hours with the medium containing the cell viability dye and then with a Desorb Solution that allows to obtain a cell lysate. The optical density was then calculated after a 540 nm spectrophotometric reading. Cells treated with test sample have shown a celi viability reduction of 94%.

On the basis of the results, interpreted according to ISO 10993-5:2009, the test item "NOVOX  5ml" must be considered CYTOTOXIC.

 

 

MTT Test

 

Cytotoxicity by MTT test: cell survival assay using cultured on human fibroblasts in monolayer cultures to assess biocompatibility with skin. The cel lmodel used was murine fibroblasts (Balb/c 3T3 cells, clone A31-1-1)

Aim of the test
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.

The MTT cytotoxicity assay

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 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.

Expression and evaluation of results

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.

Key value for chemical safety assessment

Acute toxicity: via oral route

Link to relevant study records

Reference

Reference 1

Endpoint:

acute toxicity: oral

Remarks:

Test performed in 2010 for purpose of medical device

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

from March 07, 2018 to March 09, 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: ISO 10993-5:2009 Biolorgical evaluation of medical devices Part 5: Tests for in vitro cytotoxicity

Principles of method if other than guideline:

For the cytotoxicity test by direct contact, a subconfluent Balb/c 3T3 cell culture in exponential phase of growth was used.
A qualitative evaluation was performed observing cell culture by means of an inverted microscope, while a quantitative evaluation was performed using the Neutral Red Uptake method (NRU).
The NRU is a method that allows to measure cell viability using their capacity to incorporate and to bind a cellular viability dye, the Neutral Red.
The test item was applied to the monolayer of Balb/c 3T3 cells and was incubated at 37°C ±1°C in CO2 atmosphere for 24 hours.
Qualitative evaluation:
After 24 hours of incubation the cells were observed under the microscope (qualitative evaluation) to evaluate the biological reaction.
Quantitative evatuation:
After the qualitative evaluation cells were treated for 3 hours with the medium containing the cell viability dye and then with a Desorb Solution that allows to obtain a cell lysate. The optical density was then calculated after a 540 nm spectrophotometric reading.

GLP compliance:

yes

Species:

other: Mammal fibroblasts BALB/3T3 clone A31 (ATCC® CCL163™)

Strain:

Balb/c

Details on test animals or test system and environmental conditions:

For the cytotoxicity test by direct contact, a subconfluent Balb/c 3T3 cell culture in exponential phase of growth was used.
The test item was applied to the monolayer of Balb/c 3T3 cells and was incubated at 37°C ±1°C in CO2 atmosphere for 24 hours.

Route of administration:

other: After 24 hours, verified that a subconfluent monolayer was present, supplemented culture medium was replaced and the test sample was added through deposition on inert filter paper.

Vehicle:

other: Supplemented culture medium with inert filter paper placed in the middle of each well.

Doses:

The sample was used neat, 50 µI were placed on inert paper filter put in the middle of each well.

No. of animals per sex per dose:

3 replicate for single dose

Control animals:

yes

Remarks:

3 replicate for Negative control 3 replicate for Positive control

Details on study design:

EXPERIMENTAL DESIGN
The experimental design included two 12 wells plates containing a subconfluent cell monolayer (approximately 80% confluence) subdivided in the following groups:
3 replicate for Vehicle
3 replicate for Negative control
3 replicate for Positive control
3 replicate for Test Sample

PLATE PREPARATION
Vehicle:
Supplemented culture medium with inert filter paper placed in the middle of each well.
Test sample preparation:
Before application, the sample was left to reach room temperature for 10 minutes to facilitate the erogation from syringe. The sample was used neat, 50 µI were placed on inert paper filter put in the middle of each well.
Negative control preparation:
The negative control was represented by 50 µI of DPBS deposed on inert filter paper placed in the middle of each well.
Positive control preparation:
The positive control was represented by 50 µI of 5% solution of SOS deposed on inert filter paper placed in the middle of each well.

TREATMENT
From a subconfluent culture (80% of confluency) 1 ml of cell suspension was pipetted to two 12 wells plates. The plates were incubated at 37°C ±1°C in a 5% C02 atmosphere, allowing cell sedimentation and the constitution of a subconfluent monolayer.
After 24 hours, verified that a subconfluent monolayer was present, supplemented culture medium was replaced and the test sample was added through deposition on inert filter paper.
The plates were incubated in a thermostat at 37°C ±1°C in a 5% C02 atmosphere for 24 hours. This procedure was repeated for positive and negative controls.

After this contact time, the plates were observed by an inverted microscope and biological reactions were evaluated following a 0 to 4 scale according to IS010993-5:2009.
Each well was treated with 2 ml of Neutral Red Medium for 3 hours at 37°C ±1°C in a 5% C02. After that each well was washed with DPBS and treated with 2 ml of Desorb Solution, the plate was put in a stirring for 10 minutes to homogenize the solution. The optical density was than calculated after a 540 nm spectrophotometric reading.

OBSERVATIONS
Qualitative evaluation
The biological reactivity (cell degeneration and malformations) were evaluated after 24 hours of incubation with a scale ranging from 0 to 4, according to ISO 10993-5 as shown hereafter:
Grade - Reactivity - Description of reactivity zone
0 none No detectable zone around or under specimen
1 Slight Some malformed or degenerateci cells under specimen
2 Mild Zone limited to area under specimen
3 Moderate Zone extending specimen size up to 1.0 cm
4 Severe Zone extending farther than 1.O cm beyond specimen

Quantitative eva/uation:
Optical density was measured at 540 nm by Gen5 software (Biotek). Percentages of cell viability will be calculated according to the formula:
% of cell viability=(ODtest product/ODvehicle)*100

Key result

Dose descriptor:

other: Biological reactivity

Effect level:

4

Based on:

test mat.

Key result

Dose descriptor:

other: % of cell viability

Effect level:

94 other: %

Based on:

test mat.

Interpretation of results:

Category 4 based on GHS criteria

Conclusions:

On the basis of the results, interpreted according to ISO 10993-5:2009, the test item "NOVOX 5ml" must be considered CYTOTOXIC.

Executive summary:

On the test item "NOVOX 5ml" a toxicological study was carried out aimed to evaluate any cytotoxic effect.

The following test was performed:

cytotoxicity direct contact according to ISO 10993-5:2009.

 

For the cytotoxicity test by direct contact, a subconfluent Balb/c 3T3 cell culture in exponential phase of growth was used.

A qualitative evaluation was performed observing cell culture by means of an inverted microscope, while a quantitative evaluation was performed using the Neutral Red Uptake method (NRU).

The NRU is a method that allows to measure cell viability using their capacity to incorporate and to bind a cellular viability dye, the Neutral Red.

The test item was applied to the monolayer of Balb/c 3T3 cells and was incubated at 37°C ±1°C in CO₂ atmosphere for 24 hours.

Qualitative evaluation

After 24 hours of incubation the cells were observed under the microscope (qualitative evaluation) to evaluate the biological reaction.

After 24 hours of contact, in the wells treated with test sample, a zone beyond specimen further than 1,0cm shown malforrned or degenerated cells (reactivity grade 4).

 

Quantitative eva/uation

 

After the qualitative evaluation cells were treated for 3 hours with the medium containing the cell viability dye and then with a Desorb Solution that allows to obtain a cell lysate. The optical density was then calculated after a 540 nm spectrophotometric reading.

 

Cells treated with test sample have shown a celi viability reduction of 94%.

 

 

On the basis of the results, interpreted according to ISO 10993-5:2009, the test item "NOVOX  5ml" must be considered CYTOTOXIC.

 

Reference 2

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

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

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.

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.

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.

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%.

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.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LD50

Value:

1 050 mg/kg bw

Justification for classification or non-classification

NRU Test

 

Qualitative evaluation

After 24 hours of contact, in the wells treated with test sample, a zone beyond specimen further than 1,0cm shown malforrned or degenerated cells (reactivity grade 4).

Quantitative eva/uation

Cells treated with test sample have shown a celi viability reduction of 94%.

On the basis of the results, interpreted according to ISO 10993-5:2009, the test item "NOVOX  5ml" must be considered CYTOTOXIC.

 MTT Test

Expression and evaluation of results
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.

log LD50[mg/kg]=0.372 log IC50[µg/ml]+2.024

LD50=10e(log LD50)=1050mg/kg

The product can be classified in category GHS Oral Acute Tox 4 (H302)