Ozonized olive oil

Endpoint summary

Description of key information

Slightly irritant

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin irritation: in vivo

Remarks:

Test performed in 2010 for purpose of medical device

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 27/07/2010 to 03/08/2010

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: ISO 10993-10:2002

GLP compliance:

yes

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Allevamento Bettinardi - Mamo (NO) - ltaly
- Weight at study initiation: 3210-3610 g at the beginning ofthe test
- Housing: Each rabbit was caged in stainless steel cages of cm 48.2x63x37 h equipped with automatic washing cycle. The housing room was lighted with fiuorescent lamps 12 hours for day. Room temperature and humidity were regulated by a conditioning pian! and were monitored daily. Recordings of the housing conditions are being retained in Eurofins Biolab S.r.l. files.
- Diet: The animals were fed with standard pellet complete diet supplied by the authorised breeder Harlan.
- Water: Filtered tap water from locai network was supplied ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature and humidity: Room temperature and humidity
- Photoperiod (hrs dark / hrs light): The housing room was lighted with fiuorescent lamps 12 hours for day.

Type of coverage:

semiocclusive

Preparation of test site:

shaved

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent negative control

Amount / concentration applied:

0,5 ml of the test product were applied with a gauze (25 mmx 25 mm) directly to the skin on cranial site of each rabbit and covered with non-occlusive dressing

Duration of treatment / exposure:

The patches were removed 4 hours alter the application

Observation period:

60min, 24h, 48h, 72h

Number of animals:

3

Details on study design:

TEST SITE
- Area of exposure: 6,25cm2
- Type of wrap if used: no irritant gauze

REMOVAL OF TEST SUBSTANCE
- Time after start of exposure: 4h

OBSERVATION TIME POINTS
- 60min, 24h, 48h, 72h

SCORING SYSTEM:
- Method of calculation: ISO 10993-10:2002

Irritation parameter:

edema score

Basis:

animal: Cranial (left area) and Caudal (right area): Animal number 317 - Animal number 320 - Animal number 354

Time point:

24/48/72 h

Score:

0

Max. score:

0

Reversibility:

fully reversible

Remarks on result:

no indication of irritation

Irritation parameter:

erythema score

Basis:

animal: Craudal (right area): Animal number 317 - Animal number 320 - Animal number 354

Time point:

72 h

Score:

0

Max. score:

0

Reversibility:

fully reversible

Remarks on result:

no indication of irritation

Irritation parameter:

erythema score

Basis:

animal: Cranial (left area): Animal number 317 - Animal number 320 - Animal number 354

Time point:

72 h

Score:

1

Max. score:

1

Reversibility:

fully reversible

Remarks on result:

no indication of irritation

Irritation parameter:

erythema score

Basis:

animal: Caudal (right area): Animal number 317 - Animal number 320 - Animal number 354

Time point:

48 h

Score:

1

Max. score:

1

Reversibility:

fully reversible

Remarks on result:

no indication of irritation

Irritation parameter:

erythema score

Basis:

animal: Cranial (left area): Animal number 317 - Animal number 320 - Animal number 354

Time point:

48 h

Score:

2

Max. score:

2

Reversibility:

fully reversible

Remarks on result:

other: On the basis of the results, interpreted according to GHS regulation, the test substance is considered SLIGHTLY IRRITANT for skin but, according to CLP regulation 1272/08, the subtance is not classified as skin irritant (score for CLP category 2 is from 2

Irritation parameter:

erythema score

Basis:

animal: Caudal (right area): Animal number 320 - Animal number 354

Time point:

24 h

Score:

1

Max. score:

1

Reversibility:

fully reversible

Remarks on result:

no indication of irritation

Irritation parameter:

erythema score

Basis:

animal: Caudal (right area): Animal number 317

Time point:

24 h

Score:

2

Max. score:

2

Reversibility:

fully reversible

Remarks on result:

other: On the basis of the results, interpreted according to GHS regulation, the test substance is considered SLIGHTLY IRRITANT for skin but, according to CLP regulation 1272/08, the subtance is not classified as skin irritant (score for CLP category 2 is from 2

Irritation parameter:

erythema score

Basis:

animal: Cranial (left area): Animal number 317 - Animal number 320 - Animal number 354

Time point:

24 h

Score:

2

Max. score:

2

Reversibility:

fully reversible

Remarks on result:

other: On the basis of the results, interpreted according to GHS regulation, the test substance is considered SLIGHTLY IRRITANT for skin but, according to CLP regulation 1272/08, the subtance is not classified as skin irritant (score for CLP category 2 is from 2

Irritation parameter:

overall irritation score

Basis:

mean

Time point:

24/48/72 h

Score:

1.21

Max. score:

2

Reversibility:

fully reversible

Remarks on result:

other: On the basis of the results, interpreted according to ISO 10993-10:2002, the test product must be considered SLIGHTLY IRRITANT for the skin, but not irritant according to CLP 1272/08.

Interpretation of results:

Category 3 (mild irritant) based on GHS criteria

Conclusions:

On the basis of the results, interpreted according to GHS regulation, the test substance is considered SLIGHTLY IRRITANT for skin but, according to CLP regulation 1272/08, the subtance is not classified as skin irritant (score for CLP category 2 is from 2,3 to 4.0)

Executive summary:

On the test substance a toxicological study was carried out to evaluate the possible locai toxic effects, throughout the following test:

 - skin irritation test according to ISO 10993-10:2002

The skinirritation test was carriedout through a semi-occlusive application; the test product was applied on the intact skin of 3 rabbits, in the dorsal region both on the left and on the right side.

Each animal had the right caudal region and left cranial region treated with the test product.

The right cranial region and the left caudal region were treated with a non irritant humidified gauze (25mmx25mm), used as control.

Reactions were evaluated 1 hours following the remaval of the patches and were evaluated againat 24, 48 and 72h after exposure.

-After 60 minutes all treated sites shown a very slight erythema.

-After 24 hours two animals shown a well defined erythema in treated cranial site and a very slight erythema in the caudal sites, one animai shown a well defined erythema in both treated sites.

Symptoms disappear completely in all animals in the caudal sites after 72 hours, in the cranial sites after 7 days.

In control regions no oedema or erythema were observed.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

an in vitro skin irritation study does not need to be conducted because adequate data from an in vivo skin irritation study are available

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Study Plan date:01. Feb. 2019
Experimental Starting Date: 11. Feb. 2019
Experimental Completion Date: 08. May 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

human

Strain:

other: EpiOcular(TM) (OCL-200, OCL-212 EIT), Keratinocyte Strain 4F1188

Details on test animals or tissues and environmental conditions:

Specification
Commercially available EpiOcularTM kit.
The EpiOcularTM tissue consists of normal, human-derived keratinocytes which have been cultured to form a stratified squamous epithelium similar to that found in the human cor-nea. It consists of highly organized basal cells. These cells are not transformed or trans-fected with genes to induce an extended life span. The EpiOcularTM tissues are cultured in specially prepared cell culture inserts with a porous membrane through which nutrients can pass to the cells. The tissue surface is 0.6 cm2.

Origin
EpiOcularTM tissues were procured from MatTek In Vitro Life Science Laboratories, Mlynské Nivy 73, 82105 Bratislava, Slovakia.

Main Test:
Designation of the kit: OCL-212-EIT
Day of delivery: 17. Feb. 2019
Batch no.: 27091

Additional Test with freeze killed tissues:
Designation of the kit: OCL-200-EIT
Day of delivery: 23. Jan. 2018
Batch no.: 27020
The tissues were placed in the freezer on the day of delivery and stored in the freezer until use.

Demonstration of Proficiency
The validity of the EpiOcularTM test at LAUS GmbH was demonstrated in a proficiency study. For this purpose 15 proficiency chemicals (indicated by the OECD 492 guideline) were tested. All of the 15 proficiency chemicals were correctly categorized. Therefore, the proficiency of the EpiOcularTM test was demonstrated.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

50 µL of the controls and the test item were applied

Duration of treatment / exposure:

28 minutes

Duration of post- treatment incubation (in vitro):

12 minutes serum-free DMEM at room temperature (post-soak).
120 minutes at 37 ± 1 °C, 5 ± 1 % CO2 and ≥ 95% relative humidity (post-treatment)

Number of animals or in vitro replicates:

2 replicates

Details on study design:

Preparations:
On the day of the start of the experiment, the MTT concentrate was thawed. The MTT concentrate was diluted with assay medium directly before use.
The assay medium was warmed in the water bath to 37 ± 1°C.
6-well-plates were labelled with test item, negative control and positive control and filled with 1 mL assay medium in the appropriate wells.
All inserts were inspected for viability and the presence of air bubbles between agarose gel and insert. Viable tissues were transferred in the prepared 6-well-plate and incubated at 37 ± 1 °C, 5 ± 1 % CO2 and ≥ 95% relative humidity for 1 hour.
After the pre-incubation, the medium was replaced and the wells were filled with 1 mL fresh assay medium. All 6-well-plates were incubated at 37 ± 1 °C, 5 ± 1 % CO2 and ≥ 95% relative humidity for 16 hours and 59 minutes.

Exposure and Post-Treatment:
After overnight incubation, the tissues were pre-wetted with 20 µL DPBS buffer and then incubated at 37 ± 1 °C, 5 ± 1 % CO2 and ≥ 95% relative humidity for 29 minutes. After that, 50 µL of the controls and the test item were applied in duplicate in one- minute- in-tervals. This was done in such a fashion that the upper surface of the tissue was covered. At the beginning of each experiment (application of negative controls), a stop watch was started. After dosing the last tissue, all plates were transferred into the incubator for 28 minutes at 37 ± 1 °C, 5 ± 1 % CO2 and ≥ 95% relative humidity.
At the end of the exposure time, the inserts were removed from the plates in one- minute-intervals using sterile forceps and rinsed immediately. The inserts were thoroughly rinsed with DPBS. Then, the tissues were immediately transferred to and immersed in 5 mL of pre-warmed assay medium in a pre-labelled 12-well plate for 12 minutes post soak at room temperature.
After that, each insert was removed from the medium, the medium was decanted off the tissue and the insert was blotted on absorbent material and transferred into the respective well of a pre-labelled 6-well plate containing 1 mL assay medium. For post-treatment incu-bation, the tissues were incubated for 120 minutes at 37 ± 1 °C, 5 ± 1 % CO2 and ≥ 95% relative humidity.
After the post-treatment incubation, the MTT assay was performed.

MTT Assay and Extraction:
A 24-well-plate was prepared with 300 µL freshly prepared MTT solution in each well. The tissue inserts were blotted on absorbent material and then transferred into the MTT solu-tion. The plate was incubated for 180 minutes at 37 ± 1 °C, 5 ± 1 % CO2 and ≥ 95% rela-tive humidity.
At last, each insert was thoroughly dried and set into a pre-labelled 6-well-plate, containing 2 mL isopropanol, taking care that no isopropanol was flowing into the tissue insert. The plate was firmly sealed to avoid evaporation of the solvent and stored in the refrigerator overnight. On the next day the plate was shaken for 2 hours at room temperature, pro-tected from light.

 
Measurement:
The inserts were removed from the 6-well plate and discarded. The content of each well was thoroughly mixed in order to achieve homogenisation.
From each well, two replicates with 200 µL solution (each) were pipetted into a 96-well-plate. Eight wells with 200 µL isopropanol were pipetted also. The plate was read in a plate spectrophotometer at 570 nm.

Irritation parameter:

other:

Remarks:

Mean viability (%)

Run / experiment:

exposure time: 28 minutes

Value:

66.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

Under the conditions of the test, Reaction Products of olive oil and ozone (NOVOX) is considered non-eye irritant in the EpiOcularTM Eye Irritation Test.
After treatment with the test item, the mean value of relative tissue viability was reduced to 66.8 %. This value is above the threshold for eye irritation potential (≤ 60%).
All validity criteria were met. The criterion for optical density of the negative control was fulfilled: The OD value was 2.1 (> 0.8 and < 2.5).
The positive control induced a decrease in tissue viability as compared to the negative control to 37.1%. Variation within the replicates of the controls and the test item was ac-ceptable (< 20%).
For these reasons, the result of the test is considered valid.

1        Findings and Results

1.1       Measured Values Main Test

As blank, the optical density of isopropanol was measured in eight wells of the 96-well-plate. The measured values and their mean are given in the following table:

Table 9.1‑a      Absorbance Values Blank Isopropanol (OD at 570 nm)

Replicate	1	2	3	4	5	6	7	8	Mean
Absorbance	0.033	0.033	0.033	0.034	0.033	0.034	0.033	0.034	0.033

The absorbance values of negative control, test item and positive control are given in the following table:

Table 9.1‑b      Absorbance Values Negative Control, Positive Control and Test Item (OD at 570 nm)

Designation	Measurement	Negative Control	Positive Control	Reaction Products of olive oil and ozone (NOVOX)
Tissue 1	1	2.095	0.804	1.623
	2	2.067	0.787	1.607
Tissue 2	1	2.090	0.787	1.339
	2	2.087	0.796	1.338

From the measured absorbances, the mean of each tissue was calculated, subtracting the mean absorbance of isopropanol as given in table 9.1-a (= corrected values).

Table 9.1‑c      Mean Absorbance Negative Control, Positive Control and Test Item

Designation	Negative Control	Positive Control	Reaction Products of olive oil and ozone (NOVOX)
Mean – blank (Tissue 1)	2.048	0.763	1.582
Mean – blank (Tissue 2)	2.056	0.859	1.306

1.2       Comparison of Tissue Viability

For the test item and the positive control, the following percentage values of tissue viability were calculated in comparison to the negative control:

Table 9.2‑a      % Viability Positive Control and Test Item

Designation	Positive Control	Reaction Products of olive oil and ozone (NOVOX)
% Viability (Tissue 1)	37.2%	77.1%
% Viability (Tissue 2)	37.0%	63.6%
% Viability Mean	37.1%	70.4%

1.3       Measured Values of the Additional Test for Direct MTT Reduction

The absorbance values of negative control and test item with freeze-killed tissues are given in the following table:

Table 9.3‑a      Absorbance Values Blank Isopropanol (OD at 570 nm)

Replicate	1	2	3	4	5	6	7	8	Mean
Absorbance	0.033	0.033	0.033	0.033	0.034	0.033	0.033	0.033	0.033

The absorbance values (freeze-killed tissues) of negative control and test item are given in the following table:

Table 9.3‑b      Absorbance Values (freeze-killed tissues) of Negative Control and Test Item

(OD at 570 nm)

Designation	Measurement	Negative Control	Reaction Products of olive oil and ozone (NOVOX)
Tissue 1	1	0.076	0.152
	2	0.074	0.151
Tissue 2	1	-	0.148
	2	-	0.146

From the measured absorbances, the mean of each tissue was calculated, subtracting the mean absorbance of isopropanol as given in table 9.3-a (= corrected values):

Table 9.3‑c      Mean Absorbance (freeze-killed tissues) of Negative Control and Test Item

Designation	Negative Control	Reaction Products of olive oil and ozone (NOVOX)
Mean – blank (Tissue 1)	0.042	0.119
Mean – blank (Tissue 2)	-	0.114

The corrected mean of the negative control (freeze-killed tissue) was subtracted from the corrected mean values of the OD of the test item (freeze-killed tissues):

Table 9.3‑c      Corrected Mean Absorbance of Test Item (freeze-killed tissues)

Designation	Reaction Products of olive oil and ozone (NOVOX)
Mean – corrected mean of the negative control (freeze-killed) (Tissue 1)	0.077
Mean – corrected mean of the negative control (freeze-killed) (Tissue 2)	0.072

 1.4       Comparison of Tissue Viability

For the test item (freeze-killed tissues) the following percentage values of tissue viability were calculated in comparison to the negative control of the main test:

Table 9.4‑a      % Viability of Test Item (freeze-killed tissues)

Designation	Reaction Products of olive oil and ozone (NOVOX)
% Viability (freeze-killed tissue 1)	3.7%
% Viability (freeze-killed tissue 2)	3.5%
% Mean viability (freeze-killed tissues)	3.6%

1.5       Corrected Tissue Viability of Test Item

The mean value of “% Viability (freeze-killed)” (table 9.4-a) was subtracted from “% Viability” of the main test (table 9.2-a).

Table 9.5‑a      % Corrected Viability of Test Item

Designation	Reaction Products of olive oil and ozone (NOVOX)
% Mean viability (main test)	70.4%
% Mean viability (freeze-killed tissues)	3.6%
% Corrected mean viability	66.8%

Note: All excel calculations were performed with unrounded values. Therefore, re-calculation with rounded values may lead to slightly different results

1.6       Assessment

Eye hazard potential is assessed using the criteria given in the following table:

Table 9.6‑a      Assessment of Eye Hazard Potential

% Viability	Assessment	UN GHS classification
> 60 %	Non eye irritant	No Category
≤ 60 %	At least eye irritant	No prediction can be made (category 1 or 2)

1.7       Validity

Validity criteria and results are stated in the following table:

Table 9.7‑a      Validity

Criterion	Demanded	Found (Main Test)
Mean OD of negative control	> 0.8 and < 2.5	2.1
% mean relative viability of positive control	< 50% of negative control	37.1%
Variation within replicates	< 20%	0.4% (negative control) 0.2% (positive control) 13.5% (test item)

Values for negative control and for positive control were within the range of historical data of the test facility (see page 22).

Therefore, the experiment is considered valid.

Interpretation of results:

other: CLP criteria for eye irritation not meet. No classification is required according to Regulation (EC) 1272/2008

Conclusions:

CLP: non eye irritant. Not classified.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Additional information

See attached information

Justification for classification or non-classification

On the basis of the results, according to CLP regulation 1272/08, the subtance is not classified as eye or skin irritant.