Ozonized olive oil

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 12/07/2010 to 21/07/2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

GLP compliance:

not specified

Type of study:

mouse local lymph node assay (LLNA)

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

In vivo test system

Test animals

Species:

mouse

Strain:

CBA:J

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Charles Rivera - Italy
- Females nulliparous and non-pregnant: not specified
- Age at study initiation: eight-weeks old
- Weight at study initiation: from 8.7 to 9.4g
- Housing: Mice were housed in Standard Pathogen Free (SPF) conditions in a microbiologically controlled animal facility.
- Diet (e.g. ad libitum):
- Water (e.g. ad libitum):
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20°C
- Photoperiod (hrs dark / hrs light): 12 hours continuous artificial light within each 24-hrs period

Study design: in vivo (LLNA)

Vehicle:

acetone/olive oil (4:1 v/v)

Remarks:

The sample is an oil and was used not diluted (100%) and at 50% and 25% dilutions in acetone/olive oil (AOO, 4/1 vol/vol).

Concentration:

100% (not diluted) - 50% - 25%

No. of animals per dose:

4

Details on study design:

PRE-SCREEN TESTS:
- Compound solubility:
- Substance Toxicity: the test substance was not expected any severe toxicity or irritancy

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Criteria used to consider a positive response: Results for each treatment groups are expressed as the mean Stimulation Index (SI). The SI is the ratio of the mean dpm/mouse within each test product treatment group and the positive control treated group against the mean dpm/mouse for the solvent/vehicle treated control group. In general, when the SI is 3 or more, the test substance is regarded as a skin sensitizer.

TREATMENT PREPARATION AND ADMINISTRATION:
Test product and controls were applied daily for three days (25 μl per each ear pinnae) with a micropipette. Five days following the initiation of exposure all mice receive an intravenous injection of 3H-labeled thymidine and five hours later animals are sacrificed and draining (auricular) lymph nodes are excised and pooled for each experimental group. A single cell suspension of lymph nodes is prepared by gentle mechanical disaggregation and the cells washed and resuspended in trichloroacetic acid (TCA) for at least 12h at 4°C. Precipitates are resuspended in TCA and transferred to an appropriate scintillation fluid. The incorporation by draining lymph nodes of 3H-labeled thymidine is measured by scintillation counting and recorded as mean disintegrations per minute (dpm) for each experimental group. For each concentration of the test material a Stimulation Index (SI) is derived relative to the concurrent vehicle control.

Positive control substance(s):

other: 1-Fluoro-2,4-dinitrobenzene (CAS 70-34-8) 0,02%. This concentration was chosen on the basis of the EC50 value of this substance, i.e. the concentration capable to elicit a 3x increase in 3H-thymidine incorporation in this test, without overt toxicity.

Results and discussion

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Remarks:

100% Substace

Value:

0.99

Remarks on result:

no indication of skin sensitisation based on QSAR/QSPR prediction

Results 2

Parameter:

SI

Remarks:

50% dilutions in acetone/olive oil (AOO, 4/1 vol/vol)

Value:

1.07

Remarks on result:

no indication of skin sensitisation based on QSAR/QSPR prediction

Results 3

Parameter:

SI

Remarks:

25% dilutions in acetone/olive oil (AOO, 4/1 vol/vol)

Value:

0.95

Remarks on result:

no indication of skin sensitisation based on QSAR/QSPR prediction

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In the above experimental conditions, the sample did not show any skin sensitizing potential.

Executive summary:

Eight-weeks old, CBA/j, female mice from Charles River Italia were used. Mice were divided into 3 groups of 4 animal each. Four mice per group were tested. Mice were housed in Standard Pathogen Free (SPF) conditions in a microbiologically controlled animal facility. Husbandry was at 20°C with 12 hours continuous artificial light within each 24-hrs period. Mice were housed at a density of 4 mice per cage since arrival to the animal facility. They were housed 7 days for acclimatization before testing.

The Animal House Facility is a barrier facility and procedures include provision of sterilized bedding, autoclaved feed, and filtered drinking water. Standard laboratory rodent diet is used (see enclosure A). All caging equipment is washed in barrier processing facilities and autoclaved. All animals are housed in filter top cages (static microisolators). Individually ventilated cages and static microisolator cages are changed every week. Mice are transferred to clean cages using disinfected forceps. The cage, food, and bedding are autoclaved and the water is filtered (autoclaved only for immunodepressed mice). Procedures for barrier facilities include sanitation or sterilization of all supplies and equipment. Personnel working in barrier facilities wear sterilized clothing including bodysuit with hood, shoe covers, caps, masks, double gloves and passage via air shower prior to entry.

Sentinels are exposed to dirty bedding from cages in their respective rooms. Sentinels are euthanized for serology, bacteriology, parassitoloy, and pathology screening every three months and/or annually as specified in the report.

Mice of each group were weighted at time zero (T0) and after the 5-days time (T5d) of the protocol. Weights of individuals animals and mean values were ranging from 8.7 to 9.4g at T0 to 9.0 to 10.0g at T5d.

Test product and controls were applied daily for three days (25 μl per each ear pinnae) with a micropipette. Five days following the initiation of exposure all mice receive an intravenous injection of 3H-labeled thymidine and five hours later animals are sacrificed and draining (auricular) lymph nodes are excised and pooled for each experimental group. A single cell suspension of lymph nodes is prepared by gentle mechanical disaggregation and the cells washed and resuspended in trichloroacetic acid (TCA) for at least 12h at 4°C. Precipitates are resuspended in TCA and transferred to an appropriate scintillation fluid. The incorporation by draining lymph nodes of 3H-labeled thymidine is measured by scintillation counting and recorded as mean disintegrations per minute (dpm) for each experimental group. For each concentration of the test material a Stimulation Index (SI) is derived relative to the concurrent vehicle control.

No signs of general toxicity were observed. No animals had any weight loss. Locally, no signs of irritation were observed in any animal, including the DNFB treated mice. This last observation is likely due to the relatively low concentration (0,02%) of DNFB we used. DNFB showed an evident sensitising effect, as expected.

In the above experimental conditions, the sample did not show any skin sensitizing potential.