Ozonized olive oil

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

REVERSE MUTATION:

The test item Reaction products of olive oil and ozone (NOVOX) was examined for the ability to induce gene mutations in tester strains of Salmonella typhimurium and Escherichia coli, as measured by reversion of auxotrophic strains to prototrophy. The five tester strains TA1535, TA1537, TA98, TA100 and WP2 uvrA were used. Experiments were performed both in the absence and presence of metabolic activation, using liver S9 fraction from rats pre-treated with phenobarbital and 5,6-benzoflavone.

The test item was used as a solution in acetone.

It is concluded that the test item Reaction products of olive oil and ozone (NOVOX) does not induce reverse mutation in Salmonella typhimurium or Escherichia coli in the absence or presence of S9 metabolism, under the reported experimental conditions.

 

MICRONUCLEUS:

The test item Reaction products of olive oil and ozone (NOVOX) was assayed for the ability to induce micronuclei in human lymphocytes, following in vitro treatment in the presence and absence of S9 metabolic activation. Three treatment series were performed. A short termtreatment, where the cellswere treated for 3 hours, was performed in the absence and presence of S9 metabolism. The harvest time of approximately 32 hours, corresponding to approximately two cell cycle lengths, was used. A long term(continuous) treatment was also performed only in the absence of S9 metabolism, until harvest at 31 hours. Solutions of the test item were prepared in acetone.

On the basis of solubility results, dose levels for all treatment series were selected as follows:

Experiment	S9	Treatment time (hours)	Harvest time (hours)	Dose level (µL/mL)
1	-	3	32	2.50, 1.67, 1.11, 0.741, 0.494, 0.329, 0.219 and 0.146
	+
2	32	31	31	2.50, 1.67, 1.11, 0.741, 0.494, 0.329, 0.219 and 0.146

Each experiment included appropriate negative and positive controls. Two replicate cell cultures were prepared at each test point. The actin polymerisation inhibitor Cytochalasin B was added prior to the targeted mitosis to allow the selective analysis of micronucleus frequency in binucleated cells. The cytokinesisblock proliferation index CBPI was calculated in order to evaluate cytotoxicity. Dose levels for the scoring of micronuclei were selected with the aim to evaluate test item concentrations exhibiting adequate levels of cytotoxicity and covering a range from the maximum (55 ± 5%) to slight or no toxicity. Based on the results obtained, the following concentrations were selected for the scoring of micronuclei:

S9	Treatment time (hours)	Harvest time (hours)	Dose level (µL/mL)
-	3	32	2.50 1.11 0.494	0 0 0	None*
+			2.50 1.11 0.494	3 0 0	None*
32	31	31	0.741 0.329 0.146	0 0 0	Yes No No

* dose dependent opacity at all concentrations

 

One thousand binucleated cells per culture were scored to assess the frequency of micronucleated cells. Adequate cell proliferation was observed in negative control cultures and the appropriate number of doses and cells was analysed. Statistically significant increases in the incidence of micronucleated cells were observed following treatments with the positive controls Cyclophosphamide and Colchicine, indicating the correct functioning of the test system. The study was accepted as valid. Following treatment with the test item, no statistically significant increase in the incidence of micronucleated cells over the concurrent solvent control value was observed at any dose level, in any treatment series. All incidences were within the distribution of historical negative control values and no concentration related increase was seen. It is concluded that the test item Reaction products of olive oil and ozone (NOVOX) does not induce micronuclei in human lymphocytes after in vitro treatment, under the reported experimental conditions.