Palm oil, epoxidized

Endpoint summary

Additional information

In vitro

Gene mutation (Bacterial Reverse Mutation Assay/Ames test):

There is no bacterial reverse mutation assay/Ames test data available for EPO. A read across approach was conducted with bacterial reverse mutation assay/Ames test studies from ESBO (EC No. 232-391-0).

Two read-across in vitro bacterial reverse mutation (Ames test) are available. The key read-across study (RL2) was conducted according to OECD 471 and GLP. In this study, strains of S. typhimurium TA98, TA100, TA 1535, TA1537 and TA102 were exposed to ESBO in acetone in experiment 1 (plate incorporation) at concentrations 8, 40, 200, 1000 and 5000 µg/plate (derived from initial toxicity range-finder with TA100) and in experiment 2 (pre-incubation) at concentrations of 312.5 - 5000 µg/plate. Both experiments were carried out in the presence and absence of S9 mammalian metabolic activation. Appropriate positive controls were used for each strain and solvent negative controls were used. ESBO was tested up to limit concentration (5000 µg/plate).

Only treatments of S. typhimurium TA102 in Experiment 2 (+S9 only) showed signs of toxicity (as indicated by thinning of the background bacterial lawn) in this study. In this case, toxic effects were seen mostly at the 3 highest doses. It would appear that the use of a pre-incubation step particularly enhanced the toxicity of the test agent to this test strain. Range-finder and Experiment 1 treatments were carried out using final concentrations of ESBO at 8, 40, 200, 1000 and 5000 µg/plate, Precipitation, in the form of oil droplets, was observed at concentrations of 1000 and 5000 µg/plate. For experiment 2, testing was again carried out up to maximum concentration of 5000 µg/plate (despite the observation of precipitation), as it was possible that the compound formed an emulsion within the test system and it was felt important to maximise the exposure of the cells to this. A narrowed dose range was also used in this experiment (312.5 - 5000 µg/plate) in order to examine those doses most likely to exhibit a mutagenic response. Oil droplets were observed on test plates in this experiment, following treatments of 1250 µg/plate and above. The mean solvent control counts fell within the normal historical range, and the positive control chemicals all induced large increases in revertant numbers in the appropriate strains. None of the treatments with ESBO on the tester strains, either in the absence or presence of S9, resulted in a significant increase in revertant numbers. Therefore ESBO was negative in the presence and absence of metabolic activation in the in vitro bacterial reverse mutation (Ames test) key study. EPO is also predicted to be negative in this study.

The supporting read-across study (RL2) was conducted according to the Ames method. In this study, strains of S. typhimurium TA98, TA100, TA 1535, TA1537 were exposed to ESBO in acetone at concentrations of 25, 75, 225, 675 and 2025 µg/0.1 mL. At the concentrations of 675 and 2025 µg/0.1 mL the substance precipitated in soft agar. The experiment was carried out in the presence and absence of rat liver microsome and co-factors metabolic activation. Appropriate positive controls were used for each strain and solvent negative controls were used. ESBO was tested up to precipitating concentrations. There was no evidence of induced mutant colonies over background. Therefore ESBO was negative in the presence and absence of metabolic activation in the in vitro bacterial reverse mutation (Ames test) supporting study. EPO is also predicted to be negative in this study.

The full read-across report justification is attached.

Gene mutation (in vitro mammalian cell gene mutation):

There is no in vitro mammalian cell gene mutation data available for EPO. A read across approach was conducted with an in vitro mammalian cell gene mutation study from ESBO (EC No. 232-391-0).

The read-across in vitro gene mutation study in mammalian cells (RL2) was conducted according to OECD 476 and GLP. In this study, mouse lymphoma L5178Y cells cultured in vitro were exposed to ESBO in acetone at concentrations of 312.5, 625, 1250, 2,500 and 5000 µg/mL (based on a preliminary toxicity test of 78.125-5000 µg/mL) in the presence and absence of Aroclor 1254-induced rat liver S9 mammalian metabolic activation. The exposure duration was 9-15 days and the expression time was 2 days. The appropriate positive and solvent negative controls were also performed. ESBO was tested up to limit concentrations (5000 µg/mL), include other details as appropriate. Mutant frequencies in negative control cultures fell within normal ranges, and statistically significant increases in mutation were induced by the positive control chemicals. In the absence of S9, reproducible statistically significant and dose-related increases in mutant frequency were not observed in the 2 experiments over the dose range 312.5 to 2500µg/ml. At 5000µg/ml, a positive point was obtained in Experiment 1 and due to heterogeneity in the data this dose was excluded from analysis in Experiment 2. However, if each of the replicate cultures at 5000 µg/mL in Experiment 2 are considered in turn, neither yields a statistically significant increase in mutant frequency. This, combined with the fact that there were no absolute increases in mutant numbers in Experiment 1 at 5000 µg/mL and that carry over of the test compound was a problem at this dose, suggests that the increased mutant frequency seen in experiment 1 was not the result of chemically induced mutation. There was no evidence of induced mutant colonies over background and ESBO was considered negative in the in vitro gene mutation study in mammalian cells. EPO is also predicted to be negative in the in vitro gene mutation study in mammalian cells.

The full read-across report justification is attached.

Chromosome aberration (in-vitro cytogenicity study in mammalian cells):

There is no in vitro cytogenicity study in mammalian cells available for EPO. A read across approach was conducted with an in vitro cytogenicity study in mammalian cells from ESBO (EC No. 232-391-0).

The read-across in-vitro cytogenicity study in mammalian cells (RL2) was conducted according to OECD 473 and GLP. In this study, Human Peripheral Blood Lymphocyte cultures were exposed to ESBO in acetone at concentrations of 1.554-55 µg/mL (experiment 1) and 5.506-55 µg/mL (experiment 2) with and without Aroclor 1254-induced rat liver S9 metabolic activation. The appropriate positive and solvent negative controls were included. ESBO was tested up to the solubility limit in cell culture medium. The test compound dose levels for chromosome analysis were selected by evaluating the effect of ESBO on mitotic index. Acceptable numbers of cells with structural aberrations were observed in solvent control cultures, slides from untreated cultures were not analysed. Positive controls induced statistically significant increases in the proportion of cells with structural aberrations. Chromosome aberrations were analysed at 3 consecutive dose levels. The highest concentration chosen for analysis at this time, 55 ug/mL, induced no mitotic inhibition in the absence of S9 and approximately 14 % in its presence, although this was not clearly dose-related. Experiment 2 included a delayed sampling time. Treatment in the absence of S9 was continuous for 20 or 44 hours. Treatment in the presence of S9 was for 3 hours followed by 17 or 41 hour recovery period. The highest concentration chosen for analysis at 20 hours was 55 ug/mL, which induced approximately 47 % and 25 % mitotic inhibition in the absence and presence of S9 respectively. The effect of this single concentration only was investigated at the delayed harvest at which time no mitotic inhibition was induced. In most cases, treatment of cultures with ESBO in either the absence or presence of S9 resulted in frequencies of cells with aberrations which were similar to and not significantly different from those seen in concurrent negative controls. Small increases in cells with aberrations were seen at the 20 hour sampling time following treatment with 26.95 ug/mL in the presence of S9 in Experiment 1 and 41.25 ug/mL in the absence of S9 in Experiment 2. In neither case, however, was the increase characterised by both statistical significance and frequencies of aberrant cells outside negative historical control ranges and could not therefore be considered biologically important. Therefore, ESBO was considered negative in the in vitro cytogenicity study in mammalian cells. EPO is also predicted to be negative in this test also.

The full read-across report justification is attached.

Justification for selection of genetic toxicity endpoint
No study is selected as all three in vitro read-across studies were negative.

Short description of key information:
In vitro
Gene mutation (Bacterial Reverse Mutation Assay/Ames test): A read-across Bacterial Reverse Mutation Assay from ESBO (EC No. 232-391-0), as the analogue of EPO (EC No. 805‐711‐7), was submitted to fill this data gap. The substance ESBO did not induce mutagenicity in S. typhimurium TA 98, TA 100, TA 1535, TA 1537 and TA 102 in the presence or absence of rat liver S9 metabolic activation (OECD 471, GLP);

Gene mutation (in vitro mammalian cell gene mutation): A read-across in vitro mammalian cell gene mutation) from ESBO (EC No. 232-391-0), as the analogue of EPO (EC No. 805‐711‐7), was submitted to fill this data gap. There was no evidence of induced mutant colonies over background when mouse lymphoma cells were exposed to ESBO in the presence and absence of Aroclor 1254-induced rat liver S9 mammalian metabolic activation (OECD 476/GLP).


Chromosome aberration (in-vitro cytogenicity study in mammalian cells): A read-across in-vitro cytogenicity study in mammalian cells from ESBO (EC No. 232-391-0), as the analogue of EPO (EC No. 805‐711‐7), was submitted to fill this data gap. ESBO did not induce any biologically important increases in aberrnat cells (OECD 473/GLP).

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on the available information in the dossier, the substance EPO (CAS No. 1006899‐79‐1) does not need to be classified for germ cell mutagenicity when the criteria outlined in Annex I of 1272/2008/EC are applied, based on the results of the read-across studies from ESBO.