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EC number: 305-962-8 | CAS number: 95370-96-0
Studies of genetic toxicity in vitro are available and report universally negative results
No further information
TK 11 278 was tested for mutagenic effects on histidine-auxotrophic mutants of Salmonella typhimurium. The investigations were performed with the following concentrations of the trial substance without and with microsomal activation: 25, 75, 225, 675, and 2025 µg/0.1 mL.
These tests permit the detection of point mutations in bacteria induced by chemical substances. Any mutagenic effects of the substances are demonstratable on comparison of the numbers of bacteria in the treated and control cultures that have undergone back-mutation to histidine-prototrophism. To ensure that mutagenic effects of metabolites of the test substane formed in mammals would also be detected, experiments were performed in which the cultures were additionally treated with an activation mixture (rat liver microsomes and co-factors).
In the experiments performed without and with microsomal activation, comparison of the number of back-mutant colonies in the controls and the cultures treated with the various concentrations of TK 11 278 revealed no marked deviations.
No evidence of the induction of point mutations by TK 11 278 or by the metabolites of the substance formed as a result of microsomal activation ws detectable in the strains of S. typhimurium used in these experiments.
Epoxidised Soyban Oil was assayed for mutation in 5 -histidine-requiring strains (TA98, TA100, TA1535, TA1537 and TA102) of Salmonella typhimurium, both in the absence and presence of metabolic activation by an Aroclor 1254 induced rat liver post-mitochondrial fraction (S-9), in two separate experiments.
It was concluded that Epoxidised Soybean Oil failed to induce mutation in 5 strains of Salmonella typhimurium, when treated up to a maximum concentration of 5000 µg/plate, in the absence and presence of a rat liver metabolic activation system.
Table 6: Mitotic Index Determinations for Experiment 1
Mitotic index (%)
NS = not scored
NM = slides not made
(Slides from solvent control cultures C and D not made)
Table 7: Mitotic index determinations for Experiment 2.
NT = not tested
Epoxidised soybean oil was tested in an in vitro cytogenetics assay using duplicate human lymphocyte cultures from a male and female donor in 2 independent experiments. The highest dose level used, 55 ug/ml, was close to the solubility limit of Epoxidised soybean oil in culture medium. Treatments covering a broad range of doses, separated by narrow intervals, were performed both in the absence and presence of metabolic activation by a rat liver post-mitochondrial fraction (S-9) from Aroclor 1254 induced animals. In Experiment 1, treatment in the absence of S-9 was continuous for 20 hours. Treatment in the presence of S-9 was for 3 hours only followed by a 17 hour recovery period prior to harvest. The test compound dose levels for chromosome analysis were selected by evaluating the effect of Epoxidised soybean oil on mitotic index. 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 S-9 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 S-9 was continuous for 20 or 44 hours. Treatment in the presence of S-9 was for 3 hours followed by 17 or 41 hour recovery period. The highest concentration chosen for analysis at 20 hours, was again 55 ug/ml which on this occasion induced approximately 47 % and 25 % mitotic inhibition in the absence and presence of S-9 respectively. The effect of this single concentration only was investigated at the delayed harvest at which time no mitotic inhibition was induced.
Appropriate negative (solvent and untreated) control cultures were included in the test system in both experiments at both sampling times. Acceptable numbers of cells with structural aberrations were observed in solvent control cultures, slides from untreated cultures were not analysed. 4-Nitroquinoline 1-oxide (NQO) and cyclophosphamide (CPA) were employed as positive control chemicals in the absence and presence of liver S-9 respectively. Cells receiving these sampled in each experiment 20 hours after the start of treatment; both compounds induced statistically significant increases in the proportion of cells with structural aberrations.
In most cases, treatment of cultures with Epoxidised soybean oil in either the absence or presence of S-9 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 Epoxidised soybean oil/ml in the presence of S-9 in Experiment 1 and 41.25 ug Epoxidised soybean oil/ml in the absence of S-9 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.
It is concluded that Epoxidised soybean oil was unable to induce chromosome aberrations in cultured human peripheral blood lymphocytes when tested to its limit of solubility in both the absence and presence of S-9.
(a) Duplicate samples per treatment. Each line represents results from each sample/
(b) Colonies developed after plating of 200 cells per plate, 3 plates/sample, immediately after treatment
(c) Colonies developed in medium without selectie agent after plating of 200 cells per plate, 3 plates/sample, after the expression period at the time of mutant selection
(d) Colonies developed in selective medium after plating of 2 x 10 ^5 cells per plate, 5 plates/sample, after the expression period
- no data due to contamination or drying of plates
The mutagenic potential of epoxidised soybean oil (ESO) and chlorinated ESO (Cl-ESO) were tested in cultured Chinese hamster ovary (CHO) cells. Mutation at the hypoxanthine guanine phosphoribosyl transferase (HGPRT) gene locus was measured. Mutagenicity testing was performed in the presence or absence of Aroclor 1254 -induced rat liver homogenate (S9). Both test chemicals were tested up to a maximum of 2 mg/mL. In the absence of S9, significant cytotoxicity (> 50 % cell killing) was observed at 0.5, 1.0 and 2.0 mg/mL of ESO and 0.2, 0.5 and 1.0 mg/mL of Cl-ESO. No significant cytotoxicity was observed in the presence of S9. No test chemical related mutagenicity was observed in the two experiments conducted in the absence or in the presence of S9. ESO and Cl-ESO are therefore concluded not to be a mutagen in CHO cells under the experimental conditions.
Table 4: raw plate counts and % relative survival for Epoxidised Soybean Oil in the cytotoxicity range-finder.
In the absence of S-9
In the presence of S-9
Survival (1) at Day 0*
% Relative survival
Survival (1) at day 0*
% relative survival
(1) 1.6 cells/well plated
* 32 wells scored
Table 5: Summary of Results Experiment 1
Absence of S-9
Presence of S-9
Table 6: Summary of Results Experiment 2
NS Not significant
$$ Treatment excluded due to excessive heterogeneity
*, **, *** Significant at 5 %, 1% and 0.1 % level respectively
Epoxidised Soybean Oil (ESBO) was assayed for its ability to induce mutation at the tk locus (5-trifluorothymidine resistance) in mouse lymphoma cells using a fluctuation protocol. The study consisted of a cytotoxicity range-finder followed by 2 independent experiments, each conducted in the absence and presence of metabolic activation by an Aroclor 1254 induced rat liver post-mitochondrial fraction (S-9).
Following a wide range of treatments in the range-finder experiment, separated by 2-fold intervals and ranging from 78.125 to 5000µg/ml, cells survived all doses of ESBO yielding 109.0 % relative survival in the absence and 100.0 % relative survival in the presence of S-9 at the top dose.
Accordingly, 5 doses were chosen for the first experiment, separated by 2-fold intervals and ranging from 312.5 to 5000µg/ml. All doses were plated for viability and 5-trifluorothymidine resistance 2 days after treatment. The top doses plated yielded 143.9 % and 178.7 % relative survival in the absence and presence of S-9. In the second experiment the same dose range was selected. The top dose plated in this experiment was again 5000µg/ml in the absence and presence of S-9, which yielded 100.7 % and 83.2 % relative survival respectively.
Negative (solvent) and positive control treatments were included in each experiment in the absence and presence of S-9. Mutant frequencies in negative control cultures fell within normal ranges, and statistically significant increases in mutation were induced by the positive control chemicals 4-nitroquinoline 1-oxide (without S-9) and benzo(a)pyrene (with S-9). Therefore the study was accepted as valid.
In the absence of S-9, 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 5000µg/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 5000µg/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.
In the presence of S-9, no statistically significant increases in mutant frequency were observed at any dose level tested in Experiment 1 or 2.
It is concluded that, under the conditions employed in this study, ESBO failed to demonstrate the ability to induce mutation at the tk locus of L5178Y mouse lymphoma cells in the absence and presence of S-9. Therefore, ESBO is not considered to be mutagenic.
No data are available and none are required based on the negative responses seen in studies of genetic toxicity in vitro.
The available data do not indicate any evidence of genetic toxicity. According to Regulation (EC) No. 1272/2008, no classification is warranted.
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