Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

A number of studies of genetic toxicity in vitro are available for the submission substances and the related substance epoxidised soybean oil (ESBO) and report universally negative results.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
completed 20 July 1981
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
There is a deviation in this study as current guidelelines recommend E. coli WP2 uvrA, E. coli WP2 uvrA (pKM101) or S. typhimurium TA102 tester strains are included but at time of study conduct this was not the case and so none of the aforementioned strains were used in this study. Read across to a study result from an investigation using a similar material is justified for members of the Epoxidised Oils and Derivatives group. Four epoxidised oils (linseed, soybean, 2-ethylhexyl tallate and fatty acids, C14-C22, 2-ethylhexylesters) have been identified as sharing common structural and functional similarities, recognised in an OECD SIDS review as a single category, and therefore justifying read-across between data for different members of the group. Consequently data sharing between ESBO epoxidised soybean oil and epoxidised linseed oil is commonly utilised in the preparation of this dossier and other read-across bridges are used for other members of the EOD group where appropriate.
Qualifier:
according to
Guideline:
other: Method described by AMES et al.
Principles of method if other than guideline:
The study design followed that of the Ames test as described in:
AMES, B.N., F.D. LEE, and W.E. DURSTON (1973), An Improved Bacterial Test System for the Detection and Classification of Mutagens and Carcinogens.
Proc, Natl. Acad. Sei. USA 70, 782-786.
AMES, B.N., W.E. DURSTON, E. YAMASAKI, and F.D. LEE (1973), Carcinogens are Mutagens: A Simple Test System Combining Liver Homogenates for Activation and Bacteria for Detection.
Proc. Natl. Acad. Sei. USA 70, 2281-2285.
AMES, B.N., J. McCANN, and E. YAMASAKI (1975), Methods for Detecting Carcinogens and Mutagens with the Salmonella/Mammalian- Microsome Mutagenicity Test.
Mutation Res. 31, 347-364.


GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Target gene:
histidine
4 histidine auxotrophic tester strains were used consistent with the study designs extant in the early 1980's.
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
other: histidine-auxotrophic
Metabolic activation:
with and without
Metabolic activation system:
rat liver microsomes and co-factors
Test concentrations with justification for top dose:
The tests 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.
Vehicle / solvent:
The substance was dissolved in acetone.
Untreated negative controls:
yes
Remarks:
Solvent acetone
Positive controls:
yes
Positive control substance:
other: TA98: Daunorubicin-HCl, 5 and 10 µg/0.1 mL phosphate buffer; TA100: 4-nitroquinoline-N-oxide, 0.125 and 0.25 µg/0.1 mL phosphate buffer; TA1535: N-methyl-N'-nitro-N-nitrosguanidine, 3 and 5 µg/0.1 mL phosphate buffer; TA1537: 9(5)aminoacridine hydrochlori
Details on test system and experimental conditions:
The substance was dissolved in acetone. Each Petri dish contained approx. 20 mL of minimum agar; 0.1 mL of the solution of the test substance or the vehicle and 0.1 mL of a bacterial culture in 2.0 mL of soft agar. The soft agar was composed of: 100 mL of 0.6 % agar solution with 0.6 % NaCl and 10 mL of a solution of 0.5 mM 1-histidine and 0.5 mM +biotin. In the experiments in which the substance was metabolically activated, 0.5 mL of an activation mixture was added. 1 mL activation mixture contained: 0.3 mL S9 fraction of liver from rats induced with Aroclor 1254 and 0.7 mL of a solution of co-factors.

Positive control experiments were carried out simultaneously with the following substances:
1) for Strain TA 98: daunorubicin-HCl (DAUNOBLASTIN , Farmitalia, Montedison Farmaceutica GmbH, Freiburg i. Br. , Germany),
5 and 10 \ig/0.1 ml phosphate buffer;
2) for Strain TA 100: 4-nitroquinoline-N-oxide (Fluka, Buchs, Switzerland) , 0.125 and 0.25 jig/0.1 ml phosphate buffer;
3) for Strain TA 1535: N-methyl-N'-nitro-N-nitrosoguanidine (Serva, Heidelberg, Germany), 3 and 5 ug/0.1 ml phosphate buffer;
4) for Strain TA 1537: 9(5)aminoacridine hydrochloride monohydrate (Fluka, Buchs, Switzerland), 50 and 100 \iq/0.l ml DMSO.
The activation mixture was tested with Strain TA 1535 and cyclo- ® phosphamide (ENDOXAN-ASTA , Asta-Werke, Bielefeld, Germany), 250 pg/O.l ml phosphate buffer.

In experiments without and with the addition of microsomal activation mixture three Petri dishes were prepared per strain and per group (i.e. per concentration or per control group).

The plates were incubated for about 48 hours at 37 °C in darkness.

Evaluation criteria:
When the colonies had been counted, the arithmetic mean was calculated. The test substance is generally considered to be non-mutagenic if the colony count in relation to the negative control is not doubled at any concentration.
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Additional information on results:
In the experiments performed without and with microsomal activation, comparison of the number of histidine-prototrophic mutants in the controls and after treatment with TK 11 278 revealed no marked differences.

At the concentrations of 675 and 2025 µg/0.1 mL the substance precipitated in soft agar.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

No further information

Conclusions:
In the experiments performed without and with microsomal activation, comparison of the number of histidine-prototrophic mutants in the controls and after treatment with TK 11 278 revealed no marked differences.
Executive summary:

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 demonstrable 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 substance 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 was detectable in the strains of S. typhimurium used in these experiments.

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
8 May 1992 to 1 June 1992
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
There is no indication of the duration of the incubation period in Experiment 2. This is not expected to effect the validity of the results. Read across to a study result from an investigation using a similar material is justified for members of the Epoxidised Oils and Derivatives group. Four epoxidised oils (linseed, soybean, 2-ethylhexyl tallate and fatty acids, C14-C22, 2-ethylhexylesters) have been identified as sharing common structural and functional similarities, recognised in an OECD SIDS review as a single category, and therefore justifying read-across between data for different members of the group. Consequently data sharing between ESBO epoxidised soybean oil and epoxidised linseed oil is commonly utilised in the preparation of this dossier and other read-across bridges are used for other members of the EOD group where appropriate.
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to
Guideline:
other: EEC Annex V Test B14
Qualifier:
according to
Guideline:
other: UKEMS Guidelines
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
histidine gene
Species / strain / cell type:
S. typhimurium, other: TA98; TA100; TA 1535; TA1537 and TA102
Additional strain / cell type characteristics:
other: biotin and histidine required for growth
Metabolic activation:
with and without
Metabolic activation system:
mammalian liver post-mitochondrial fraction (S9)
Test concentrations with justification for top dose:
Please see Table 1 and 2 below.
An initial toxicity range-finder was carried out in TA100 strain only, using final concentrations of Epoxidised Soybean Oil at 8, 40, 200, 1000 and 5000 µg/plate plus a solvent and a positive control. These treatments were non-toxic and the same dose range was used for experiment 1. For experiment 2 treatments, the dose range was narrowed to 312.5 - 5000 µg/plate in order to investigate those concentrations most likely to exhibit a mutagenic response.
Vehicle / solvent:
Test chemical solutions were prepared by dissolving Epoxidised Soybean Oil in analytical grade acetone, immediately prior to assay to give the required maximum concentration treatment solution. Further dilutions were then made using acetone. The test chemical solutions were protected from light and used within approximately 4 hours of the initial formulation of the test agent.
Untreated negative controls:
yes
Remarks:
solvent acetone
Positive controls:
yes
Remarks:
Please Table 2 below
Details on test system and experimental conditions:
An initial toxicity range-finder was carried out in TA100 strain only, using final concentrations of Epoxidised Soybean Oil at 8, 40, 200, 1000 and 5000 µg/plate plus a solvent and a positive control. These treatments were non-toxic and the same dose range was used for experiment 1.

Five strains of bacteria were used in this study. For all assays, bacteria were cultured for about 10 hours at 37 °C in nutrient broth (containing ampicillin for strains TA98 and TA100 and ampicillin and tetracycline for strain TA102). Bacteria were taken from vials of frozen cultures, which had been checked for strain characteristics (histidine dependence, rfa character and resistance to ampicillan (TA98 and TA100) or ampicillin plus tetracycline (TA102). Checks were carried out according to Maron and Ames and De Serres and Shelby. For all treatments, cultures were used within 2 hours of the end of the incubation period.

Epoxidised Soybean Oil was tested for mutation in 5 strains of Salmonella typhurium at the concentrations detailed in Table 1. Triplicate plates with and without S-9 mix were used. Negative (solvent) and positive controls were included in both assays, in quintuplicate without and with S-9 mix. In each experiment, bacterial strains were treated with diagnostic mutagens in triplicate in the absence of S-9. The activity of the S-9 mix used in each experiment was confirmed by AAN treatments (again in triplicate) of at least one strain in the presence of S-9.

Because the results of the first experiment were negative, treatments in the presence of S-9 in Experiment 2 included a pre-incubation step, where the quantities of test chemical or control solution, bacteria and S-9 mix detailed, were mixed together and incubated for 1 hour at 37 °C, before the addition of 2.5 mL molten agar at 46 °C. Plating of these treatments then proceeded as for the normal plate-incorporation procedure. In this way, it was hoped to increase the range of mutagenic chemicals that could be detected in the assay.

Colony Counting:
Colonies were counted electronically using a Seescan Colony Counter or manually where minor agar damage might have interfered with automatic counting, and the background lawn inspected for signs of toxicity.
Evaluation criteria:
Acceptance Criteria:
The assay was considered valid if the following criteria were met:
i) the mean negative control counts fell within the normal range as defined in Appendix 4
ii) the positive control chemicals induced clear increases in revertant numbers confirming discrimination between different strains, and an active S-9.
iii) no more than 5 % of the plates were lost through contamination or some other unforeseen event

Evaluation criteria:
A test compound was considered to be mutagenic if
i) the assay was valid
ii) Dunnett's test gave a significant response (p <= 0.01), and the data set showed a significant dose-correlation
iii) the positive responses described in (ii) were reproducible
Statistics:
The m-statistic was first calculated to check that the data were Poisson-distributed and then Dunnett's test was used to compare the counts of each dose with the control. The presence or otherwise of a dose response was then checked by linear regression analysis.
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 102
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Toxicity:
Only treatments of strain TA102 in Experiment 2 (+ S-9 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 Epoxidised SOybean Oil 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 compund 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.

Mutation:
The individual plate counts were averaged to give mean values. From the data it can be seen that mean solvent control counts fell within the normal historical range, that the positive control chemicals all induced large increases in revertant numbers in the appropriate strains, and that < 5 % of plates were lost, leaving adequate numbers of plates at all treatments. The study was accepted as valid.

The mutation data were evaluated as follows:
No treatment with Epoxidised Soybean Oil of any of the tester strains, earlier in the absence or presence of S-9, resulted in a significant increase in revertant numbers. The data obtained therefore gave no indication of an ability of the test agent to induce mutation.
Remarks on result:
other: strain/cell type: S. typhimurium TA 102
Remarks:
Migrated from field 'Test system'.

No further information

Conclusions:
It is 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.
Executive summary:

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.

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
24 November 2004 to 10 December 2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP and guideline compliant study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Principles of method if other than guideline:
In the pre-incubation assay 50 µL test solution /solvent control, or 100 µL positive control, 500 µL S9 mix / S9 mix substitution buffer and 100 µL bacterial suspension were mixed in a test tube and incubated at 37°C for 60 minutes. After pre-incubation 2.0 mL overlay agar (45° C) was added to each tube. The mixture was poured on minimal agar plates. In the more sensitive pre-incubation assay ethanol is toxic to the bacteria. Therefore, a lower amount of test solution was used.
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Target gene:
The Salmonella typhimurium histidine (his) and the E. coli tryptophan (trp) reversion system measures his- to his+ and trp- to trp+ reversions, respectively. The S. typhimurium and Escherichia coli strains are constructed to differentiate between base pair (TA 1535, TA 100, and WP2 uvrA) and frameshift (TA 1537, TA 98) mutations.
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes/no
Additional strain / cell type characteristics:
other: histidine independence
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
Regular checking of the properties of the strains regarding the membrane permeability and ampicillin resistance as well as spontaneous mutation rates is performed
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes
Additional strain / cell type characteristics:
other: tryptophan dependence
Metabolic activation:
with and without
Metabolic activation system:
liver mitochondrial S-9
Test concentrations with justification for top dose:
In the pre-experiment the concentration range of the test item was 3 – 5000 µg/plate. This was reported as Experiment I since no toxic effects were observed and the maximum concentration was selected for the high dose, 5000 µg/plate.
The following concentrations were tested:
Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
Experiment II: 33; 100; 333; 1000; 2500; and 5000 µg/plate
Vehicle / solvent:
Fatty acids, tall-oil, epoxidized, 2-ethylhexylesters (ETP) was dissolved in ethanol (purity > 99.8 %). The solvent was chosen because of its solubility properties and its relative non-toxicity to the bacteria .
The test item precipitated in the overlay agar with and without S9 mix at 5000 µg/plate in Experiment I and from 2500 µg/plate up to 5000 µg/plate in Experiment II. The undissolved particles had no influence on the data recording.
Untreated negative controls:
yes
Remarks:
concurrent untreated
Negative solvent / vehicle controls:
yes
Remarks:
solvent control - ethanol
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
Migrated to IUCLID6: without S-9, for strains TA1535 and TA100
Positive controls:
yes
Positive control substance:
other: 4-nitro-o-phenylene diamine without S-9, for strains TA1537 and TA98
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
Migrated to IUCLID6: For E.coli WP2 uvrA without S-9
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene, with S-9, all tester strains
Details on test system and experimental conditions:
For each strain and dose level including the controls, three plates were used.
The following materials were mixed in a test tube and poured onto the selective agar plates:
100 µL Test solution at each dose level, solvent (negative control) or reference mutagen solution (positive control),
500 µL S9 mix (for test with metabolic activation) or S9 mix substitution buffer (for test without metabolic activation),
100 µL Bacteria suspension (cf. test system, pre-culture of the strains),
2000 µL Overlay agar

In the pre-incubation assay 50 µL test solution /solvent control, or 100 µL positive control, 500 µL S9 mix / S9 mix substitution buffer and 100 µL bacterial suspension were mixed in a test tube and incubated at 37°C for 60 minutes. After pre-incubation 2.0 mL overlay agar (45° C) was added to each tube. The mixture was poured on minimal agar plates.

After solidification the plates were incubated upside down for at least 48 hours at 37° C in the dark
Evaluation criteria:
Acceptability criteria
The Salmonella typhimurium and Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:
-regular background growth in the negative and solvent control
-the spontaneous reversion rates in the negative and solvent control are in the range of our historical data
-the positive control substances should produce a significant increase in mutant colony
frequencies

Evaluation criteria
A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment
Statistics:
No statistical evaluation of the data was required
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Fatty acids, tall-oil, epoxidized, 2-ethylhexylesters (ETP) was assessed for its potential to induce gene mutations in the plate incorporation test (Experiment I) and the pre-incubation test (Experiment II) using Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escherichia coli strain WP2 uvrA.
The assay was performed in two independent experiments, with and without liver microsomal activation. Each concentration and the controls were tested in triplicate. The test item was tested at the following concentrations:
Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
Experiment II: 33; 100; 333; 1000; 2500; and 5000 µg/plate

The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without S9 mix in both experiments.
No toxic effects, evident as a reduction in the number of revertants, occurred in the test groups with and without metabolic activation.
No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with Fatty acids, tall-oil, epoxidized, 2-ethylhexylesters (ETP) at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency for higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.
Appropriate reference mutagens were used as positive controls. They showed a distinct increase of induced revertant colonies.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Tables of results for plate incorporation and pre-incubation test results are attached as word files

Conclusions:
During this mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains of S. typhimurium and E.coli used.
Therefore, Fatty acids, tall-oil, epoxidized, 2-ethylhexylesters (ETP) is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.
Executive summary:

This study was performed to investigate the potential of Fatty acids, tall-oil, epoxidized, 2-ethylhexylesters (ETP) to induce gene mutations in the plate incorporation test (Experiment I) and the pre-incubation test (Experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escherichia coli strain WP2 uvrA.

The assay was performed in two independent experiments both with and without liver microsomal activation.

Each concentration, including the controls, was tested in triplicate.

Fatty acids, tall-oil, epoxidized, 2-ethylhexylesters (ETP)

was tested at the following concentrations:

Experiment I:              3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

Experiment II:             33; 100; 333; 1000; 2500; and 5000 µg/plate

The plates incubated with the test item showed normal back­ground growth up to 5000 µg/plate with and without metabolic activation in both independent experiments.

No toxic effects, evident as a reduction in the number of revertants, occurred in the test groups with and without metabolic activation.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with Fatty acids, tall-oil, epoxidized, 2-ethylhexylesters (ETP) at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency for higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies. Relevant historical control data were also presented in the sudy report but are not reproduced in this summary.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
August 3, 1992 - October 16, 1992
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
GLP compliant, according to guidelines Read across to a study result from an investigation using a similar material is justified for members of the Epoxidised Oils and Derivatives group. Four epoxidised oils (linseed, soybean, 2-ethylhexyl tallate and fatty acids, C14-C22, 2-ethylhexylesters) have been identified as sharing common structural and functional similarities, recognised in an OECD SIDS review as a single category, and therefore justifying read-across between data for different members of the group. Consequently data sharing between ESBO epoxidised soybean oil and epoxidised linseed oil is commonly utilised in the preparation of this dossier and other read-across bridges are used for other members of the EOD group where appropriate.
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Principles of method if other than guideline:
Not relevant
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
This test evaluated the effect of Epoxidised Soybean Oil on mitotic index/chromosomal aberrations.
Species / strain / cell type:
lymphocytes: Human Peripheral Blood Lymphocytes
Details on mammalian cell type (if applicable):
Two healthy, non-smoking volunteers (male in Experiment 1 and female in Experiment 2) were used in this study. Neither donor was suspected of any virus infection nor had been exposed to high levels of radiation or hazardous chemicals. For each experiment an appropriate volume of whole blood was drawn from the peripheral circulation on the day of culture. Whole blood cultures were established in sterile disposable centrifuge tubes by placing 0.4 mL heparinised blood into 9.0 mL Hepes-buffered RPMI medium containing 20 % (v/v) foetal calf serum and 50 ug/mL gentamycin. Photohaemagglutinin (PHA, reagent grade) was included at a concentration of 37.5 uL per ml of culture to stimulate the lymphocytes to divide. Cultures were rocked continuously during incubation. The blood cultures were incubated at 37 C for approx. 48 hours.
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S-9 mammalian liver post-mitochondrial fraction
Test concentrations with justification for top dose:
Preliminary solubility data indicated that limited precipitation occurred when an acetone solution at 6 mg/mL was diluted 100-fold into culture medium. No precipitation was observed when a 5.3 mg/ml acetone solution was diluted similarly to give 53 ug/ml. A concentration of 55 ug/ml was therefore considered close to the solubility limit and an appropriate maximum concentration for the main study. Test chemical stock solutions were prepared by dissolving Epoxidised soybean oil in analytical grade acetone to give 5.5 mg/ml. Stock solutions were not membrane filter-sterilised (because of the nature of the solvent ) and further dilutions made in acetone. Solutions were then used within 2 hours of initial dissolution as seen in Table 1 and 2.
Vehicle / solvent:
Acetone
Negative solvent / vehicle controls:
yes
Remarks:
Acetone was added to cultures designated as negative controls
Positive controls:
yes
Remarks:
Positive control chemicals were dissolved in sterile anhydrous analytical grade dimethyl sulphoxide immediately prior to use
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
Please see table 3 for concentration levels
Positive controls:
yes
Remarks:
Positive control chemicals were dissolved in sterile anhydrous analytical grade dimethyl sulphoxide immediately prior to use
Positive control substance:
cyclophosphamide
Remarks:
Please see table 3 for concentration levels
Details on test system and experimental conditions:
METHOD OF APPLICATION:
Test chemical stock solutions were prepared by dissolving epoxidised soybean oil in analytical grade acetone to give 5.5 mg/ml. Solutions were made according to Tables 1 and 2.

Glucose-6-phosphate (180 mg/ml), NADP (25 mg/ml), 150 mM KCl and rat liver S-9 were mixed in the ratio 1:1:1:2. A 0.5 ml aliquot of the resulting S-9 mix was added to each cell culture 99.5 mL) containing the test chemical to achieve the required final concentration in a total of 10 mL. Cultures treated in the absence of S-9 received 0.5 ml 150 mM KCl.

DURATION
- Preincubation period: approx 48 hours
- Exposure duration: 3 hours
- Expression time (cells in growth medium): 20 or 44 hours. Please see Table 5.

SPINDLE INHIBITOR (cytogenetic assays): Colchicine
STAIN (for cytogenetic assays): the cells were stained for 5 minutes in 4 % (v/v) filtered Giemsa stain in pH 6.8 buffer.

NUMBER OF REPLICATIONS: Please see Table 4

DETERMINATION OF CYTOTOXICITY
Slides were examined, uncoded, for mitotic index or percentages of cells in mitosis

Evaluation criteria:
Slides were examined, uncoded, for mitotic index or percentages of cells in mitosis.
Scoring of aberrations - where possible 25 cells from each of the selected NQO and CPA positive control treatments were analysed to ensure that the system was operating satisfactorily. One hundred metaphases from each culture were analysed for chromosome aberrations. Only cells with 44-46 chromosomes were considered accepatble for analysis of structural aberrations. Any cell with more than 46 chromosomes, that is polyploid, endoreplicated and hyperdiploid cells, observed during this search was noted and recorded seperately.
Statistics:
None reported
Species / strain:
lymphocytes: Human Peripheral Blood Lymphocytes
Metabolic activation:
with and without
Genotoxicity:
other: not required
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
The results of mitotic index determinations for the treatments in Experiment 1, without and with S-9 sampled at 20 hours are shown in Table 6. No mitotic inhibition was observed in the absence of S-9 and little (approx. 14 %) in its presence, although this was not clearly dose-related. The following doses were then chosen:

Mitotic Inhibition *
20 hours, -S-9: 26.95, 38.5, 55 ug/mL 0 %
20 hours, +S-9: 26.95, 38.5, 55 ug/mL 14 %

* = at highest analysed dose

The results of mitotic index determinations for the treatments in Experiment 2, without and with S-9 sampled at 20 and 44 hours are seen in Table 7.


In contrast to Experiment 1, mitotic inhibition was observed in the absence of S-9 at the 20 hour sampling in Experiment 2. Approximately 47 % mitotic inhibition was seen at the highest level in the absence of S-9 and 25 % in its presence. No mitotic inhibition was seen at the 44 hour sampling time. The following doses were selcted for analysis:

Mitotic Inhibition *
20 hours, -S-9: 30.94, 41.25, 55 ug/mL 47 %
20 hours, +S-9: 30.94, 41.25 55 ug/mL 25 %
44 hours, - and + S-9; 55 ug/mL 0%

* = at highest analysed dose
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Table 6: Mitotic Index Determinations for Experiment 1

Treatment (ug/ml)

Mitotic index (%)

-S-9

 

+S-9

 

A

B

A

B

Untreated

NS

NS

NS

NS

Solvent

3.2

2.3

4.3

3.5

1.554

NM

NM

NM

NM

2.219

NM

NM

NM

NM

3.171

NM

NM

NM

NM

4.530

NM

NM

NM

NM

6.471

NM

NM

NM

NM

9.244

NM

NM

NM

NM

13.21

NM

NM

NS

NS

18.87

NS

NS

NS

NS

26.95

3.1

2.2

3.6

2.7

38.5

2.1

2.3

2.9

4.0

55

2.8

2.5

3.0

3.7

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.

Treatment (ug/mL)

Mitotic index (%)

20 hours

44 hours

A

B

A

B

A

B

A

B

Untreated

NS

NS

NS

NS

NS

NS

NS

NS

Solvent

4.7

3.8

5.0

6.9

3.8

3.5

5.9

6.7

5.506

NT

NT

NT

NT

NM

NM

NT

NT

7.342

NT

NT

NT

NT

NM

NM

NT

NT

9.789

NT

NT

NT

NT

NM

NM

NT

NT

13.05

NT

NT

NT

NT

NM

NM

NM

NM

17.4

NT

NT

NT

NT

NM

NM

NM

NM

23.2

NS

NS

NS

NS

NS

NS

NM

NM

30.94

3.2

3.7

5.5

6.8

NS

NS

NS

NS

41.25

4.2

3.8

5.4

6.0

NS

NS

NS

NS

55

1.5

3.0

3.6

5.3

3.8

4.1

6.0

6.7

NS = not scored

NM = slides not made

NT = not tested

Conclusions:
Some differences in response were seen between experiment 1 and 2 but no consistent indication of an effect on structure or frequency of chromosomal aberrations, with or without metabolic activation. 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.
Executive summary:

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.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
15 September 2004 to 15 December 2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP and Guideline compliant assay with no significant deviations
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
This in vitro test is a test for the detection of structural chromosomal aberrations. Such aberrations are frequently lethal to the damaged cells. However, cytogenetic damage in somatic cells is an indicator of a potential to induce more subtle chromosomal damage that may be compatible with cell division. Similar damage induced in germ cells may lead to heritable cytogenetic abnormalities. Heritable cytogenetic abnormalities are known to have deleterious effects in man, e.g. induction of neoplastic events or birth defects. Also, chromosome abnormalities in somatic cells may become one of the reasons why a transformed cell population develops, possibly leading to neoplastic processes.

Structural chromosomal aberrations are generally evaluated in first post treatment mitoses. With the majority of chemical mutagens induced structural aberrations are of the chromatid type, but chromosome type aberrations also occur.
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Metabolic activation system:
liver mitochondrail S-9
Test concentrations with justification for top dose:
Range finding assay - A pre-test on cell growth inhibition with 4 hrs and 24 hrs treatment was performed in order to determine the toxicity of the test item.Cytotoxicity was determined using concentrations separated by no more than a factor of 2 - √10.

Main experiments
The highest concentration used in the cytogenetic experiments was chosen with regard to OECD Guideline for in vitro mammalian cytogenetic tests requiring the top concentration to show clear toxicity with reduced cell numbers or mitotic indices below 50 % of control, whichever is the lowest and/or the occurrence of precipitation. In case of nontoxicity the maximum concentration should be 5 mg/mL, 5 μL/mL or 10 mM, whichever is the lowest, if an appropriate solvent is available.
Allowing for the molecular weight of the test item, 4200 μg/mL (approx. 10 mM) of ETP were applied as top concentration for treatment of the cultures in the pre-test. Test item concentrations between 32.8 and 4200 μg/mL (with and without S9 mix) were chosen for the evaluation of cytotoxicity. In the pre-test on toxicity, precipitation of the test item after 4 hrs treatment was observed at 32.8 μg/mL and above, in the absence and the presence of S9 mix.
Using reduced cell numbers as an indicator for toxicity in the pre-test, clear toxic effects were observed after 4 hrs treatment with 4200 μg/mL in the absence of S9 mix. Considering the toxicity data of the pre-test, 4200 μg/mL was chosen as top concentration in Exp I in the absence of S9 mix.
100 μg/mL was chosen as top concentration in Exp I in the presence of S9 mix.

Experiment II was similarly influenced by toxicity and precipitation. In the range finding experiment no clearly reduced cell numbers were observed after 24 hrs exposure with 4200 μg/mL. However, based on data after 4 hrs exposure, 4200 μg/mL was chosen as top concentration for continuous
exposure in the absence of S9 mix. 100 μg/mL was chosen as top treatment concentration with S-9 in Exp.II.




Vehicle / solvent:
Ethanol
Untreated negative controls:
yes
Remarks:
untreated
Negative solvent / vehicle controls:
yes
Remarks:
ethanol
Positive controls:
yes
Remarks:
see below
Positive control substance:
ethylmethanesulphonate
Remarks:
Migrated to IUCLID6: without metabolic activation
Positive control substance:
cyclophosphamide
Remarks:
Migrated to IUCLID6: with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
On the day of the experiment (immediately before treatment), the test item was diluted with ethanol ( purity 99.8 %). The final concentration of ethanol in the culture medium was 0.5 % (v/v). The solvent was chosen for its solubility properties and relative nontoxicity to the cell cultures

Large stocks of the V79 cell line were stored in liquid nitrogen in the cell bank of RCC Cytotest Cell Research GmbH allowing the repeated use of the same cell culture batch in experiments. Before freezing each batch was screened for mycoplasm contamination and checked for karyotype stability.

Thawed stock cultures were propagated at 37° C in 80 cm² plastic flasks. About 5 x 10E5 cells per flask were seeded into 15 mL of MEM
(Minimal Essential Medium) supplemented with 10 % foetal calf serum. The cells were subcultured twice weekly. The cell cultures were incubated at 37° C in a humidified atmosphere with 1.5 % carbon dioxide (98.5 % air).
Exponentially growing stock cultures more than 50 % confluent are treated with trypsin-EDTA-solution at 37° C for approx. 5 minutes. Then the enzymatic treatment is stopped by adding complete culture medium and a single cell suspension is prepared. The trypsin concentration for all subculturing steps is 0.5 % (w/v) in Ca-Mg-free salt solution

The cells were seeded into Quadriperm dishes (at least 2 chambers per dish and test group). In each chamber 1 x 10E4 to 6 x 10E4 cells were seeded with regard to the preparation time. The medium was MEM with 10 % FCS (complete medium).

4 hour exposure period - The culture medium of exponentially growing cell cultures was replaced with serum-free medium (for treatment with S9 mix) or complete medium (for treatment without S9 mix) with 10 % FCS (v/v), containing the test item. For the treatment with metabolic activation 50 μL
S9 mix per mL medium were used. Concurrent negative, solvent, and positive controls were performed. After 4 hrs the cultures were washed twice with "Saline G" and then the cells were cultured in complete medium for the remaining culture time.

18 and 28 hour exposure period - The culture medium of exponentially growing cell cultures was replaced with complete medium (with 10 % FCS) containing different concentrations of the test item without S9 mix. The medium was not changed until preparation of the cells.
All cultures were incubated at 37° C in a humidified atmosphere with 1.5 % CO2 (98.5 % air).

15.5 hrs and 25.5 hrs, respectively after the start of the treatment colcemid was added (0.2 μg/mL culture medium) to the cultures. 2.5 hrs later, the cells on the slides were treated in the chambers with hypotonic solution (0.4 % KCl) for 20 min at 37° C. After incubation in the hypotonic solution the cells were fixed with a mixture of methanol and glacial acetic acid (3:1 parts, respectively).

DURATION
- Exposure duration:4, 18 or 28 hours
- Expression time (cells in growth medium): 14 hours

- Fixation time (start of exposure up to fixation or harvest of cells): preparation interval of 18 or 28 hours

SELECTION AGENT (mutation assays):
SPINDLE INHIBITOR (cytogenetic assays): colcemid
STAIN (for cytogenetic assays): Giemsa - duplicate slides prepared at each treatment

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: Breaks, fragments, deletions, exchanges, and chromosome disintegrations were recorded as structural chromosome aberrations. Gaps were recorded as well but not included in the calculation of the aberration rates. 100 well spread metaphase plates per culture were scored for cytogenetic damage on coded slides.

Only metaphases with characteristic chromosome numbers of 22 ± 1 were included in the analysis. To describe a cytotoxic effect the mitotic index (% cells in mitosis) was determined. In addition, the number of polyploid cells in 500 metaphase cells per culture was determined (% polyploid metaphases; in the case of this aneuploid cell line polyploid means a near tetraploid karyotype).

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; In a quantitative assessment, exponentially growing cell cultures (seeding about 40,000 cells/ slide, with regard to the culture time 48 hrs) were treated with the test item for simulating the conditions of the main experiment. A qualitative evaluation of cell number and cell morphology was made 4 hrs and 24 hrs after start of treatment. 24 hrs after start of treatment the cells were stained. Using a 400 fold microscopic magnification the cells were counted in 10 coordinate defined fields of the slides (2 slides per treatment group). The cell number of the treatment groups is given as % cells in relation to the control.
Evaluation criteria:
Assay acceptability
The chromosome aberration test is considered acceptable if it meets the following criterion:
a) The number of structural aberrations found in the negative and/or solvent controls falls within the range of historical laboratory control data: 0.0 - 4.0 %.
b) The positive control substances should produce significant increases in the number of cells with structural chromosome aberrations, which are within the range of the laboratory´s historical control data:
without S9
EMS 100 – 1000 μg/mL - 8.0-99.0% aberrant cells
with S9
CPA 0.7 – 1.0 μg/mL - 7.5-62.0% aberrant cells

Evaluation criteria:
The test material is considered non-clastogenic if:
-the number of induced structural chromosome aberrations in all evaluated dose groups is in the range of historical control data (0.0 - 4.0 % aberrant cells, excluding gaps).
and/or
− no significant increase of the number of structural chromosome aberrations is observed.

The test material is considered clastogenic if:
− the number of induced structural chromosome aberrations is not in the range of historical control data (0.0 - 4.0 % aberrant cells, exclusive gaps).
and
− either a concentration-related or a significant increase of the number of structural chromosome aberrations is observed.

Although the inclusion of the structural chromosome aberrations is the purpose of this study, it is important to include the polyploids and endoreduplications. The following criteria is valid:
A test item can be classified as aneugenic if:
− the number of induced numerical aberrations is not in the range of historical control data (0.0 - 8.5 % polyploid cells).



Statistics:
No details
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
In the pre-experiment, precipitation of the test item in culture medium was observed after treatment with 32.8 μg/mL and above in the absence and in the presence of S9 mix. No relevant influence of the test item on the pH value or osmolarity was observed (solvent control 410 mOsm, pH 7.3 versus 339 mOsm and pH 7.3 at 4200 μg/mL).

In this study, in the absence of S9 mix, precipitation of the test item in culture medium was observed in Experiment I at preparation interval 18 hrs at 25 μg/mL and above and in Experiment II at preparation interval 18 hrs and 28 hrs at 50 μg/mL and above. In the presence of S9 mix, test item precipitates in culture medium were observed in Experiment I at preparation interval 18 hrs at 50 μg/mL and above and in Experiment II at preparation interval 28 hrs at 100 μg/mL.

In the cytogenetic experiments, toxic effects indicated by reduced cell numbers of about and below 50 % of control were observed Experiment II, in the absence of S9 mix after 18 hrs and 28 hrs continuous treatment with 4200 μg/mL (49 % and 52 % of control, respectively). In these experimental parts the mitotic indices where not distinctly reduced up to the highest applied concentration. In all additional experimental parts no cytotoxicity indicated by reduced cell numbers or mitotic indices were observed after treatment with the test item.

Remarks on result:
other: strain/cell type: V79 lung fibroblasts
Remarks:
Migrated from field 'Test system'.

The test item ETP, diluted with ethanol, was assessed for its potential to induce structural chromosome aberrations in V79 cells of the Chinese hamster in vitro in the absence and the presence of metabolic activation by S9 mix. Two independent experiments were performed. In Experiment I, the exposure period was 4 hrs with and without metabolic activation. In Experiment II the exposure period was 4 hrs with S9 mix and 18 hrs and 28 hrs without S9 mix. The chromosomes were prepared 18 hrs (Exps. I and II) and 28 hrs (Exp. II) after start of treatment with the test item. In each experimental group two parallel cultures were set up. 100 were scored for structural chromosome aberrations.

Conclusions:
Under the experimental conditions reported, the test item did not induce structural chromosome aberrations as determined by the chromosome
aberration test in V79 cells (Chinese hamster cell line) in vitro.
Therefore, Fatty acids, tall-oil, epoxidized, 2-ethylhexylesters (ETP) is considered to be non clastogenic in this chromosome aberration test with and without S9 mix when tested up to cytotoxic and/or precipitating concentrations.

By read -across, fatty acids C12-20 and C12-20 unsaturated, 2-ethylhexyl esters are also considerred to be non-clastogenic
Executive summary:

ETP, diluted with ethanol, was assessed for its potential to induce structural chromosome aberrations in V79 cells of the Chinese hamster in vitro in two independent experiments.

In each experimental group two parallel cultures were set up. 100 plates were scored for structural chromosome aberrations. The highest applied concentration in the pre-test on toxicity (4200 μg/mL; approx. 10 mM) was chosen with regard to the molecular weight of the test item with respect to the current OECD Guideline 473. Dose selection for the cytogenetic experiments was performed considering the toxicity data and the occurrence of precipitation. The chosen treatment concentrations are described in Table 2 (page 17). The evaluated experimental points and the results are summarised in Table 1 (page 10 and 11). In Experiment I in the absence of S9 mix no toxic effects indicated by reduced mitotic indices or reduced cell numbers were observed after treatment with the test item. Cytotoxicity indicated by reduced cell numbers of about and below 50 % of control was observed in Experiment II, after 18 and 28 hrs continuous treatment in the absence of S9 mix. Since no relevant toxicity was observed in the pre-test on toxicity in the presence of S9 mix, the test item was tested up to a concentration exhibiting clear test item precipitation as recommended in the OECD Guideline 473. In both independent experiments, neither a statistically significant nor a biologically relevant increase in the number of cells carrying structural chromosomal aberrations was observed after treatment with the test item. No relevant increase in the frequencies of polyploid metaphases was found after treatment with the test item as compared to the frequencies of the controls. Appropriate mutagens were used as positive controls. They induced statistically significant increases.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
28 May 1986 to 26 June 1986
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Test and control substance concentrations in carner were not determined by analytical method. The stability of test substances in carrier were not determined. Solutions of test substance were prepared on each day of use Read across to a study result from an investigation using a similar material is justified for members of the Epoxidised Oils and Derivatives group. Four epoxidised oils (linseed, soybean, 2-ethylhexyl tallate and fatty acids, C14-C22, 2-ethylhexylesters) have been identified as sharing common structural and functional similarities, recognised in an OECD SIDS review as a single category, and therefore justifying read-across between data for different members of the group. Consequently data sharing between ESBO epoxidised soybean oil and epoxidised linseed oil is commonly utilised in the preparation of this dossier and other read-across bridges are used for other members of the EOD group where appropriate.
Qualifier:
no guideline followed
Principles of method if other than guideline:
The study design followed the principles of the widely accepted mammalian mutation assay CHO/HGPRT, although the guideline had not been formally adopted at the time of study conduct. The assay had been reviewed by the EPA Gene-Tox program (Hsie et al, 1981) and the method was subsequently adopted by the OECD as Test Method 476.
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
Target gene:
hypoxanthine guanine phosphoribosyl transferase (HGPRT) in Chinese hamster ovary (CHO) cells
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
The subclone K1BH$ of CHO cells, originally obtained from Dr. A.W. Hale of Oak Ridge National Laboratory was used. The cells were routinely maintained in the laboratory as exponentially growing cultures in Ham's F12 medium (K. C. Biological) supplemented with heat-inactivated 10 % newborn calf serum (K.C. Biological) in an incubator-controlled environment of a 95 % humidified atmosphere of 5 % ± 1 % CO2 and 95 % air at 37.5 °C.
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254- induced rat liver homogenate (S9)
Test concentrations with justification for top dose:
200, 500, 1000 and 2000 µg/mL of both ESO and Cl-ESO.
Vehicle / solvent:
Cytoxicity Determination: Ham's F12 medium without serum (5 mL) with or without activation.
Mutagenesis: Subculture medium (hypoxanthine-free Ham's F12 medium (K.C. Biological) supplemented with 5 % dialysed foetal calf serum).
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Remarks:
ethylmethane sulfonate and benzo(a)pyrene
Details on test system and experimental conditions:
CYTOXICITY DETERMINATION
CHO cells were seeded in 25 cm2 plastic culture flasks at 0.5 x 10 ^6 cells per flask in growth medium 18-24 hours before treatment. On the day of treatment, the medium was changed to Ham's F12 medium without serum (5 mL) with or without S-9 activation. Volumes of 25 µL of different concentrations of the test chemical were added. After an incubation of 3 hours at 37.5 °C ± 2 °C, the treatment medium was discarded. The cells were washed with 5 mL Hank's balanced salt solution and the cells were removed from the flasks by trypsinization and counted. Three aliquots of approximately 200 cells were plated per sample for determination of cloning efficiency. The plates were returned to incubation for 7 - 9 days. The colonies developed were fixed with 70 % methanol, stained with 10 % Giemsa and counted by hand. Cytoxicity was expressed as relative survival.

MUTAGENESIS
CHO cells were plated on the day before treatment. The next day, they were treated with test chemicals, positive controls and negative solvent control as described for cytotoxicity above. The cells were then processed, as described above for the cytoxicity determination, except that in addition to plating 200 cells for cloning efficiency, 10 ^6 cells per sample were plated in 10 mL of subculture medium (hypoxanthine-free Ham's F12 medium (K.C. Biological) supplemented with 5 % dialysed foetal calf serum). The cells were subcultured every 2 - 3 days for 7 -9 days for the expression of the mutant phenotype. Mutant selection was performed as previously described (Li, 1982) using selective medium consisting of hypoxanthine-free Ham's F12 medium supplemented with 10 µM 6 TG and 5 % dialysed fetal calf serum. Approximately 10 ^6 cells per sample were plated in 100 mm plates (5 plates, 2 x 10 ^5 cells per plate) in 8 mL of selective medium per plate for mutant selection. Three aliquots of approximately 200 cells per sample were plated in 3 mL of selective medium without 6TG for the determination of cloning efficiency (C.E.). The plates were returned to the incubator for 7-9 days. The colonies that developed were fixed, stained and counted. Results were expressed as mutant frequency.

ACTIVATION SYSTEM
Aroclor 1254-induced rat liver homogenate (S9), lot No. 03830, commercially purchased from Microbiological Associates, was used as an exogenous activation system. The S9/cofactor mixture consisted of 50 mM sodium phosphate (pH 7.5), 4 mM NADP, 5 mM glucose-6-phosphate, 30 mM KCl, 10 mM MgCl2, 10 mM CaCl2 and different amounts of liver S9. The 5 % S9 in this report represents the percent of S9 (v/v) in the S9/cofactor mixtures. One mL of the S9/cofactor mixture was added to 4 mL of medium for cytoxicity and mutagenicity testing.
Evaluation criteria:
The general experimental design used to determine the mutagenicity of the test chemical in the CHO-HPGRT assay is described below:
1 - Mutagenicity Determination in the Absence of Aroclor 1254-Induced Rat Liver Homogenate (S9):
This experiment was designed to test for mutagenicity of ESO and Cl-ESO in the absence of an exogenous activating system. Duplicate treatment s amples of solvent control, 4 doses (0.2, 0.5, 1 and 2 mg/mL) of the test chemicals, and positive control were used with a treatment time of 3 hours.
2 - Mutagenicity Determination in the presence of S9:
This experiment, using information obtained from experiments discussed previously, served to determine if the mutagenicity of the test chemical can be modified by exogenous activation. A concentration of 5 % S9 was used for this experiment. This level of S9 has been shown to provide signi ficant mutagenic responses when tested with a variety of promutagens used in this assay (A.P. Li, 1984). As in the first experiment, testing was per formed using duplicate samples with a treatment time of 3 hours.
Statistics:
Mutagenicity data were analysed according to the statistical method of Snee and Irr (1981) designed specifically for the CHO/HGPRT mutation assay. Mutant frequency values were transformed according to the equation Y=(X-1)^0.15, where Y=transformed mutant frequency and X= observed mutant frequency. Student's t-test was then used to compare treatment data to solvent control data. The Snee and Irr analysis also allowed the determination of dose-response relationship as linear, quadratic or higher-order.
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Positive controls validity:
valid
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Positive controls validity:
valid
Additional information on results:
ESO and Cl-ESO were tested for mutagenicity in the absence and presence of exogenous activation in two separate experiments.

In the first experiment, no exogenous activating system was used. Four concentrations of each test chemical were tested, with 2000 µg/mL as the highest concentration. Dose-related cytoxicity was observed for both ESO and Cl-ESO. The lowest relative survival values (highest cell killing) observed for the two test chemicals were 0.32 (at 2000 µg/mL ESO) and 0.17 (at 1000 µg/mL Cl-ESO). No statistically significant mutagenicity was observed for either chemical at any of the doses tested.

In the second experiment, the chemicals were tested for mutagenicity in the presence of activation, using Aroclor 1254-induced rat liver homogenate (S9) as the exogenous activating system. As in the first experiment, four doses of ESO and Cl-ESO were tested up to the highest concentration of 2000 µg/mL. No significant cytoxicity (> 50 % cell killing) or statistically significant mutagenicity was observed for the test chemicals.

The positive controls, ethylmethane sulfonate and benzo(a)pyrene, for testing in the absence and presence of S9, respectively, yielded the expected strong positive responses indicating that the experimental conditions were adequate for the detections of mutagens.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Table 1        Colony Counts for the Mutagenicity Determination of ESO and Cl-ESO in the Absence of S9

Treatment (a)(µg/mL)

Cytoxicity (b)

Mutagenicity

Survival (c)

Selection (d)

DMSO Control

112

125

103

119

103

123

1

1

2

1

1

DMSO Control

142

154

160

107

105

117

0

0

0

0

1

ESO

200

105

111

127

110

118

137

4

1

0

0

0

200

80

83

98

132

131

150

2

0

0

0

0

500

56

59

70

134

126

105

0

0

0

0

0

500

60

73

57

146

135

129

0

0

1

0

0

1000

73

57

70

124

146

120

4

3

3

2

3

1000

52

66

55

115

120

115

2

0

0

0

1

2000

40

52

54

102

119

101

0

1

0

0

0

2000

28

45

36

153

146

160

0

0

0

0

0

Cl-ESO

200

61

62

59

-

-

-

2

0

3

0

0

200

58

76

52

127

111

118

1

0

0

1

0

500

73

78

79

100

97

100

2

3

0

1

2

500

51

56

56

119

122

116

1

0

0

0

0

1000

23

23

19

134

113

131

0

0

0

0

1

1000

13

26

30

141

124

139

0

0

0

0

0

2000

89

98

82

143

147

120

0

1

1

2

0

2000

69

76

78

109

139

128

1

0

0

0

0

2

105

99

100

65

83

77

9

13

18

20

12

2

87

117

97

91

107

88

8

14

15

13

19

(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

Conclusions:
ESO and Cl-ESO were tested in CHO cells in the absence and presence of S9. Significant cytoxicity (> 50 % cell death) was observed in the absence of S9. No statistically significant mutagenicity was observed. The test chemicals are, therefore, concluded to be non-mutagenic in CHO cells under the experimental conditions.
Executive summary:

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.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
April 15, 1992 - June 25, 1992
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
GLP compliant. According to guidelines. Read across to a study result from an investigation using a similar material is justified for members of the Epoxidised Oils and Derivatives group. Four epoxidised oils (linseed, soybean, 2-ethylhexyl tallate and fatty acids, C14-C22, 2-ethylhexylesters) have been identified as sharing common structural and functional similarities, recognised in an OECD SIDS review as a single category, and therefore justifying read-across between data for different members of the group. Consequently data sharing between ESBO epoxidised soybean oil and epoxidised linseed oil is commonly utilised in the preparation of this dossier and other read-across bridges are used for other members of the EOD group where appropriate.
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Principles of method if other than guideline:
Not relevant
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
Target gene:
tk gene locus
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
L5178Y TK +/- mouse lymphoma cells were obtained from the American Type Culture Collection. They were stored as frozen stocks in liquid nitrogen. Each batch of cells was purged of TK mutants, checked for spontaneous mutant frequency and that they were mycoplasma free. For each experiment the vials were thawed rapidly, the cells were diluted in RPMI 10 and incubated in a humidified atmosphere of 5 % v/v CO2 in air. When the cells were growing well, subcultures were established in an appropriate number of flasks.
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S-9 mammalian liver post-mitochondrial fraction
Test concentrations with justification for top dose:
The concentrations were selected using a cytotoxicity range-finder. See Table 1 below.
Vehicle / solvent:
Acetone was used as a solvent. The test item was added and the culture was then vortexed for approx. 10 seconds to obtain a good emulsion. A top dose of 5000 ug/ml was achievable using an emulsion.
Negative solvent / vehicle controls:
yes
Remarks:
Comprised of solvent, acetone, diluted 100-fold in the treatment medium.
Positive controls:
yes
Remarks:
Please see Below
Positive control substance:
benzo(a)pyrene
Remarks:
The positive controls were also made to certain concentrations please see table 2 below Migrated to IUCLID6: Please see Below
Positive controls:
yes
Remarks:
Please see Below
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
The positive controls were also made to certain concentrations please see table 2 below Migrated to IUCLID6: Please see Below
Details on test system and experimental conditions:
METHOD OF APPLICATION:

Preparations of cofactor solutions with and without S-9

Quantity (ml)
With S-9 Without S-9
Glucose-6-phosphate (180 mg/ml) 1.0 -
NADP (25 mg/ml) 1.0 -
150 mM KCL 1.0 5.0
Rat Liver S-9 2.0 -

The above were used at the rate of 1.0 ml per 19 ml of cell culture containing the test chemical (to achieve the required final concentration in a total of 20 ml).


Three types of RPMI 1640 medium were prepared as follows:
Final Concentration in:
RPMI A RPMI 10 RPMI 20
Horse Serum (heat inactivated) 0 % v/v 10 % v/v 20 % v/v
Gentamycin 100 ug/ml 100 ug/ml 100 ug/ml
Fungizone 2.5 ug/ml 2.5 ug/ml 2.5 ug/ml
Pluronic 0.5 ug/ml 0.5 ug/ml -


DURATION
- Exposure duration: 9- 15 days
- Expression time (cells in growth medium): 2 days

NUMBER OF CELLS EVALUATED: 1 x E7 cells per flask

DETERMINATION OF CYTOTOXICITY
Following adjustment of the cultures to 2 x E5 cells/ml after treatment, samples from these were diluted to 8 cells/ml as seen in table 3. using a 8-channel pipette, 0.2 ml of concentration C of each culture was placed into each well of two 96-well microtitre plates (192 wells, at an average of 1.6 cells per wall). The plates were incubated at 37 C in a humidified incubator gassed with 5 % v/v CO2 in air until scorable (9-15 days). Wells containing viable clones were identified by eye using background illumination and counted.

Evaluation criteria:
At the end of the expression period the cell densities in the selected cultures were adjusted to 1 x E4/ml. TFT (300 ug/ml) was diluted 100-fold into these suspensions to give a final concentration of 3 ug/ml. Using a 8-channel pipette, 0.2 ml of each suspension was placed into each well of four 96-well microtitre plates (384 wells at 2 x E3 cells per well). Plates were incubated until scorable (13 days) and wells containing clones were identified as above and counted. In addition, the number of wells containing large colonies and the number containing small colonies were scored for the negative and positive controls and for doses of test chemical showing a significant increase in mutant frequency over the negative control.
Statistics:
All calculations were performed on a microcomputer.

Determination of survival or viability: Poison distribution, plating efficiency, % relative survival
Determination of mutant frequency: Mutant frequency, plating efficiency,
Statistical significance of mutant frequencies.
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
Please see Tables 5 & 6 below
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
No marked toxicity was seen after the application of the highest dose. Please see Table 4
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES: Please see table 4 below
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Table 4: raw plate counts and % relative survival for Epoxidised Soybean Oil in the cytotoxicity range-finder.

Treatment ug/mL)

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

0

18

100.0

23

100.0

78.125

21

129.2

21

84.2

156.25

16

83.8

17

59.7

312.5

18

100.0

19

71.0

625

15

76.5

15

49.9

1250

15

76.5

15

49.9

2500

16

83.8

18

65.2

5000

19

109.0

23

100.0

(1) 1.6 cells/well plated

* 32 wells scored

Table 5: Summary of Results Experiment 1

Treatment (ug/ml)

Absence of S-9

Treatment (ug/mL)

Presence of S-9

% RS

Mutant Frequency

% RS

Mutant Frequency

0

100.0

312.57

0

100.0

401.83

312.5

95.8

389.82 NS

312.5

97.9

484.86 NS

625

86.6

326.20 NS

625

87.9

563.19 NS

1250

79.9

439.82 NS

1250

86.0

518.57 NS

2500

131.9

520.41 *

2500

152.9

426.40 NS

5000

143.9

467.96 *

5000

178.7

433.74 NS

Linear Trend

**

Linear Trend

NS

NQO

BP

 

 

0.05

83.5

1676.58

2

55.2

1794.55

0.1

45.3

2078.73

3

39.4

2122.84

 

Table 6: Summary of Results Experiment 2

Treatment (ug/ml)

Absence of S-9

Treatment (ug/mL)

Presence of S-9

% RS

Mutant Frequency

% RS

Mutant Frequency

0

100.0

410.98

0

100.0

304.98

312.5

112.9

334.82 NS

312.5

91.7

331.46 NS

625

87.7

466.23 NS

625

82.6

331.03 NS

1250

71.4

404.74 NS

1250

87.7

313.73 NS

2500

84.5

461.15 NS

2500

84.5

283.11 NS

5000 $$

100.7

 

5000

83.2

311.46 NS

Linear Trend

NS

Linear Trend

NS

NQO

BP

 

 

0.05

67.5

1136.27

2

59.5

883.78

0.1

37.7

1143.11

3

32.1

1443.04

 

NS Not significant

$$ Treatment excluded due to excessive heterogeneity

*, **, *** Significant at 5 %, 1% and 0.1 % level respectively

Conclusions:
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.
Executive summary:

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.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

The genotoxic effects of epoxidised soybean oil, ESBO, were investigated in a battery of in-vitro mutagenicity assays. Read across to these results for ETP and consequently for fatty acids, C12-20 and C12-20 unsaturated, 2-ethylhexyl esters, is justified on similarity of structure and function. Mutagenic effects on histidine-auxotrophic mutants of Salmonella typhimurium were investigated using methods similar to that of OECD test guideline 471. The chromosome damaging potential and effects on cultured human peripheral blood lymphocytes was investigated according to OECD test guideline 473. Mutagenic effects on the thymidine kinase locus in mouse lymphoma cells were investigated according to test guideline 476. The test substance was not found to be genotoxic in any of the tests performed.

Justification for classification or non-classification

No classification is proposed in the absence of any indication of genetic toxicity.