Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Genetic toxicity in vitro (OECD TG 471 - Ames): not mutagenic

In vitro gene mutation study in mammalian cells (OECD TG 476): negative (read across from Fatty acids, C16-C18 and C18-unsatd., ME esters, epoxidized)

In vitro cytogenicity / micronucleus study (OECD TG 487): clastogenic

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23 Oct 2016 - 07 Nov 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
Adopted July 21, 1997).
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
L142, 31 May 2008.
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
His, Trp gene
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: Trinova Biochem GmbH, Germany [Master culture from Dr. Bruce N. Ames (TA1535: 2006, TA1537: 2016, TA98: 2015, TA100: 2015; and Master culture from The National Collections of Industrial and Marine Bacteria, Aberdeen, UK (WP2uvrA: 2008)]
- Suitability of cells: Recommended test system in international guidelines (e.g. OECD, EC).
- Methods for maintenance in cell culture if applicable: Samples of frozen stock cultures of bacteria were transferred into enriched nutrient broth (Oxoid LTD, Hampshire, England) and incubated in a shaking incubator (37 ± 1°C, 150 rpm), until the cultures reached an optical density of 1.0 ± 0.1 at 700 nm (109 cells/ml). Freshly grown cultures of each strain were used for testing.

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: enriched nutrient broth (Oxoid LTD, Hampshire, England)
- Properly maintained: yes

Additional strain / cell type characteristics:
not applicable
Cytokinesis block (if used):
not applicable
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9-mix induced by Aroclor 1254
Test concentrations with justification for top dose:
Selection of an adequate range of doses was based on a dose range finding test with the strains TA100 and WP2uvrA, both with and without 5% (v/v) S9-mix. Eight concentrations, 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate were tested in triplicate. The highest concentration of the test item used in the subsequent mutation assay was 5000 μg/plate.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol (Merck, Darmstadt, Germany
- Justification for choice of solvent/vehicle: Test item preparation was performed with approved procedures and documented in detail. Preparations were visually inspected for homogeneity prior to use and all preparations were used within 2.5 hours after adding vehicle to the test item.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
2-nitrofluorene
sodium azide
methylmethanesulfonate
other: ICR-191, 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium; in agar (plate incorporation)
- Cell density at seeding (if applicable): 0.1 ml of a fresh bacterial culture (109 cells/ml)

DURATION
- Exposure duration: 48 ± 4 h

DETERMINATION OF CYTOTOXICITY
- Method: Bacterial background lawn evaluation
- Any supplementary information relevant to cytotoxicity:
Normal = Distinguished by a healthy microcolony lawn.
Slightly reduced = Distinguished by a slight thinning of the microcolony lawn.
Moderately reduced = Distinguished by a moderate thinning of the microcolony lawn.
Extremely reduced = Distinguished by an extreme thinning of the microcolony lawn and an increase in the size of the microcolonies compared to the solvent control plate.
Absent = Distinguished by a complete lack of any microcolony background lawn.

Rationale for test conditions:
The study procedures described in this report are in compliance with the guidelines
Evaluation criteria:
A Salmonella typhimurium reverse mutation assay and/or Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:
a) The vehicle control and positive control plates from each tester strain (with or without S9-mix) must exhibit a characteristic number of revertant colonies when compared against relevant historical control data generated at Charles River Den Bosch.
b) The selected dose range should include a clearly toxic concentration or should exhibit limited solubility as demonstrated by the preliminary toxicity range-finding test or should extend to 5 mg/plate.
c) No more than 5% of the plates are lost through contamination or some other unforeseen event. If the results are considered invalid due to contamination, the experiment will be repeated.
Statistics:
A test item is considered negative (not mutagenic) in the test if:
a) The total number of revertants in the tester strain TA100 or WP2uvrA is not greater than two (2) times the concurrent vehicle control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three (3) times the concurrent vehicle control.
b) The negative response should be reproducible in at least one follow-up experiment.

A test item is considered positive (mutagenic) in the test if:
a) The total number of revertants in the tester strain TA100 or WP2uvrA is greater than two (2) times the concurrent vehicle control, or the total number of revertants in tester strains TA1535, TA1537, TA98 is greater than three (3) times the concurrent vehicle control.
b) In case a follow up experiment is performed when a positive response is observed in one
of the tester strains, the positive response should be reproducible in at least one follow up
experiment.
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Only in the presence of S9-mix at the highest tested concentration 5000µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Only in the presence of S9 mix at the highet tested concentration
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation:The test item precipitated on the plates at 2800 and the top dose of 5000 μg/plate.

RANGE-FINDING/SCREENING STUDIES: yes

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data:

TA1535
Without S9: Range=78 - 1381 Mean=785 SD=167 N=1684
With S9: Range=78 - 1058 Mean=228 SD=105 N=1662
TA1537
Without S9: Range=55 – 1565 Mean=653 SD=290 N=1448
With S9: Range=55 – 1112 Mean=387 SD=143 N=1536
TA98
Without S9: Range=410 – 2057 Mean= 1155 SD=370 N=1646
With S9: Range=263 – 1907 Mean=860 SD=323 N=1686
TA100
Without S9: Range=549 – 1848 Mean=892 SD=178 N=1650
With S9: Range=620 - 2651 Mean=1404 SD=327 N=1677
WP2uvrA
Without S9: Range= 127 – 1951 Mean=1263 SD=461 N=1370
With S9: Range= 127 – 1951 Mean=342 SD=165 N=1410

- Negative (solvent/vehicle) historical control data:

TA1535
Without S9: Range=4-36 Mean=14 SD=6 N=1662
With S9: Range=3-34 Mean=13 SD=5 N=1677
TA1537
Without S9: Range=3-25 Mean=7 SD=3 N=1548
With S9: Range=3-28 Mean=9 SD=4 N=1547
TA98
Without S9: Range=9-50 Mean=17 SD=5 N=1662
With S9: Range=9-57 Mean=25 SD=7 N=1703
TA100
Without S9: Range=63-153 Mean=100 SD=16 N=1659
With S9: Range=60-156 Mean=103 SD=18 N=1691
WP2uvrA
Without S9: Range=12-68 Mean=26 SD=7 N=1421
With S9: Range=12-70 Mean=32 SD=8 N=1424

Conclusions:
Under the conditions of this study, the test substance was determined to be not mutagenic and does not need to be classified for mutagenicity in accordance with the criteria outline in Annex I of CLP (1272/2008/EC).
Executive summary:

The mutagenic activity of Soybean oil, epoxidized, Me ester, reaction products with propylene glycol was evaluated in accordance with OECD 471 (1997) guideline and according to GLP principles.

The test was performed in two independent experiments in the presence and absence of S9-mix (rat liver S9-mix induced by Aroclor 1254). In the dose range finding test, the test item was tested up to concentrations of 5000 μg/plate in the absence and presence of S9-mix in the strains TA100 and WP2uvrA. The test item precipitated on the plates at the top dose of 5000 μg/plate. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.

Based on the results of the dose range finding test, the test item was tested in the first mutation assay at a concentration range of 52 to 5000 μg/plate in the absence and presence of 5% (v/v) S9-mix in the tester strains TA1535, TA1537 and TA98. The test item precipitated on the plates at the top dose of 5000 μg/plate. Cytotoxicity, as evidenced by a decrease in the number of revertants, was observed in tester strains TA1535 and TA1537 in the presence of S9-mix at the highest tested concentration. In a follow-up experiment of the assay with additional parameters, the test item was tested at a concentration range of 275 to 5000 μg/plate in the absence and presence of 10% (v/v) S9-mix in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA. The test item precipitated on the plates at the dose levels of 2800 and 5000 μg/plate. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed. The test item did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in the tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in a follow-up experiment. Based on the results of this study it is concluded that the test item is not mutagenic and does not need to be classified for mutagenicity in accordance with the criteria outline in Annex I of CLP (1272/2008/EC).

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
08 May 2012 - 20 Feb 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OTS 798.5300 (Detection of Gene Mutations in Somatic Cells in Culture)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
30 March 2009
Type of assay:
other: in vitro mammalian cell gene mutation test using the Hprt and xprt genes (migrated information)
Target gene:
HPRT (hypoxanthine-guanine phosphoribosyl transferase)
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: Laboratory for Mutagenicity Testing; Technical University, 64287 Darmstadt, Germany
- Suitability of cells: The V79 cell line has been used successfully in in vitro experiments for many years.Especially the high proliferation rate and a good cloning efficiency of untreated cells (as a rule more than 50%) both necessary for the appropriate performance of the study, recommend the use of this cell line.
- Cell cycle length, doubling time or proliferation index: doubling time 12 - 16 h in stock cultures
- Methods for maintenance in cell culture if applicable: Thawed stock cultures are propagated at 37 °C in 80 cm2 plastic flasks. About 5x10^5 cells were seeded into each flask with 15 mL of culture medium. The cells were sub-cultured twice weekly. The cell cultures were incubated at 37 °C in a humidified atmosphere with 1.5% CO2.
- Modal number of chromosomes: The cells have a stable karyotype with a modal chromosome number of 22.

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: MEM (minimal essential medium) containing Hank’s salts supplemented with 10% foetal bovine serum (FBS), neomycin (5 μg/mL) and
amphotericin B (1%).
- Properly maintained: yes
- Periodically checked: Each batch is screened for mycoplasm contamination and
checked for karyotype stability and spontaneous mutant frequency. Consequently, the
parameters of the experiments remain similar because of the reproducible characteristics
of the cells.
Additional strain / cell type characteristics:
not specified
Cytokinesis block (if used):
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
Based on cytotoxicity and phase separation noted in the pre-experiment, the individual
concentrations of the main experiments were selected. The individual concentrations were spaced by a factor of 2. Narrower spacing at high concentrations was used in experiment II without metabolic activation to cover the cytotoxic range more closely.

Two independent experiments
Experiment I
4 hours -S9: 3.8, 7.5, 15.0, 30.0, 60.0 and 120.0 (PS) µg/mL
4 hours +S9: 3.8, 7.5, 15.0, 30.0, 60.0 and 120.0 (PS) µg/mL

Experiment II
24 hours -S9: 3.8, 7.5, 15.0, 30.0, 60.0 and 90.0 (PS) µg/mL
4 hours +S9: 7.5, 15.0, 30.0, 60.0 and 120.0 (PS) µg/mL

(PS) = Phase Separation = phase separation visible at the end of treatment
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
ethylmethanesulphonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): 1.5x10^6 (single culture)

DURATION
- Preincubation: 24 hours
- Exposure duration: 4 hours or 24 hours
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): 8 days
- Stain: 10% methylene blue in 0.01% KOH solution.

SELECTION AGENT (mutation assays): 6-TG (6-thioguanine)

CRITERIA FOR MICRONUCLEUS IDENTIFICATION:

DETERMINATION OF CYTOTOXICITY
- Method: relative cloning efficiency I or cell density below 50%
Rationale for test conditions:
According to guideline
Evaluation criteria:
The gene mutation assay is considered acceptable if it meets the following criteria:
The number of mutant colonies per 10^6 cells found in the solvent controls falls within the laboratory historical control data (see Annex II).
The positive control substances should produce a significant increase in mutant colony frequencies (see Annex II).
The cloning efficiency II (absolute value) of the solvent controls should exceed 50%.
The data of this study comply with the above mentioned criteria [see Annex I (tables of results, mutation rate and factor calculated referring to the cloning efficiency of the untreated cultures) and Annex II (Historical data)].

test item is classified as positive if it induces either a concentration-related increase of the mutant frequency or a reproducible and positive response at one of the test points.
A test item producing neither a concentration-related increase of the mutant frequency nor a reproducible positive response at any of the test points is considered non-mutagenic in this system.

A positive response is described as follows:
- A test item is classified as mutagenic if it reproducibly induces a mutation frequency that is three times above the spontaneous mutation frequency at least at one of the concentrations in the experiment.
- The test item is classified as mutagenic if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be considered also in the case that a threefold increase of the mutant frequency is not observed.
However, in a case by case evaluation this decision depends on the level of the corresponding solvent control data. If there is by chance a low spontaneous mutation rate within the laboratory´s historical control data range, a concentration-related increase of the mutations within this range has to be discussed. The variability of the mutation rates of solvent controls within all experiments of this study was also taken into consideration.
Statistics:
A linear regression (least squares) was performed to assess mutant frequencies.
A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological and statistical significance was considered together.
Key result
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:
not examined
Positive controls validity:
valid
Additional information on results:
The increased mutation frequency observed in the second experiment without metabolic activation at 60.0 μg/mL in culture I and at 7.5 to 60.0 μg/mL in culture II was considered an irreproducible fluctuation because:
- Only the increase observed in culture I at 60.0 μg/mL exceeded the historical range of solvent controls (2.6 - 40.3 mutant colonies/10^6 cells).
- The apparent increase of the mutation frequency in culture II is based on the low solvent control of just 3.9 colonies per 10^6 cells.
- The absolute values of the mutation frequency remained within the historical range of solvent controls.
- The apparent increase in culture II was not dose dependent as indicated by the lacking statistical significance.
A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of the mutation frequency. A significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was determined in the second culture of experiment I without metabolic activation and in the first culture of experiment II without metabolic activation. However, the trends were judged as biologically irrelevant as they were not reproduced in the parallel cultures. Furthermore, the mutation frequency of the second culture of the first experiment without metabolic activation remained within the historical range of solvent controls.

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no pH effects were seen
- Effects of osmolality: no osmolality effects were seen
- Precipitation: Phase separation occurred at 62.5 μg/mL and above with and without metabolic activation following 4 and
24 hours treatment.

RANGE-FINDING/SCREENING STUDIES:
Test item concentrations between 31.3 μg/mL and 4000 μg/mL were used to evaluate toxicity in the presence (4 hours treatment) and absence (4 hours and 24 hours treatment) of metabolic activation. Relevant cytotoxic effects indicated by a relative suspension growth below 50 were noted at 62.5 μg/mL and above without metabolic activation and 500.0 μg/mL and above with metabolic activation following 4 hours treatment. Following 24 hours treatment without metabolic activation cytotoxic effects as described above occurred at 62.5 μg/mL and above.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data:
- Negative (solvent/vehicle) historical control data:

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: [complete, e.g. CBPI or RI in the case of the cytokinesis-block method; RICC, RPD or PI when cytokinesis block is not used]
- Other observations when applicable: [complete, e.g. confluency, apoptosis, necrosis, metaphase counting, frequency of binucleated cells]
Conclusions:
Under the experimental conditions the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, “Fatty acids, C16-C18 and C18-unsatd., ME esters, epoxidized”is considered to be non-mutagenic in this HPRT assay.
Executive summary:

The potential to induce gene mutations at the HPRT locus using V79 cells of the Chinese hamster was examined for “Fatty acids, C16-C18 and C18-unsatd., ME esters, epoxidized” in a OECD TG 476 study under GLP conditions. The study was executed in two independent experiments, using identical experimental procedures. The test item was dissolved in ethanol. Cells were exposed for 4 or 24 hours, with and without metabolic activation. The concentration range of the main experiments was limited by phase separation of the test item and cytotoxic effects. The tested concentrations of the main experiments ranged from 3.8 to 120. No substantial and reproducible dose dependent increase of the mutation frequency was observed up to the maximum concentration with and without metabolic activation. Appropriate reference mutagens (EMS and DMBA), used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system. In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, “Fatty acids, C16-C18 and C18-unsatd., ME esters, epoxidized”is considered to be non-mutagenic in this HPRT assay.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
The full read across justification report is attached under "Attached justification".
30 April 2018 READ-ACROSS STUDY / SOYBEAN OIL, EPOXIDIZED, ME ESTER, REACTION PRODUCTS WITH PROPYLENE GLYCOL/ IN VITRO GENE MUTATION STUDY IN MAMMALIAN CELLS I&BBE2863R001F1.0

Executive Summary
According to Annex VIII, 8.4 of the REACh Regulation (EC) No 1907/2006, In vitro gene mutation study in mammalian cells is standard information required for the registration of substances manufactured or imported in quantities of ten tonne per year or more. However, according to Annex XI, 1.5 of the REACH Regulation, Read-across and grouping approaches can be used to adapt the standard testing regime. This read-across study report follows notably the recommendations made by the European Chemicals Agency in its “Guidance on information requirements and chemical safety assessment Chapter R.6 – QSARs and grouping of chemicals” (ECHA, 2008) and in its document “Read-Across Assessment Framework (RAAF)” (ECHA, 2017).

A read-across approach appears appropriate to predict the endpoint “, In vitro gene mutation study in mammalian cells” for the substance Soybean oil, epoxidized, methyl ester, reaction products with propylene glycol because:

Both the source and target substances are part of a chemical group consisting of Epoxidized Oils and Derivatives (EOD). The composition of the source chemical: Fatty acids, C16-C18 and C18-unst, Me esters, epoxidized, closely resembles the target substance.

The epoxide value for the source substance is higher than for the target substance indicating that this substance is potentially more reactive and thus an acceptable (worst-case) candidate regarding the prediction of possible genotoxic effects.

This report follows the RAAF method and so presents:
1) The hypothesis: analogue read-across approach, based on the similarity of the substances and the absence of mutagenicity for these types of structures;
2) The scientific justifications (“Assessment Elements”) and their evaluation (“Assessment Options”); which demonstrate the confidence that can be put in this prediction.
3) The conclusions, usable for classification assessment or risk assessment, which are summarised hereafter.

Reason / purpose:
read-across source
Key result
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:
not examined
Positive controls validity:
valid
Additional information on results:
The increased mutation frequency observed in the second experiment without metabolic activation at 60.0 μg/mL in culture I and at 7.5 to 60.0 μg/mL in culture II was considered an irreproducible fluctuation because:
- Only the increase observed in culture I at 60.0 μg/mL exceeded the historical range of solvent controls (2.6 - 40.3 mutant colonies/10^6 cells).
- The apparent increase of the mutation frequency in culture II is based on the low solvent control of just 3.9 colonies per 10^6 cells.
- The absolute values of the mutation frequency remained within the historical range of solvent controls.
- The apparent increase in culture II was not dose dependent as indicated by the lacking statistical significance.
A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of the mutation frequency. A significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was determined in the second culture of experiment I without metabolic activation and in the first culture of experiment II without metabolic activation. However, the trends were judged as biologically irrelevant as they were not reproduced in the parallel cultures. Furthermore, the mutation frequency of the second culture of the first experiment without metabolic activation remained within the historical range of solvent controls.

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no pH effects were seen
- Effects of osmolality: no osmolality effects were seen
- Precipitation: Phase separation occurred at 62.5 μg/mL and above with and without metabolic activation following 4 and
24 hours treatment.

RANGE-FINDING/SCREENING STUDIES:
Test item concentrations between 31.3 μg/mL and 4000 μg/mL were used to evaluate toxicity in the presence (4 hours treatment) and absence (4 hours and 24 hours treatment) of metabolic activation. Relevant cytotoxic effects indicated by a relative suspension growth below 50 were noted at 62.5 μg/mL and above without metabolic activation and 500.0 μg/mL and above with metabolic activation following 4 hours treatment. Following 24 hours treatment without metabolic activation cytotoxic effects as described above occurred at 62.5 μg/mL and above.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data:
- Negative (solvent/vehicle) historical control data:

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: [complete, e.g. CBPI or RI in the case of the cytokinesis-block method; RICC, RPD or PI when cytokinesis block is not used]
- Other observations when applicable: [complete, e.g. confluency, apoptosis, necrosis, metaphase counting, frequency of binucleated cells]
Conclusions:
Based on the read across results of the mammalian gene mutation assay for the source substance "Fatty acids, C16-C18 and C18-unsatd., ME esters, epoxidized", to the target "Soybean oil, epoxidized, methyl ester, reaction products with propylene glycol". The substance is considered to be non-mutagenic.
Executive summary:

The potential for "Soybean oil, epoxidized, methyl ester, reaction products with propylene glycol" to induce gene mutations was assessed using the read across source substance: "Fatty acids, C16-C18 and C18-unsatd., ME esters, epoxidized" in a OECD TG 476 study under GLP conditions. The study was executed in two independent experiments, using identical experimental procedures. The test item was dissolved in ethanol. Cells were exposed for 4 or 24 hours, with and without metabolic activation. The concentration range of the main experiments was limited by phase separation of the test item and cytotoxic effects. The tested concentrations of the main experiments ranged from 3.8 to 120. No substantial and reproducible dose dependent increase of the mutation frequency was observed up to the maximum concentration with and without metabolic activation. Appropriate reference mutagens (EMS and DMBA), used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system. In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, the substance is considered to be non-mutagenic in this HPRT assay.

Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
02 Mar 2018 - 23 May 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Version / remarks:
29 July 2016
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian cell micronucleus test
Species / strain / cell type:
lymphocytes:
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: healthy adult, non-smoking volunteers (aged 18 to 35 years)
- Suitability of cells: in line with guideline
- Average Generation Time (AGT) of the cells: 14.1-15.3h
- Sex, age and number of blood donors if applicable: 4 donors aged 23 - 32
- Whether whole blood or separated lymphocytes were used if applicable: whole blood

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Culture medium consisted of RPMI 1640 medium (Life Technologies), supplemented with 20% (v/v) heat-inactivated (56°C; 30 min) fetal calf serum (Life Technologies), L-glutamine (2 mM) (Life Technologies), penicillin/streptomycin (50 U/mL and 50 µg/mL respectively) (Life Technologies) and 30 U/mL heparin (Sigma, Zwijndrecht, The Netherlands).
- Properly maintained: yes
Cytokinesis block (if used):
Cytochalasine B
Metabolic activation:
with and without
Metabolic activation system:
S9
Vehicle / solvent:
First assay: 78, 156 and 313 µg/mL culture medium based on dose range finder
Second assay: 50, 150, 250, 275, 300 and 312.5 µg/mL culture medium based on dose range finder
Assay 2a: 10, 50, 175, 200, 225, 250, 275 and 300 µg/mL culture medium based on cytotoxicity percentages observed in the second assay
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
other: Colchicine
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 46 ± 2 hours
- Exposure duration: 3 hrs or 24 hours
- Expression time (cells in growth medium): 24 hours
- Fixation time (start of exposure up to fixation or harvest of cells): 24 - 27 hours

SPINDLE INHIBITOR (cytogenetic assays): cytochalasin B

STAIN (for cytogenetic assays): Giemsa

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Cell cultures were centrifuged (5 min, 365 g) and the supernatant was removed. Cthe pellet was re-suspended in 1% Pluronic F68 (Applichem, Darmstadt, Germany). After centrifugation (5 min, 250 g), cells were swollen by hypotonic 0.56% (w/v) potassium chloride (Merck) solution. Then, ethanol (Merck): acetic acid (Merck) fixative (3:1 v/v) was added. Cells were collected by centrifugation (5 min, 250 g) and cells in the pellet were fixated with 3 changes of ethanol: acetic acid fixative (3:1 v/v).
Fixed cells were dropped onto cleaned slides, and were allowed to dry and thereafter stained for 10 - 30 min with 5% (v/v) Giemsa (Merck) solution in Sörensen buffer pH 6.8. Thereafter slides were rinsed in water and allowed to dry. The dry slides were automatically embedded and mounted with a coverslip in an automated cover slipper (ClearVue Coverslipper, Thermo Fisher Scientific, Breda, The Netherlands).

NUMBER OF CELLS EVALUATED: 500

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): 1000

CRITERIA FOR MICRONUCLEUS IDENTIFICATION:
The following criteria for scoring micronuclei were adapted from Fenech, 1996:
• The diameter of micronuclei should be less than one-third of the main nucleus.
• Micronuclei should be separate from or marginally overlap with the main nucleus as long as there is clear identification of the nuclear boundary.
• Micronuclei should have similar staining as the main nucleus.

DETERMINATION OF CYTOTOXICITY
- Method: Cytokinesis-Block Proliferation Index (CBPI)
- Any supplementary information relevant to cytotoxicity:
Rationale for test conditions:
In accordance with the guideline
Evaluation criteria:
A test item is considered positive (clastogenic or aneugenic) in the in vitro micronucleus test if all of the following criteria are met:
a) At least one of the test concentrations exhibits a statistically significant (Chi-square test, one-sided, p < 0.05) increase compared with the concurrent negative control.
b) The increase is dose-related in at least one experimental condition when evaluated with a Cochran Armitage trend test.
c) Any of the results are outside the 95% control limits of the historical control data range.
A test item is considered negative (not clastogenic or aneugenic) in the in vitro micronucleus test if:
a) None of the test concentrations exhibits a statistically significant (Chi-square test, one-sided, p < 0.05) increase compared with the concurrent negative control.
b) There is no concentration-related increase when evaluated with a Cochran Armitage trend test.
c) All results are inside the 95% control limits of the negative historical control data range.
Statistics:
First cytogenetic assay Chi-square test
First cytogenetic assay Cochran Armitage Trend test
Second cytogenetic assay (2a) Chi-square test
Key result
Species / strain:
lymphocytes: Primary
Metabolic activation:
with
Genotoxicity:
positive
Remarks:
3 hour exposure
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
lymphocytes: Primary
Metabolic activation:
without
Genotoxicity:
negative
Remarks:
3 hour exposure
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
lymphocytes: Primary
Metabolic activation:
without
Genotoxicity:
negative
Remarks:
24 hour exposure
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: At a concentration of 312.5 µg/mL Soybean oil, epoxidized, Me ester, reaction products with propylene glycol precipitated in the culture medium.
- Definition of acceptable cells for analysis:
The following criteria for scoring of binucleated cells were used (1 - 2, 6):
• Main nuclei that were separate and of approximately equal size.
• Main nuclei that touch and even overlap as long as nuclear boundaries are able to be distinguished.
• Main nuclei that were linked by nucleoplasmic bridges.
The following cells were not scored:
• Trinucleated, quadranucleated, or multinucleated cells.
• Cells where main nuclei were undergoing apoptosis (because micronuclei may be gone already or may be caused by apoptotic process).
The following criteria for scoring micronuclei were adapted from Fenech, 1996 (1):
• The diameter of micronuclei should be less than one-third of the main nucleus.
• Micronuclei should be separate from or marginally overlap with the main nucleus as long as there is clear identification of the nuclear boundary.
• Micronuclei should have similar staining as the main nucleus.

CYTOKINESIS BLOCK
- Distribution of mono-, bi- and multi-nucleated cells: The number of mono- and binucleated cells with micronuclei found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database, except for the positive response in the presence of S9-mix which was above the 95% control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture: refer to tables

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: refer to table
- Negative (solvent/vehicle) historical control data: refer to table

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: CBPI

First mutagenicity assay

Without metabolic activation (-S9-mix)

 

3 hours exposure time, 27 hours harvest time

 

 

 

Concentration (µg/mL)

Cytostasis (%)

Number of mononucleated cells with micronuclei1)

Number of binucleated cells with micronuclei1)

1000

1000

2000

1000

1000

2000

A

B

A+B

A

B

A+B

0

0

2

4

6

4

13

17

78

12

0

0

0

4

10

14

156

24

0

1

1

2

7

9

  3132)

28

1

0

1

3

2

5

0.25-C

41

2

1

3

43

47

    90***

0.1 Colch

34

16

15

   31***

7

8

15

 

With metabolic activation (+S9-mix)

 

3 hours exposure time, 27 hours harvest time

 

 

 

Concentration (µg/mL)

Cytostasis (%)

Number of mononucleated cells with micronuclei1)

Number of binucleated cells with micronuclei1)

1000

1000

2000

1000

1000

2000

A

B

A+B

A

B

A+B

0

0

3

1

4

4

10

14

78

7

2

3

5

7

10

17

156

15

1

0

1

7

10

17

  3132)

29

2

4

6

15

17

  32**

15 CP

62

1

3

4

51

46

    97***

*)           Significantly different from control group (Chi-square test), * P < 0.05, ** P < 0.01 or *** P < 0.001.
1)            1000 bi- and mononucleated cells were scored for the presence of micronuclei. 
2)                The test item precipitated in the culture medium

Duplicate cultures are indicated by A and B.

Mutagenicity assay 2A

Without metabolic activation (-S9-mix)

 

24 hours exposure time, 24 hours harvest time

 

 

 

Concentration (µg/mL)

Cytostasis (%)

Number of mononucleated cells with micronuclei1)

Number of binucleated cells with micronuclei1)

1000

1000

2000

1000

1000

2000

A

B

A+B

A

B

A+B

0

0

1

1

2

1

2

3

50

4

0

0

0

0

1

1

175

28

0

0

0

1

1

2

 2253)

52

2

1

3

2

2

4

0.15-C

48

1

1

2

20

14

  34***

0.05 Colch

94

18

23

   41***

 42

 32

   7***

*)  Significantly different from control group (Chi-square test), * P < 0.05, ** P < 0.01 or *** P < 0.001.

1)            1000 bi- and mononucleated cells were scored for the presence of micronuclei. 
2)            328 and 272 binucleated cells were scored for the presence of micronuclei, respectively. 
3)                The test item precipitated in the culture medium

Duplicate cultures are indicated by A and B.

Historical control data

 

Mononucleated

Binucleated

 

+ S9-mix

- S9-mix

+ S9-mix

- S9-mix

 

3 hour exposure

3 hour exposure

24 hour exposure

3 hour exposure

3 hour exposure

24 hour exposure

Mean number of micronucleated cells
(per 1000 cells)

0.83

1.00

0.86

3.67

3.57

3.58

SD

0.95

1.01

1.12

2.53

2.20

2.51

n

112

114

110

112

114

110

Upper control limit

(95% control limits)

2.85

3.37

3.60

9.42

8.94

9.64

Lower control limit

(95% control limits)

-1.19

-1.38

-1.87

-2.08

-1.79

-2.48

 

SD = Standard deviation

n = Number of observations

Distribution historical negative control data from experiments performed between October 2014 and October 2017. 


 

Conclusions:
Based on the results of this study, Soybean oil, epoxidized, Me ester, reaction products with propylene glycol induces the formation of micronuclei in human lymphocytes in the presence of S9 metabolic activation. In the absence of S9-mix no genotoxic effect was found. Therefore the test substance may be considered a clastogenic compound.
Executive summary:

Soybean oil, epoxidized, Me ester, reaction products with propylene glycolwas evaluated for its ability to induce micronuclei in cultured human lymphocytes according to OECD TG 487. The possible clastogenicity and aneugenicity of the test item was tested in two independent experiments. In the first cytogenetic assay, the test item was tested up to 312.5 µg/mL for a 3 hours exposure time with a 27 hours harvest time in the absence and presence of S9-fraction. The test item precipitated in the culture medium at this dose level. 

In the second cytogenetic assay, the test item was tested up to 225 µg/mL for a 24 hours exposure time with a 24 hours harvest time in the absence of S9-mix. All the positive and negative controls were considered valid and it was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly. 

In the first cytogenetic assay, in the absence of S9-mix, the test item did not induce a statistically significant or biologically relevant increase in the number of mono- and binucleated cells with micronuclei. In the presence of S9-mix, at the 3 hours exposure time, the test item induced a statistically significant increase in the number of binucleated cells with micronuclei at the highest concentration tested. The number of mononucleated cells with micronuclei was within the 95% control limits of the distribution of the historical negative control database and the number of binucleated cells with micronuclei was above the 95% control limits of the distribution of the historical negative control database. Moreover a statistical significant dose related trend was observed and therefore this increase was considered biologically relevant. These results indicate that the test item is positive in the in vitro micronucleus study and might be considered a clastogenic compound. It is described in the literature that some aneugens increase the micronuclei frequencies in mononucleated cells as well as in binucleated cells whereas clastogens induce micronuclei solely in binucleated cells. 

In the second cytogenetic assay with a 24 hours continuous exposure time, the test item did not induce a statistically significant or biologically relevant increase in the number of mono- and binucleated cells with micronuclei.

 

In conclusion, Soybean oil, epoxidized, Me ester, reaction products with propylene glycol induces the formation of micronuclei in human lymphocytes in the presence of S9 metabolic activation under the experimental conditions described in this report. Since the test item induces the micronuclei frequency in the presence of S9-mix in binucleated cells only, it may be considered a clastogenic compound. 

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

Genetic toxicity in vivo

Description of key information

genetic toxicity in vivo (OECD TG 489, comet assay): test proposed

Link to relevant study records
Reference
Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Type of information:
experimental study planned
Study period:
to be planned
Justification for type of information:
TESTING PROPOSAL ON VERTEBRATE ANIMALS

NON-CONFIDENTIAL NAME OF SUBSTANCE:
- Name of the substance on which testing is proposed to be carried out : Soybean oil, epoxidized, Me ester, reaction products with propylene glycol

CONSIDERATIONS THAT THE GENERAL ADAPTATION POSSIBILITIES OF ANNEX XI OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION:
- Available GLP studies : 2 in vitro studies on the target substance (OECD TG 471, negative and OECD TG 487, positive), 1 in vitro read-across study (OECD TG 476, negative)
- Available non-GLP studies : none
- Historical human data": none
- (Q)SAR: not sufficiently reliable for the prediction of repeated dose toxicity
- In vitro methods: performed, and the study result provide sufficient base for further in vivo examination
- Weight of evidence: no data are available
- Grouping and read-across: A read-across is not sufficiently reliable for the prediction of this in vivo effect
- Substance-tailored exposure driven testing: not applicable
- Approaches in addition to above: not applicable

CONSIDERATIONS THAT THE SPECIFIC ADAPTATION POSSIBILITIES OF ANNEXES VI TO X (AND COLUMN 2 THEREOF) OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION:
The provision of a n in vivo study is a standard REACH requirement.Appropriate in vivo mutagenicity studies shall be considered in case of a positive result in any of the genotoxicity studies in Annex VII or VIII. It is not possible to meet this data requirement through any non-testing methods or any Column 2 or Annex XI adaptations

FURTHER INFORMATION ON TESTING PROPOSAL IN ADDITION TO INFORMATION PROVIDED IN THE MATERIALS AND METHODS SECTION:
- Details on study design / methodology proposed: For Soybean oil, epoxidized, Me ester, reaction products with propylene glycol it is proposed by the registrant to perform an OECD TG 489 assay as a follow up study. This “In vivo alkaline single-cell gel electrophoresis assay for DNA strand breaks (Comet assay)” can provide conclusive information on both the clastogenicity as well as possible gene mutations of the test substance.
Qualifier:
according to
Guideline:
OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)
Version / remarks:
current
Deviations:
no
Endpoint conclusion
Endpoint conclusion:
no study available (further information necessary)

Additional information

OECD TG 471 - Ames

The mutagenic activity of Soybean oil, epoxidized, Me ester, reaction products with propylene glycol was evaluated in accordance with OECD 471 (1997) guideline and according to GLP principles.

The test was performed in two independent experiments in the presence and absence of S9-mix (rat liver S9-mix induced by Aroclor 1254). In the dose range finding test, the test item was tested up to concentrations of 5000 μg/plate in the absence and presence of S9-mix in the strains TA100 and WP2uvrA. The test item precipitated on the plates at the top dose of 5000 μg/plate. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.

Based on the results of the dose range finding test, the test item was tested in the first mutation assay at a concentration range of 52 to 5000 μg/plate in the absence and presence of 5% (v/v) S9-mix in the tester strains TA1535, TA1537 and TA98. The test item precipitated on the plates at the top dose of 5000 μg/plate. Cytotoxicity, as evidenced by a decrease in the number of revertants, was observed in tester strains TA1535 and TA1537 in the presence of S9-mix at the highest tested concentration. In a follow-up experiment of the assay with additional parameters, the test item was tested at a concentration range of 275 to 5000 μg/plate in the absence and presence of 10% (v/v) S9-mix in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA. The test item precipitated on the plates at the dose levels of 2800 and 5000 μg/plate. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed. The test item did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in the tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in a follow-up experiment. Based on the results of this study it is concluded that the test item is not mutagenic and does not need to be classified for mutagenicity in accordance with the criteria outline in Annex I of CLP (1272/2008/EC).

OECD TG 476 (read across from Fatty acids, C16-C18 and C18-unsatd., ME esters, epoxidized)

The potential for "Soybean oil, epoxidized, methyl ester, reaction products with propylene glycol" to induce gene mutations was assessed using the read across source substance: "Fatty acids, C16-C18 and C18-unsatd., ME esters, epoxidized" in a OECD TG 476 study under GLP conditions. The study was executed in two independent experiments, using identical experimental procedures. The test item was dissolved in ethanol. Cells were exposed for 4 or 24 hours, with and without metabolic activation. The concentration range of the main experiments was limited by phase separation of the test item and cytotoxic effects. The tested concentrations of the main experiments ranged from 3.8 to 120. No substantial and reproducible dose dependent increase of the mutation frequency was observed up to the maximum concentration with and without metabolic activation. Appropriate reference mutagens (EMS and DMBA), used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system. In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, the substance is considered to be non-mutagenic in this HPRT assay.

OECD TG 487 (In vitro cytogenicity / micronucleus study)

Soybean oil, epoxidized, Me ester, reaction products with propylene glycolwas evaluated for its ability to induce micronuclei in cultured human lymphocytes according to OECD TG 487. The possible clastogenicity and aneugenicity of the test item was tested in two independent experiments. In the first cytogenetic assay, the test item was tested up to 312.5 µg/mL for a 3 hours exposure time with a 27 hours harvest time in the absence and presence of S9-fraction. The test item precipitated in the culture medium at this dose level. 

In the second cytogenetic assay, the test item was tested up to 225 µg/mL for a 24 hours exposure time with a 24 hours harvest time in the absence of S9-mix. All the positive and negative controls were considered valid and it was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly. 

In the first cytogenetic assay, in the absence of S9-mix, the test item did not induce a statistically significant or biologically relevant increase in the number of mono- and binucleated cells with micronuclei. In the presence of S9-mix, at the 3 hours exposure time, the test item induced a statistically significant increase in the number of binucleated cells with micronuclei at the highest concentration tested. The number of mononucleated cells with micronuclei was within the 95% control limits of the distribution of the historical negative control database and the number of binucleated cells with micronuclei was above the 95% control limits of the distribution of the historical negative control database. Moreover a statistical significant dose related trend was observed and therefore this increase was considered biologically relevant. These results indicate that the test item is positive in the in vitro micronucleus study and might be considered a clastogenic compound. It is described in the literature that some aneugens increase the micronuclei frequencies in mononucleated cells as well as in binucleated cells whereas clastogens induce micronuclei solely in binucleated cells. 

In the second cytogenetic assay with a 24 hours continuous exposure time, the test item did not induce a statistically significant or biologically relevant increase in the number of mono- and binucleated cells with micronuclei. In conclusion, Soybean oil, epoxidized, Me ester, reaction products with propylene glycol induces the formation of micronuclei in human lymphocytes in the presence of S9 metabolic activation under the experimental conditions described in this report. Since the test item induces the micronuclei frequency in the presence of S9-mix in binucleated cells only, it may be considered a clastogenic compound.

Test proposal (OECD TG 489)

Justification for classification or non-classification

Based on the available information, Soybean oil, epoxidized, Me ester, reaction products with propylene glycol induces the formation of micronuclei in human lymphocytes in the presence of S9 metabolic activation. Therefore the test substance may be a clastogenic compound and is proposed for further in vivo mutagenicity testing, in accordance with paragraph 8.4 of the regulation (EC) No 1907/2006.