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Toxicological information

Genetic toxicity: in vitro

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Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guidelineopen allclose all
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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
Identification: Soybean oil, epoxidized, Me ester, reaction products with propylene glycol
Test item 207811/B
Appearance: Yellow liquid (determined by Charles River Den Bosch)
Purity/Composition: UVCB
Test item storage: At room temperature
Stable under storage conditions until: 17 May 2018 (expiry date)
Test item handling: No specific handling conditions required
Chemical name (IUPAC), synonym or trade name: Not indicated

Method

Target gene:
His, Trp gene
Species / strain
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.
Controls
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.

Results and discussion

Test resultsopen allclose all
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

Applicant's summary and conclusion

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).