Reaction products of furan-2,5-dione and octadec-1-ene

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Guideline study under GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

3-[(2E)-octadec-2-en-1-yl]dihydrofuran-2,5-dione

Cas Number:

1628163-42-7

Molecular formula:

C22H38O3

IUPAC Name:

3-[(2E)-octadec-2-en-1-yl]dihydrofuran-2,5-dione

Constituent 2

Reference substance name:

3-[(2Z)-octadec-2-en-1-yl]dihydrofuran-2,5-dione

Molecular formula:

C22H38O3

IUPAC Name:

3-[(2Z)-octadec-2-en-1-yl]dihydrofuran-2,5-dione

Constituent 3

Reference substance name:

3-(octadecenyl)dihydrofuran-2,5-dione excluding 3-(2-octadecen-1-yl)dihydrofuran-2,5-dione

Molecular formula:

C22H38O3

IUPAC Name:

3-(octadecenyl)dihydrofuran-2,5-dione excluding 3-(2-octadecen-1-yl)dihydrofuran-2,5-dione

Constituent 4

Reference substance name:

Hexadecenylsuccinic anhydride

EC Number:

250-911-4

EC Name:

Hexadecenylsuccinic anhydride

Molecular formula:

C20H34O3

IUPAC Name:

3-(hexadecenyl)dihydrofuran-2,5-dione

Constituent 5

Reference substance name:

3-icosenylsuccinic anhydride

EC Number:

258-603-1

EC Name:

3-icosenylsuccinic anhydride

Molecular formula:

C24H42O3

IUPAC Name:

3-icosenyldihydrofuran-2,5-dione

Constituent 6

Reference substance name:

Side products from the reaction of octadec-1-ene and maleic anhydride

Molecular formula:

Not applicable - UVCB component

IUPAC Name:

Side products from the reaction of octadec-1-ene and maleic anhydride

Constituent 7

Reference substance name:

C16-20 (even numbered) alkenes (branched and linear)

Molecular formula:

C16H32, C18H36, C20H40

IUPAC Name:

C16-20 (even numbered) alkenes (branched and linear)

Constituent 8

Reference substance name:

Maleic anhydride

EC Number:

203-571-6

EC Name:

Maleic anhydride

Cas Number:

108-31-6

Molecular formula:

C4H2O3

IUPAC Name:

furan-2,5-dione

Test material form:

solid

Details on test material:

off-white solid, amber liquid at 80 °C. Expected to be stable for duration of testing; Expiration date: not applicable.

Specific details on test material used for the study:

As a result of increasingly rigorous criteria being applied to the analysis of commercial material used in physical property/toxicity testing, the identity of the material has been modified to reveal a more accurate and precise depiction of the commercial substance. This enhancement is reflected in changes in chemical identifiers such as EC and/or CAS numbers from those noted in earlier versions of data records or in study reports.

Method

Target gene:

histidine

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9 fraction, 5%

Test concentrations with justification for top dose:

0.5, 0.75, 1.0, 1.58, 2.5, 5.0, 7.5, 15.8, 50, 158, 500, 1580, and 5000 µg/plate

Vehicle / solvent:

DMSO

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-acetylaminofluorene

9-aminoacridine

sodium azide

methylmethanesulfonate

other: daunomycin, 60 ug/ml

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation); with confirmatory tests using the preincubation method.
Rat liver S9 fraction was obtained from Molecular Toxicology, Inc., and used at 5%. The prepared S9 mix contained the following sterile cofactors (Maron & Ames, 1983): 8 mM MgCl2, 33 mM KCl, 100 mM sodium phosphate buffer pH 7.4, 5 mM glucose-6-phosphate and 4 mM NADP.

The test substance was formulated as a solution in DMSO (0.0050, 0.0075, 0.01, 0.0158, 0.025, 0.05, 0.075, 0.158, 0.5, 1.58, 5, 15.8 and 50 mg/mL) to provide corresponding dose levels of up to 5000 µg/plate. The solutions were vortexed prior to use.

The confirmatory test employed the pre-incubation modification of the plate incorporation test. The test or control substances, bacteria suspension, and S9/substitution buffer were incubated under agitation for approximately 30 minutes at 37±2°C prior to mixing with the overlay agar and pouring onto the minimal agar plates before proceeding as described for the initial test. The study design for the confirmatory test, including strains, dose levels etc. was as described above for the initial (main) test.

A supplemental test (plate incorporation and pre-incubation) was performed to adequately interpret assay results, due to the findings of toxicity (incomplete bacterial lawn) and contamination in the original studies. The study was performed with strains TA 1535 and 1538, at 8 doses ranging from 0.5 to 15.8 µg/plate. Appropriate vehicle and positive controls were included. This supplemental data and the data collected during original phase of testing are presented in body of the report.

NUMBER OF REPLICATIONS:

The background lawn for vehicle control plates should appear normal (i.e., slightly hazy with abundant microscopic non-revertant bacterial colonies). The mean revertant colony counts for each strain treated with the vehicle should lie close to or within the expected range taking into account the laboratory historical control range and/or published values (Mortelmans & Zeiger, 2000; Gatehouse, 2012). The positive controls (with S9 where required) should produce substantial increases in revertant colony numbers with the appropriate bacterial strain as specified in the Evaluation of Mutagenicity Section below.
In the case where part of the study is invalid based on these criteria, detailed results for that part of the study will not be reported and the affected part of the study would normally be subjected to an automatic repeat as described in an amendment, if appropriate.

Toxic effects of the test substance are indicated by the partial or complete absence of a background lawn of non-revertant bacteria (colony counts, if any, should not be reported) or a substantial dose-related reduction in revertant colony counts compared with lower dose levels and concurrent vehicle control taking into account the laboratory historical control range. Where precipitation obscures observations on the condition of the background lawn, the lawn can be considered normal and intact if the revertant colony counts are within the expected range based on results for lower dose levels and historical control counts for that strain.

Evaluation criteria:

For each experimental point, the Mutation Factor (MF) was calculated by dividing the mean revertant colony count by the mean revertant colony count for the corresponding concurrent vehicle control group. The mutagenic activity of the test item was assessed by applying the following criteria:

The results were considered positive (i.e., indicative of mutagenic potential) if:

The results for the test item showed a substantial increase in revertant colony counts, i.e., response MF ≥ 2 for strains TA98, TA100, and WP2 uvrA or MF ≥ 3 for strains TA1535 and TA1537, with mean value(s) outside the laboratory historical control range. Otherwise, results were considered negative.
The above increase must be dose related and/or reproducible, i.e., increases must be obtained at more than one experimental point (at least one strain, more than one dose level, more than one occasion or with different methodologies).

If the second criterion is not met, the results may be classified as equivocal, and further testing may be appropriate.
A test substance that produces neither a concentration related increase in the number of revertant colonies nor a reproducible substantial increase in revertant colonies is considered to be non-mutagenic in this test system.

Statistics:

The testing laboratory calculated means and standard deviations for all quantitative data collected.

Results and discussion

Test results

Key result

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

incomplete bacterial lawn was observed; additional data was generated.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

The mean revertant colony counts for each strain treated with the vehicle were close to or within the expected range, considering the laboratory historical control range and/or published values (Mortelmans & Zeiger, 2000; Gatehouse, 2012). The positive control substances caused the expected substantial increases in revertant colony counts in both the absence and presence of S9 in each phase of the test confirming the sensitivity of the test and the activity of the S9 mix. Therefore, each phase of the test is considered valid.

Signs of precipitation were observed at 5000 µg/plate for all strains tested, for both methods, and with and without metabolic activation. Contamination was noted with strain TA1535 at dose levels of 500 and/or 1580 µg/plate in both the absence and presence of S9 using either method.

Evidence of toxicity was observed by presence of an incomplete lawn for strain TA98 at 500 µg/plate and with strains TA1535 and TA1537 at doses ≥ 50 µg/plate. To further investigate toxicity, supplemental testing was performed for strains TA1535 and TA1537 using eight dose levels in a range of 0.5 to 15.8 µg/plate.

Single plate contamination, that did not obscure plate counts, was observed for strain TA1357 at 0.5 µg/plate, without the presence of S9. No evidence of toxicity, contamination or precipitation was observed in the supplemental testing performed for stains TA1535 or TA1537.

For all strains, at least five non-toxic dose levels without precipitation or plate contamination were evaluated, therefore bacterial mutagenicity was adequately assessed.

There was no concentration-related or substantial test substance related increases in the number of revertant colonies observed with strains TA1535, TA1537, TA98 , TA100 or E. coli WP2 uvrA in both the absence and presence of S9 using either the plate incorporation or the pre-incubation method. Based on these findings and on the evaluation system used, n-Octadecenyl Succinic Anhydride (n-ODSA), CAS# 67066-88-0, did not elicit evidence of bacterial mutagenicity in the Ames assay.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

n-Octadecenyl Succinic Anhydride (n-ODSA) was not mutagenic in the Ames assay.

Executive summary:

The Ames test was conducted with n-Octadecenyl Succinic Anhydride (n-ODSA), CAS# 67066-88 0 at levels of 0.5, 0.75, 1.0, 1.58, 2.5, 5.0, 7.5, 15.8, 50, 158, 500, 1580, and 5000 µg/plate, with the high level being the standard limit for this test. The main test was conducted using the plate incorporation method in both the absence and presence of metabolic activation (chemically-induced rat liver S9 mix). The results of the test were confirmed using a similar study design but employing the pre-incubation modification of the Ames test.

Signs of precipitation were observed at 5000 µg/plate for all strains tested, for both methods, and with and without metabolic activation. Contamination was noted with strain TA1535 at dose levels of 500 and/or 1580 µg/plate in both the absence and presence of S9 using either method. In addition, individual plate contamination was noted with strain TA1537 in the 0.5 µg/plate dose level without metabolic activation; however this contamination did not obscure the counts.

Evidence of toxicity was observed by presence of an incomplete lawn for strain TA98 at 500 µg/plate and with strains TA1535, TA1537 at doses ≥ 50 µg/plate. To further investigate toxicity supplemental testing was performed for strains TA1535, TA1537 using eight dose levels in a range of 0.5 to 15.8 µg/plate.

Single plate contamination, that did not obscure plate counts, was observed for strain TA1357 at 0.5 µg/plate, without the presence of S9. No other evidence of toxicity, contamination or precipitation was observed in the supplemental testing performed for stains TA1535, TA1537. For all strains, at least five non-toxic dose levels without precipitation or plate contamination were evaluated, therefore bacterial mutagenicity was adequately assessed.

In conclusion, based on these findings and on the evaluation system used, n-Octadecenyl Succinic Anhydride (n-ODSA), CAS# 67066 -88 -0 did not elicit evidence of bacterial mutagenicity in the Ames assay.