Reaction product with n-butanol of high-boiling stream resulting from the hydroxylation, oxidative cleavage and hydrolysis of vegetable oil saturated and unsaturated C16-C22 triglycerides.

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

There is one recent K1 study (Oberto, 2013) that did not report any adverse effects in the Ames test. Beside that, there are a number of publications cited that did not observe any adverse effects in genetic toxicity either.

Link to relevant study records

Reference

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:

2013-09-24 - 2013-09-25

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Recent GLP study according to relevant OECD guideline with detailed reporting

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial gene mutation assay

Species / strain / cell type:

E. coli WP2 uvr A

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Metabolic activation system:

liver S9 fraction from rat

Test concentrations with justification for top dose:

Toxicity test: 50, 158, 500, 1580 and 5000 µg/plate. Precipitation of the test item, which did not interfere with the scoring, was observed at the end of the incubation period at the highest concentration in the absence of metabolic activation and at the two highest concentrations in the presence of metabolic activation. No toxicity was observed with any tester strain at any dose level, in the absence or presence of S9 metabolism. On the basis of toxicity test results, in Main Assay I, using the plate incorporation method, the test item was assayed at the following dose levels: 5000, 2500, 1250, 625 and 313 μg/plate. For Main Assay II, The test item was assayed at the same dose levels used in Main Assay I.

Vehicle / solvent:

The test item was used as a solution in acetone

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

acetone, DMSO

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: 2-aminoanthracene

Details on test system and experimental conditions:

Four strains of Salmonella typhimurium (TA1535, TA1537, TA98 and TA100) and a strain of Escherichia coli (WP2 uvrA) were used in this study. Permanent stocks of these strains are kept at -80°C in RTC. Overnight subcultures of these stocks were prepared for each day’s work PRELIMINARY TOXICITY TEST A preliminary toxicity test was undertaken in order to select the concentrations of the test item to be used in the main assays. In this test a wide range of dose levels of the test item, set at half-log intervals, were used. Treatments were performed both in the absence and presence of S9 metabolism using the plate incorporation method; a single plate was used at each test point and positive controls were not included. Toxicity was assessed on the basis of a decline in the number of spontaneous revertants, a thinning of the background lawn or a microcolony formation. MAIN EXPERIMENTS Two experiments were performed including negative and positive controls in the absence and presence of an S9 metabolising system. Three replicate plates were used at each test point. In addition, plates were prepared to check the sterility of the test item solutions and the S9 mix and dilutions of the bacterial cultures were plated on nutrient agar plates to establish the number of bacteria in the cultures. The first experiment was performed using a plate-incorporation method. The components of the assay (the tester strain bacteria, the test item and S9 mix or phosphate buffer) were added to molten overlay agar and vortexed. The mixture was then poured onto the surface of a minimal medium agar plate and allowed to solidify prior to incubation. The second experiment was performed using a pre-incubation method. The components were added in turn to an empty test-tube. The incubate was vortexed and placed at 37°C for 30 minutes. Two mL of overlay agar was then added and the mixture vortexed again and poured onto the surface of a minimal medium agar plate and allowed to solidify. INCUBATION AND SCORING The prepared plates were inverted and incubated for approximately 72 hours at 37°C. After this period of incubation, plates were scored by counting the number of revertant colonies on each plate.

Evaluation criteria:

For the test item to be considered mutagenic, two-fold (or more) increases in mean revertant numbers must be observed at two consecutive dose levels or at the highest practicable dose level only. In addition, there must be evidence of a dose-response relationship showing increasing numbers of mutant colonies with increasing dose levels.

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

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

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TOXICITY TEST The test item FAV-ES was assayed in the toxicity test at a maximum dose level of 5000 μg/plate and at four lower concentrations spaced at approximately half-log intervals: 1580, 500, 158 and 50.0 μg/plate. Precipitation of the test item, which did not interfere with the scoring, was observed at the end of the incubation period at the highest concentration in the absence of metabolic activation and at the two highest concentrations in the presence of metabolic activation. No toxicity was observed with any tester strain at any dose level, both in the absence or presence of S9 metabolism. ASSAY FOR REVERSE MUTATION Two experiments were performed. On the basis of toxicity test results, in Main Assay I, using the plate incor- poration method, the test item was assayed at the following dose levels: 5000, 2500, 1250, 625 and 313 μg/plate. No toxicity was observed with any tester strain at any dose level both in the absence or presence of S9 metabolism. Precipitation of the test item, which did not interfere with the scoring, was observed at the end of the incubation period at the two highest concentrations, both in the absence and presence of metabolic activation. As no relevant increase in revertant numbers was observed at any concentration tested, a pre-incubation step was included for all treatments of Main Assay II. The test item was assayed at the same dose levels used in Main Assay I. No toxicity was observed with any tester strain at any dose level in the absence or presence of S9 metabolism. Precipitation of the test item, which did not interfere with the scoring, was observed at the end of the incubation period at the two highest concentrations only in the presence of S9 metabolism. However it must be noted that at the end of the pre-incubation period, in the absence of metabolic activation, test item precipitate stuck to the vials was observed at the three highest concentrations. No relevant increase in the number of revertant colonies was observed in the plate incorporation or pre-incubation assay, at any dose level, with any tester strain, in the absence or presence of S9 metabolism. The sterility of the S9 mix and of the test item solutions was confirmed by the absence of colonies on additional agar plates spread separately with these solutions. Marked increases in revertant numbers were obtained in these tests following treatment with the positive control items, indicating that the assay system was functioning correctly.

Conclusions:

Interpretation of results (migrated information):
negative

Executive summary:

The test item FAV-ES was examined for the ability to induce gene mutations in tester strains of Salmonella typhimurium and Escherichia coli, as measured by reversion of auxotrophic strains to prototrophy. The five tester strains TA1535, TA1537, TA98, TA100 and WP2 uvrA were used. Experiments were performed both in the absence and presence of metabolic activation, using liver S9 fraction from rats pre-treated with phenobarbitone and betanaphthoflavone. The test item was used as a solution in acetone.

No toxicity was observed with any tester strain at any dose level, in the absence or presence of S9 metabolism. On the basis of toxicity test results, in Main Assay I, using the plate incorporation method, the test item was assayed at the following dose levels: 5000, 2500, 1250, 625 and 313 μg/plate.

As no relevant increase in revertant numbers was observed at any concentration tested in Main Assay I, a pre-incubation step was included for all treatments of Main Assay II. The test item was assayed at the same dose levels used in Main Assay I.

The test item did not induce two-fold increases in the number of revertant colonies in the plate incorporation or pre-incubation assay, at any dose level, in any tester strain, in the absence or presence of S9 metabolism.

It is concluded that the test item FAV-ES does not induce reverse mutation in Salmonella typhimurium or Escherichia coli under the reported experimental conditions.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

Genotox data are available for the target substance and for a number of read-across substances.

The test item FAV-ES was examined for the ability to induce gene mutations in an Ames test on tester strains of Salmonella typhimurium and Escherichia coli . Five tester strains (TA1535, TA1537, TA98, TA100 and WP2 uvrA) were used, and the experiments were performed both in the absence and presence of metabolic activation using the plate incorporation method and the pre-incubation method. Tested dose levels were 5000, 2500, 1250, 625 and 313 μg for both assays.

 

FAV-ES was found to not induce reverse mutation in Salmonella typhimurium or Escherichia coli under the reported experimental conditions.

 

Furthermore, literature publications are available describing Ames tests that are performed on the following read-across substances:

 

Dibutyl adipate (OECD SIDS, 1996a) does not induce reverse mutations in Salmonella typhimurium or Escherichia coli under the conditions of the Ames test.

 

Dibutyl sebacate (Hachiya, 1994) does not induce reverse mutations in Salmonella typhimurium or Escherichia coli under the conditions of the Ames test.

 

Medium- and long-chain triacyl glycerol (MLCT) (Matulka, 2006) does not induce reverse mutation in Salmonella typhimurium or Escherichia coli under the conditions of the Ames test.

 

 

With regard to chromosome aberrations and gene mutations in mammalian cells, the following publications are available on some of the read-across substances:

 

The OECD SIDS report on dibutyl adipate describes a chromosome aberration test in Chinese Hamster lung cells. The maximum tested dose was 2.6 mg/mL, which corresponds to 10 mM. Structural chromosome aberrations were observed in the presence of metabolic activation, but not in the absence of the metabolic activation.

 

Following this positive in-vitro test, an in-vivo test was conducted. In the in-vivo micronucleus assay (Honarvar, 2002) 5 male and 5 female mice were dosed with 500, 1000 or 2000 mg/kg bw of dibutyl adipate. The test did not show a biologically relevant or statistically significant enhancement in the frequency of micronuclei at any dose level. The ratio PCE/NCE was not substantially different compared to the vehicle control group. It can therefore be concluded that dibutyl adipate does not induce chromosome aberrations in vivo. The positive in vitro test is to be considered a false-positive.

 

The genotoxic potential of short- and long-chain acyl triglycerides (SLCT) was assessed by Hayes et al. (Hayes, 1994). The researchers performed a chromosome aberration test, a HPRT assay, a hepatocyte unscheduled DNA synthesis assay and an in-vivo rat micronucleus assay.

The chromosome aberration assay was carried out on Chinese hamster ovary cells (CHO) with doses of 250.0, 500.0 and 1000 µg/mL. The tested substance was found to be not clastogenic nor cytotoxic under the conditions of the test, both in the absence and in the presence of metabolic activation.

The gene mutation HPRT assay used Chinese hamster ovary cells (CHO) with doses of 31.25, 62.5, 125, 250, 500 and 1000 µg/mL. The tested substance did not alter the relative cloning efficiency nor the mutation frequency of the cells in the absence and presence of metabolic activation.

The Unscheduled DNA Synthesis Assay used freshly obtained rat hepatocytes with doses of 0.5, 1.0, 5.0, 10.0, 25.0, 50.0, 100.0, 250.0, 500.0 and 1000 µg/mL.

The test substance did not induce Unschedules DNA Synthesis in isolated hepatocytes under the conditions of the assay.

 

Finally, the in-vivo chromosome aberration study was examined by the collection of bone marrow from rats that received a daily dose (ca. 7000 mg/kg bw) of SLCT via the diet over a period of 13 weeks. The treatment did not increase the incidence of micronucleated polychromatic erythrocytes. As a consequence, it was concluded that SLCT lacks genotoxic potential.

The results obtained for the read-across substances are in line with those found for FAV-ES, thus substantiating the read-across approach.No genotoxic effects were observed. 

Justification for selection of genetic toxicity endpoint
This is a recent study according to GLP and international guidelines.

Justification for classification or non-classification

Based on the information available on the substance itself and the read-across substances, the substance is not to be classified according to the criteria described in EU Regulation No. 12 72/2008 on the Classification, Labelling and Packaging of Substances and Mixtures (CLP) or Directive 67/548/EEC (Dangerous Substances Directive).