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Toxicological information

Genetic toxicity: in vitro

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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
Study period:
14-07-2009 to 31-08-2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study has been performed according to OECD and/or EC guidelines and according to GLP principles.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Fatty acids, tall-oil, reaction products with polyethylene-polyamines
- Substance type: Paste/Liquid
- Physical state: Lower phase: yellow to light brown paste; Upper phase: clear amber liquid with flakes at the bottom
- Stability under test conditions: Stable
- Storage condition of test material: At room temperature in the dark under Nitrogen


Method

Target gene:
- S. typhimurium: Histidine gene
- E. coli: Tryptophan gene
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone
Test concentrations with justification for top dose:
Experiment 1:
Preliminary test (without and with S9) TA100 and WP2uvrA: 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate
Main study: TA1535, TA1537 and TA98:
Without S9-mix: 1, 3, 10, 33, 66 and 100 µg/plate
With S9-mix: 1, 3, 10, 33, 100 and 200 µg/plate.
Experiment 2:
TA1535, TA1537, TA98, TA100 and WP2uvrA
Without S9-mix: 1, 3, 10, 33, 66 and 100 µg/plate
With S9-mix: 1, 3, 10, 33, 100 and 200 µg/plate

Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: Accepted and approved by authorities and international guidelines
Controlsopen allclose all
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
without S9 mix

Migrated to IUCLID6: 5 µg/ml in saline for TA1535
Positive control substance:
9-aminoacridine
Remarks:
without S9 mix

Migrated to IUCLID6: 60 µg/plate in water for TA1537
Positive control substance:
2-nitrofluorene
Remarks:
without S9 mix

Migrated to IUCLID6: 10 µg/plate in DMSO for TA98
Positive control substance:
methylmethanesulfonate
Remarks:
without S9 mix

Migrated to IUCLID6: 650 µg/plate in DMSO for TA100
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
without S9 mix

Migrated to IUCLID6: 10 µg/plate in DMSO for WP2uvrA
Positive control substance:
other: 2-aminoanthracene in DMSO for all tester strains
Remarks:
with S9 mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 hour

NUMBER OF REPLICATIONS:
- Doses of the test substance were tested in triplicate in each strain. Two independent experiments were conducted.

NUMBER OF CELLS EVALUATED: 10E8 per plate

DETERMINATION OF CYTOTOXICITY
- Method: The reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies

OTHER EXAMINATIONS:
- The presence of precipitation of the test compound on the plates was observed.

Evaluation criteria:
A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is not greater than three (3) times the concurrent control.
b) The negative response should be reproducible in at least one independently repeated experiment.

A test substance is considered positive if:
a) A two-fold (TA100) or more or a three-fold (TA1535, TA1537, TA98, WP2uvrA) or more increase above solvent control in the mean number of revertant colonies is observed in the test substance group.
b) The increase in the mean number of revertant colonies follows the concentration of test substance (dose-response relationship).

Results and discussion

Test resultsopen allclose all
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:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation was observed at dose levels of 3330 and 5000 µg/plate

RANGE-FINDING/SCREENING STUDIES:
- In tester strain TA100, toxicity was observed at dose levels of 100 μg/plate and above in the absence and presence of S9-mix. In tester strain WP2uvrA, toxicity was observed at dose levels of 333 μg/plate and above in the absence and presence of S9-mix.

COMPARISON WITH HISTORICAL CONTROL DATA:
- The negative and strain-specific positive control values were within our laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
TA1535: without S9: 66 µg/plate and above and with S9: 200 µg/plate and above
TA1537: without S9: 100 µg/plate and above and with S9: 200 µg/plate and above
TA98: without S9: 100 µg/plate and above and with S9: 200 µg/plate and above
TA100: without S9: 66 µg/plate and above and with S9: 100 µg/plate and above
WP2uvrA: without S9: 333 µg/plate and above and with S9: 333 µg/plate and above

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

Based on the results of this study it is concluded that Fatty acids, tall-oil, reaction products with polyethylene-polyamines is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.
Executive summary:

Evaluation of the mutagenic activity of Fatty acids, tall-oil, reaction products with polyethylenepolyamines (Amidoamine) in theSalmonella typhimuriumreverse mutation assay and theEscherichia colireverse mutation assay (with independent repeat).

 

Fatty acids, tall-oil, reaction products with polyethylene-polyamines (Amidoamine) was tested in theSalmonella typhimuriumreverse mutation assay with four histidine-requiring strains ofSalmonella typhimurium(TA1535, TA1537, TA98 and TA100) and in theEscherichia colireverse mutation assay with a tryptophan-requiring strain ofEscherichia coli(WP2uvrA). The test was performed in two independent experiments in the presence and absence of S9-mix (rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone).

 

The study procedures described in this report were based on the most recent OECD and EC guidelines. The test substance, batch S001681 was dissolved in ethanol. The dose range finding study was reported as part of the first experiment of the mutation assay. Table 1 gives an overview of the experiments conducted.

 

Table 1 Overview of experiments

Experiment

Tester strains

Metabolic activation

Concentration range (μg/plate)

Dose range finding

TA100 and WP2uvrA

-

5% (v/v) S9

Up to 5000 μg/plate

Up to 5000 μg/plate

Experiment 1

TA1535, TA1537 and TA98

-

5% (v/v) S9

Up to 100 μg/plate

Up to 200 μg/plate

Experiment 2

TA1535, TA1537, TA98, TA100 and WP2uvrA

-

10% (v/v) S9

Up to 100 μg/plate

Up to 200 μg/plate

- no S9-mix added.

 

Fatty acids, tall-oil, reaction products with polyethylene-polyamines (Amidoamine) precipitated on the plates at dose levels of 3330 and 5000μg/plate. Cytotoxicity, as evidenced by a decrease in the number of revertants and/or a reduction of the bacterial background lawn, was observed in all tester strains in the absence and presence of S9-mix. With the exception of tester strain WP2uvrA (second experiment), in which no cytotoxicity was observed. However, since toxicity was observed in the first mutation experiment, in the dose level just above the highest dose level tested in this experiment, the validity of the test was considered to be not affected by this lack of toxicity in this tester strain.

 

Fatty acids, tall-oil, reaction products with polyethylene-polyamines (Amidoamine) 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 tester strain WP2uvrA both in the absence and presence of S9-metabolic activation.

These results were confirmed in an independently repeated experiment.

 

In this study, the negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

 

Based on the results of this study it is concluded that Fatty acids, tall-oil, reaction products with polyethylene-polyamines (Amidoamine) is not mutagenic in theSalmonella typhimuriumreverse mutation assay and in theEscherichia colireverse mutation assay.