[No public or meaningful name is available]

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1998-06-26 - 1998-10-13

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1998

Report date:

1998

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

OECD Guidelines for Testing of Chemicals. (1997) No. 471: Genetic Toxicology: Bacterial Reverse Mutation Test

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

EEC Annex to Directive 92/69/EEC. (1992) Part B : Methods for Determination of Toxicity, B.13. Other effects - Mutagenicity: Escherichia coli - Reverse Mutation Assay. O.J. No. L 383 A, 157

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

EEC Annex to Directive 92/69/EEC. (1992) Part B : Methods for Determination of Toxicity, B. 14. Other effects - Mutagenicity: Salmonella typhi murium - Reverse Mutation Assay. O.J. No. L 383 A, 160.

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Version / remarks:

US EPA Office of Prevention, Pesticides and Toxic Substances (1998) Health Effects Test Guidelines No. OPPTS 870.5100: "Bacterial Reverse Mutation Test".

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Fatty acids, C16-18 (even numbered), reaction products with tetraethylenepentamine

Molecular formula:

not applicable (UVCB substance)

IUPAC Name:

Fatty acids, C16-18 (even numbered), reaction products with tetraethylenepentamine

Details on test material:

- Substance type: pure substance
- Storage condition of test material: room temperature, dry
- Appearance: Pale yellow granules

Method

Target gene:

his (S. typhimurium strains) / trp (E. coli strain)

Species / strain

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: see "any other information on materials and methods"

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced rat liver S9, 10% in first, 20% in second test)

Test concentrations with justification for top dose:

0, 5, 15, 50, 150, 500, 1500, 5000 µg/plate (± S9, Test 1)
0, 50, 150, 500, 1500, 5000 µg/plate (± S9, Test 2)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol (Fisher, analytical reagent grade, lot No. 9629150386, purity 99.8%)
- Justification for choice of solvent/vehicle: solubility

Controls

Untreated negative controls:

yes

Remarks:

solvent control

Negative solvent / vehicle controls:

yes

Remarks:

0.1 ml ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

other: 2-Aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: none
- Exposure duration: 72 h

SELECTION AGENT (mutation assays): histidine- / tryptophan-free media

NUMBER OF REPLICATIONS: 2 independent tests, each 3 replicates per concentration

DETERMINATION OF CYTOTOXICITY
- Method: relative growth

Evaluation criteria:

For a test to be considered valid the mean of the solvent control revertant colony numbers for each strain should lie in the range stated in the appropriate Standard Operating Procedure. Also, the positive control compounds must cause at least a doubling of mean revertant colony numbers over the negative control.
The mean number of revertant colonies for all treatment groups were compared with those obtained for the solvent control groups. The mutagenic activity of a test substance was assessed by applying the following criteria:
a) if treatment with a test substance produces an increase in revertant colony numbers of at least twice the concurrent solvent controls, with some evidence of a positive dose-relationship, in two separate experiments, with any bacterial strain either in the presence or absence of S9 mix, it is considered to show evidence of mutagenic activity in this test system. No statistical analysis is performed.
b) If treatment with a test substance does not produce reproducible increases of at least 1.5 times the concurrent solvent controls in either mutation test it is considered to show no evidence of mutagenic activity in this test system. No statistical analysis is performed.
c) If the results obtained fail to satisfy the criteria for a clear "positive" or "negative" response given in paragraphs a) and b), additional testing may be performed in order to resolve the issue of the test substance's mutagenic activity in this test system. Should an increase in revertant colony numbers then be observed which satisfies paragraph (a) the substance is considered to show evidence of mutagenic activity in this test system. No statistical analysis is performed.

Statistics:

If no clear "positive" response can be obtained, the test data may be subjected to analysis to determine the statistical significance of any observed increases in revertant colony numbers. The statistical procedures used will be those described by Mahon el al (1989) and will usually be analysis of variance followed by Dunnett's test.

Results and discussion

Test results

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:

a reduction in revertant colony numbers occurred in all plates with the test item at 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: none stated

COMPARISON WITH HISTORICAL CONTROL DATA:
The mean revertant colony counts for the solvent controls were within the ranges stated in the appropriate Standard Operating Procedure or quoted by Gatehouse et al (1990) and within historical control data.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

See attachment due to limitations of the entry field

Applicant's summary and conclusion

Conclusions:

The study was performed according to the OECD TG 471 and EU Method B.13/14 (GLP) without deviations and therefore considered to be of the highest quality (reliability Klimisch 1). The validity criteria of the test system are fulfilled. In both experiments no significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation. The test material was therefore considered to be non-mutagenic under the conditions of this test.

Executive summary:

In this in vitro assessment of the mutagenic potential according to OECD TG 471of Octadecanoic acid, reaction products with tetraethylenepentamine, histidine dependent auxotrophic mutants of Salmonella typhimurium, strains TA1535, TA1537, TA98 and TA100, and a tryptophan dependent mutant of Escherichia coli strain CM891 (WP2uvrA/pKM101), were exposed to the test substance diluted in ethanol, which was also used as a negative control.

Two independent mutation tests were performed in the presence and absence of liver preparations from Aroclor 1254-induced rats (S9 mix). Both were standard plate incorporation assays, but in the second, the proportion of S9 fraction in the S9 mix was increased from 10% to 20%.

Concentrations of up to 5000 µg/plate were tested in the mutation tests. This is the standard limit concentration recommended in the regulatory guidelines this assay follows. Other concentrations used were a series of ca half-log10 dilutions of the highest concentration.

Inhibition of bacterial growth, observed as a reduction in revertant colony numbers, occurred in all strains following exposure to the test item at 5000 µg/plate.

No evidence of mutagenic activity was seen at any concentration of the test item in either mutation test.

The concurrent positive controls demonstrated the sensitivity of the assay and the metabolising activity of the liver preparations.

It is concluded that, when tested in ethanol, the test item shows no evidence of mutagenic activity in this bacterial system.