Registration Dossier

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 guidelineopen allclose all
Qualifier:
according to
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
Qualifier:
according to
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
Qualifier:
according to
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
Qualifier:
according to
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

Reference
Name:
Unnamed
Type:
Constituent
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.