Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018-01-15 to 2018-02-22
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted July 21, 1997.
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
May 30, 2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid

Method

Target gene:
Histidine locus
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
microsomal preparation derived from Aroclor 1254-induced rat liver
Test concentrations with justification for top dose:
Concentrations of 10.0, 31.6, 100, 316, 1000 and 3160 μg test substance per plate were chosen for the main experiments based on priliminary cytotoxicity tests.
Vehicle / solvent:
The test substance was dispersed in highly purified water to stable suspensions for all tested concentrations. The test item was not soluble in dimethyl sulfoxide (DMSO) or ethanol.
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
no
Remarks:
not applicable water
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
benzo(a)pyrene
mitomycin C
other: 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: 1st experiment: plate incorporation method
2nd experiment: preincubation method

- Cell density at seeding: Overnight culture final cell density was approximately 10E8 - 10E9 cells/mL.

DURATION
- Preincubation period: 20 min
- Exposure duration: 48 hours

SELECTION AGENT (mutation assays): Histidine

NUMBER OF REPLICATIONS: duplicate

DETERMINATION OF CYTOTOXICITY
Cytotoxicity is defined as reduction in the number of colonies by more than 50% compared to the solvent control and/or a scarce background lawn.
Evaluation criteria:
A test item is considered to show a positive response if
- the number of revertants is significantly increased (p ≤ 0.05, U-test according to MANN and WHITNEY, compared to the solvent control to at least 2-fold of the solvent control for TA98, TA100, TA1535 and TA1537 and 1.5-fold of the solvent control for TA102 in both independent experiments.
- a concentration-related increase over the range tested in the number of the revertants per plate is observed. The Spearman's rank correlation coefficient may be applied.
- Biological relevance of the results should be considered first.

Positive results from the bacterial reverse mutation test indicate that a substance induces point mutations by base substitutions or frameshifts in the genome of Salmonella typhimurium.
A test item for which the results do not meet the above mentioned criteria is considered as non-mutagenic in the AMES test.
Statistics:
No statistical analysis was performed, as all fold changes were below the evaluation criteria of mutagenicity.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
not applicable
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
not applicable
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
not applicable
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
not applicable
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
not applicable
Untreated negative controls validity:
valid
Positive controls validity:
valid

Applicant's summary and conclusion

Conclusions:
Under the present test conditions, Octadecanoic acid, reaction products with acetic acid and tetraethylenepentamine did not cause a mutagenic effect in the Salmonella typhimurium strins TA97, TA100, TA102, TA1535 and TA1537 neither in the presence nor absence of mammaliean metabolic activation when tested up to a cytotoxic and precipitation concentration of 3160 µg/plate.
Executive summary:

In a reverse gene mutation assay in bacteria according to OECD guideline 471, strains TA 98, TA 100, TA 102, TA 1535 and TA 1537 of S. typhimurium were exposed to Octadecanoic acid, reaction products with acetic acid and tetraethylenepentamine, at concentrations of 10.0, 31.6, 100, 316, 1000 and 3160µg/plate without metabolic activation. The first experiment was conducted as plate incorporation assay, the second as pre-incubation test, both in the absence and presence of a mammalian metabolic activation.

Precipitation of the test item was observed at 1000 µg/plate and higher in all experiments.

Pronounced cytotoxicity, evaluated by reduction in the number of revertants by more than 50%, was noted in the plate incorporation test and in the pre-incubation tests, each carried out without and with metabolic activation at the top concentration of 3160 μg /plate in the following test strains:

 

Experiments without metabolic activation: TA98, TA1535 and TA1537;

Plate incorporation test with metabolic activation: all test strains;

Preincubation test with metabolic activation: TA98, TA102 and TA1537

No biological relevant increases in revertant colony numbers of any of the five tester strains were observed following treatment with Octadecanoic acid, reaction products with acetic acid and tetraethylenepentamine at any concentration level, neither in the presence nor absence of metabolic activation in experiment I and II.

The reference mutagen induced a distinct increase of revertant colonies indicating the validity of the experiments.