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Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017

Materials and methods

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

Test material

Constituent 1
Chemical structure
Reference substance name:
Potassium isooctadecanoate
EC Number:
266-369-7
EC Name:
Potassium isooctadecanoate
Cas Number:
66469-15-6
Molecular formula:
C18H36O2.K
IUPAC Name:
potassium 16-methylheptadecanoate
Test material form:
solid: crystalline
Details on test material:
Product description: Potassium isooctadecanoate/potassium isostearate/potassium 16-methylheptadecanoate
Name: Isooctadecanoic acid, potassium salt (1:1)
CAS No.: 66469-15-6
Physical state: off white to yellowish solid at 20 °C
Batch No.: PFS-755-169
Re-certification date of batch: 12 December 2017
Purity: 100 % (mono constituent substance)
pH, 10% in DI water 8.0 - 9.0
Stability: stable under test conditions
Storage condition of test material: Room temperature, protected from light


Specific details on test material used for the study:
Product description: Potassium isooctadecanoate/potassium isostearate
Name: Isooctadecanoic acid, potassium salt
CAS No.: 66469-15-6
Physical state: off white to yellowish solid at 20 °C
Batch No.: PFS-755-169
Re-certification date of batch: 12 December 2017
Purity: 100 % (UVCB)
pH, 10% in DI water 8.0 - 9.0
Stability: stable under test conditions
Storage condition of test material: Room temperature, protected from light


Method

Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
Mammalian Microsomal Fraction S9 Mix
Test concentrations with justification for top dose:
The toxicity of the test item was determined with tester strains TA 98 and TA 100 in a pre-experiment. Eight concentrations were tested for toxicity and induction of mutations with three plates each. The experimental conditions in this pre-experiment were the same as described below for the main experiment I (plate incorporation test). Toxicity may be detected by a clearing or rather diminution of the background lawn or a reduction in the number of revertants down to a mutation factor of approximately ≤ 0.5 in relation to the solvent control.
The test item was tested in the pre-experiment at the following concentrations: 3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate

The test item concentrations to be applied in the main experiments were chosen according to the results of the pre-experiment. 5000 µg/plate was selected as the maximum concentration. The concentration range covered two logarithmic decades. Two independent experiments were performed at the following concentrations:

Experiment I:
31.6, 100, 316, 1000, 2500 and 5000 µg/plate

Experiment II:
5.0, 15.8, 50.0, 158, 500, 1580 and 5000 µg/plate

As the results of the pre-experiment were in accordance with the criteria described above, these were reported as a part of the main experiment I.
Vehicle / solvent:
Water; The test item was completely dissolved in aqua ad iniectabilia at the evaluated concentrations.
Controls
Untreated negative controls:
yes
Remarks:
Aqua dest.
Negative solvent / vehicle controls:
yes
Remarks:
ethanol
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: 4-NOPD; 4-nitro-o-phenylene-diamine: TA 98, TA 1537 (without metabolic activation) 2-AA; 2-aminoanthracene: S. typhimurium: TA 98, TA 100, TA 1535, TA 1537 and E. coli WP2 uvrA (with metabolic activation)
Details on test system and experimental conditions:
Test System

Tester strains TA 98, TA 1535 and E. coli were obtained from MOLTOX, INC., NC 28607, USA. Tester strains TA 100 and TA 1537 were obtained from Xenometrix AG, Switzerland. All Salmonella strains contain mutations in the histidine operon, thereby imposing a requirement for histidine in the growth medium. They contain the deep rough (rfa) mutation, which deletes the polysaccharide side chain of the lipopolysaccharides of the bacterial cell surface. The other mutation is a deletion of the uvrB gene coding for a protein of the DNA nucleotide excision repair system resulting in an increased sensitivity in detecting many mutagens. This deletion also includes the nitrate reductase (chl) and biotin (bio) genes (bacteria require biotin for growth). The tester strains TA 98 and TA 100 contain the R-factor plasmid, pkM101. These strains are reverted by a number of mutagens that are detected weakly or not at all with the non R-factor parent strains. pkM101 increases chemical and spontaneous mutagenesis by enhancing an error-prone DNA repair system which is normally present in these organisms. The tester strain E. coli WP2 uvrA carries the defect in one of the genes for tryptophan biosynthesis. Tryptophan-independent mutants (revertants) can arise either by a base change at the site of the original alteration or by a base change elsewhere in the chromosome so that the original defect is suppressed.

Mammalian Microsomal Fraction S9 Mix

The bacteria most commonly used in these reverse mutation assays do not possess the enzyme system which, in mammals, is known to convert promutagens into active DNA damaging metabolites. In order to overcome this major drawback an exogenous metabolic system is added in the form of mammalian microsome enzyme activation mixture.

S9 Homogenate

The S9 liver microsomal fraction was prepared at Eurofins Munich. Male Wistar rats were induced with phenobarbital (80 mg/kg bw) and β-naphthoflavone (100 mg/kg bw) for three consecutive days by oral route.
The following quality control determinations are performed:
a) Biological activity in the Salmonella typhimurium assay using 2-aminoanthracene and benzo[a]pyrene
b) Sterility Test
A stock of the supernatant containing the microsomes was frozen in aliquots of 2 and 4 mL and stored at ≤-75 °C.

The protein concentration in the S9 preparation (Lot: 280716) was 30.3 mg/mL. In Lot 190416 the protein concentration was 34.4 mg/mL and was adjusted to 30 mg/mL.

Preparation of S9 Mix
The S9 mix preparation was performed according to Ames et al.
Evaluation criteria:
Criteria of Validity

A test is considered acceptable if for each strain:
- the bacteria demonstrate their typical responses to ampicillin (TA 98, TA 100)
- the negative control plates (A. dest.) with and without S9 mix are within the following ranges (mean values of the spontaneous reversion frequency are within the historical control data range (2013 -2015).
- corresponding background growth on both negative control and test plates is observed.
- the positive controls show a distinct enhancement of revertant rates over the control plate.
- at least five different concentrations of each tester strain are analysable.

Evaluation of Mutagenicity

The Mutation Factor is calculated by dividing the mean value of the revertant counts by the mean values of the solvent control (the exact and not the rounded values are used for calculation).
A test item is considered as mutagenic if:
- a clear and dose-related increase in the number of revertants occurs and/or
- a biologically relevant positive response for at least one of the dose groups occurs in at least one tester strain with or without metabolic activation.

A biologically relevant increase is described as follows:
- if in tester strains TA 98, TA 100 and E. coli WP2 uvrA the number of reversions is at least twice as high
- if in tester strains TA 1535 and TA 1537 the number of reversions is at least three times higher as compared to the reversion rate of the solvent control.

Statistics:
According to the OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary. A test item producing neither a dose related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups is considered to be non-mutagenic in this system.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Remarks:
ethanol
Untreated negative controls validity:
valid
Remarks:
aqua dest.
Positive controls validity:
valid
Remarks:
4-NOPD (-S9) and 2-AA (+S9)
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Remarks:
ethanol
Untreated negative controls validity:
valid
Remarks:
aqua dest.
Positive controls validity:
valid
Remarks:
NaN3 (-S9) and 2-AA (+S9)
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Remarks:
ethanol
Untreated negative controls validity:
valid
Remarks:
aqua dest.
Positive controls validity:
valid
Remarks:
NaN3 (-S9) and 2-AA (+S9)
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Remarks:
ethanol
Untreated negative controls validity:
valid
Remarks:
aqua dest.
Positive controls validity:
valid
Remarks:
NaN3 (-S9) and 2-AA (+S9)
Key result
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
Remarks:
ethanol
Untreated negative controls validity:
valid
Remarks:
aqua dest.
Positive controls validity:
valid
Remarks:
MMS (-S9) and 2-AA (+S9)
Remarks on result:
other: Plate-incorporation Test

Any other information on results incl. tables

Results

The test item isooctadecanoic acid, potassium salt was investigated for its potential to induce gene mutations according to the plate incorporation test (experiment I and II) using Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and tester strain E. coli WP2 uvrA. In two independent experiments several concentrations of the test item were used. Each assay was conducted with and without metabolic activation. The concentrations, including the controls, were tested in triplicate. The following concentrations of the test item were prepared and used in the experiments:

Experiment I:

31.6, 100, 316, 1000, 2500 and 5000 µg/plate

Experiment II:

5.0, 15.8, 50.0, 158, 500, 1580 and 5000 µg/plate

Precipitation of the test item was observed in all tester strains used in experiment I at concentrations of 2500 µg/plate and higher (without metabolic activation) and at concentrations of 1000 µg/plate and higher (with metabolic activation). In experiment II precipitation of the test item was observed in all tester strains used at concentrations of 1580 µg/plate and higher (with and without metabolic activation). No toxic effects of the test item were noted in any of the five tester strains used up to the highest dose group evaluated with and without metabolic activation in experiment I and II. The reduction in

the number of revertants down to a mutation factor of ≤ 0.5 found in experiment I in tester strain TA 1537 at concentrations of 2500 and 5000 µg/plate (without metabolic activation) was regarded as not biologically relevant due to lack of concomitant clearing of the background lawn and the fact that this effect was not reproducible in experiment II. No biologically relevant increases in revertant colony numbers of any of the five tester strains were observed following treatment with isooctadecanoic acid, potassium salt at any concentration level, neither in the presence nor absence of metabolic activation in experiment I and II. All criteria of validity were met.

Applicant's summary and conclusion

Conclusions:
In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, Isooctadecanoic acid, potassium salt did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used. Therefore, isooctadecanoic acid, potassium salt is considered to be non-mutagenic in this bacterial reverse mutation assay.
Executive summary:

In an OECD 471 GLP study isooctadecanoic acid, potassium salt did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used. Therefore, Isooctadecanoic acid, potassium salt is considered to be non-mutagenic in this bacterial reverse mutation assay.

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