Phosphoric acid, C14-15-branched and linear alkyl esters, potassium salts

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:

2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

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

Test material information

Constituent 1

Reference substance name:

Phosphoric acid, C14-15 branched and linear alkyl esters, potassium salts

EC Number:

812-497-9

Cas Number:

1893414-79-3

Molecular formula:

n/a for an UVCB substance

IUPAC Name:

Phosphoric acid, C14-15 branched and linear alkyl esters, potassium salts

Constituent 2

Reference substance name:

Water

EC Number:

231-791-2

EC Name:

Water

Cas Number:

7732-18-5

Molecular formula:

H2O

IUPAC Name:

water

Test material form:

solid: crystalline

Specific details on test material used for the study:

Name: Phosphoric acid, C14-15 branched and linear alkyl esters, potassium salts
CAS No.: 1893414-79-3
Physical state: white solid at 20 °C
Batch No.: PU61810016
Re-certification date of batch: 09 March 2018
Purity: 100 % (UVCB, lyophilized solid, water content 0.85 % (w/w))
Stability: stable under test conditions
Storage condition of test material: Room temperature, protected from light

Method

Target gene:

The Salmonella typhimurium histidine (his) reversion system measures his- → his+ reversions. The S. typhimurium strains are constructed to differentiate between base pair (TA 100, TA 1535, TA 102) and frameshift (TA 98, TA 1537) mutations.

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:

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 with the following concentrations:
3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate

2500 µg/plate was selected as the maximum concentration. The concentration range covered two logarithmic decades. Two independent experiments were performed with the following concentrations:

Experiment I:
1.00, 3.16, 10.0, 31.6, 100, 316, 1000 and 2500 µg/plate
(except TA 98 and TA 100 with and without metabolic activation)

0.316, 1.00, 3.16, 10.0, 31.6, 100, 316 and 1000 µg/plate
(only TA 98 without metabolic activation)

3.16, 10.0, 31.6, 100, 316, 1000 and 2500 µg/plate
(only TA 98 with metabolic activation and TA 100 with and without metabolic activation)

Experiment II:
1.00, 3.16, 10.0, 31.6, 100, 316 and 1000 µg/plate
(TA 98 and TA 1535 with metabolic activation)

1.00, 3.16, 10.0, 31.6, 100, 316, 1000 and 2500 µg/plate
(TA 100 with metabolic activation and TA 102 with and without metabolic activation)

0.0316, 0.100, 0.316, 1.00, 3.16, 10.0, 31.6 and 100 µg/plate
(TA 98, TA 100, TA 1535 without metabolic activation and TA 1537 with and without metabolic activation)

Vehicle / solvent:

Aqua destillata

Controls

Untreated negative controls:

yes

Remarks:

Aqua destillata

Negative solvent / vehicle controls:

yes

Remarks:

Aqua destillata

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-NOPD (4-nitro-o-phenylene-diamine); 2-AA (2-aminoanthracene)

Details on test system and experimental conditions:

Negative as well as positive controls were included in each experiment. Strain specific positive controls were included in the assay, which demonstrated the effective performance of the test. Negative/solvent controls were treated in the same way as all dose groups.

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. This increases cell
permeability of larger substances. 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, TA 100 and TA 102 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 properties of the S. typhimurium strains with regard to membrane permeability, ampicillin- and tetracycline-resistance as well as normal spontaneous mutation rates are checked regularly according to Ames et al.. In this way it is ensured that the experimental conditions set up by Ames are fulfilled.

Evaluation criteria:

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 TA 102 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 than the reversion rate of the solvent control.

Statistics:

According to 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.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Background lawn reduced

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks:

4-NOPD (-S9) & 2-AA (+S9)

Test results 2

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Background lawn reduced & precipitation

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks:

NaN3 (-S9) & 2-AA (+S9)

Test results 3

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

precipitation

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks:

MMS (-S9) & 2-AA (+S9)

Test results 4

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Background lawn reduced & precipitation

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks:

NaN3 (-S9) & 2-AA (+S9)

Test results 5

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Background lawn reduced & precipitation

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks:

4-NOPD (-S9) & 2-AA (+S9)

Any other information on results incl. tables

The test item Phosphoric acid, C14-15-branched and linear alkyl esters, potassium salts was investigated for its potential to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 102.

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:

1.00, 3.16, 10.0, 31.6, 100, 316, 1000 and 2500 µg/plate

(except TA 98 and TA 100 with and without metabolic activation)

0.316, 1.00, 3.16, 10.0, 31.6, 100, 316 and 1000 µg/plate

(only TA 98 without metabolic activation)

3.16, 10.0, 31.6, 100, 316, 1000 and 2500 µg/plate

(only TA 98 with metabolic activation and TA 100 with and without metabolic activation)

Experiment II:

1.00, 3.16, 10.0, 31.6, 100, 316 and 1000 µg/plate

(TA 98 and TA 1535 with metabolic activation)

1.00, 3.16, 10.0, 31.6, 100, 316, 1000 and 2500 µg/plate

(TA 100 with metabolic activation and TA 102 with and without metabolic activation)

0.0316, 0.100, 0.316, 1.00, 3.16, 10.0, 31.6 and 100 µg/plate

(TA 98, TA 100, TA 1535 without metabolic activation and TA 1537with and without metabolic activation)

Precipitation of the test item was observed in all tester strains used in experiment I and II at a concentration of 2500 µg/plate and higher (with and without metabolic activation), if tested. Toxic effects of the test item were noted in all tester strains evaluated in experiment I and II. In experiment I toxic effects of the test item were observed in tester strain TA 98 at concentrations of 31.6 µg/plate and higher (without metabolic activation) and at concentrations of 1000 µg/plate (with metabolic activation). In tester strain TA 100 toxic effects of the test item were noted at concentrations of 100 µg/plate and higher (without metabolic activation) and at a concentration of 2500 µg/plate (with metabolic activation). In tester strains TA 1535 and TA 1537 toxic effects of the test item were seen at concentrations of 100 µg/plate and higher (with and without metabolic activation). In tester strain TA 102 toxic effects of the test item were observed at a concentration of 2500 µg/plate (without metabolic activation). In experiment II toxic effects of the test item were noted in tester strain TA 98 at concentrations of 31.6 µg/plate and higher (without metabolic activation) and at concentrations of 316 µg/plate and higher (with metabolic activation). In tester strain TA 100 toxic effects of the test item were observed at concentrations of 31.6 µg/plate and higher (without metabolic activation) and at a concentration of 2500 µg/plate (with metabolic activation). In tester strain TA 1535 toxic effects of the test item were seen at concentrations of 3.16 µg/plate and higher (without metabolic activation) and at concentrations of 316 µg/plate and higher (with metabolic activation). In tester strain TA 1537 toxic effects of the test item were observed at concentrations of 3.16 µg/plate and higher (without metabolic activation) and at a concentration of 100 µg/plate (with metabolic activation). In tester strain TA 102 toxic effects of the test item were noted at concentrations of 316 µg/plate and higher (with and without metabolic activation). The reduction in the number of revertants down to a mutation factor of ≤ 0.5 found in experiment II in tester strain TA 1537 at a concentration of 0.316 µg/plate (without metabolic activation) was regarded as not biologically relevant due to lack of a dose-response relationship. No biologically relevant increases in revertant colony numbers of any of the five tester strains were observed following treatment with Phosphoric acid, C14-15-branched and linear alkyl esters, potassium salts 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:

Phosphoric acid, C14-15-branched and linear alkyl esters, potassium salts is considered to be non-mutagenic in this bacterial reverse mutation assay.

Executive summary:

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, Phosphoric acid, C14-15-branched and linear alkyl esters, potassium salts did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used. Therefore Phosphoric acid, C14-15-branched and linear alkyl esters, potassium salts is considered to be non-mutagenic in this bacterial reverse mutation assay.