Phosphoric acid, mono- and di-C12-18-(even numbered)-alkyl esters, sodium 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:

From July 25, 2016 to August 31, 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

in the study plan but study integrity was not adversely affected by the deviation

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

in the study plan but study integrity was not adversely affected by the deviation

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Phosphoric acid, mono- and di-C 12 -18-(even numbered)-alkyl esters, sodium salts

Molecular formula:

C12H25O4P1Na2 (representative mono ester - mono- C12 PSE, Na+); C24H50O4P1Na1 (representative di ester - di- C12 PSE, Na+);

IUPAC Name:

Phosphoric acid, mono- and di-C 12 -18-(even numbered)-alkyl esters, sodium salts

Constituent 2

Reference substance name:

Alcohols, coco

EC Number:

270-351-4

EC Name:

Alcohols, coco

Cas Number:

68425-37-6

Molecular formula:

Not applicable

Constituent 3

Reference substance name:

Sodium dihydrogenorthophosphate

EC Number:

231-449-2

EC Name:

Sodium dihydrogenorthophosphate

Cas Number:

7558-80-7

Molecular formula:

H3O4P.Na

IUPAC Name:

Sodium phosphate

Constituent 4

Reference substance name:

Water

EC Number:

231-791-2

EC Name:

Water

Cas Number:

7732-18-5

Molecular formula:

H2O

IUPAC Name:

Dihydrogen oxide

Test material form:

other: solid

Details on test material:

- Name of test material (as cited in study report): Phosphoric acid, mono- and di-C 12 -18-(even numbered)-alkyl esters, sodium salts
- Batch number: 2011-104-06-01
- ZS internal substance number: 0038
- ZS name: PHOSPHORSÄUREPARTIALESTER, NA-SALZ C12-18
- Appearance: White solid

Specific details on test material used for the study:

Batch no.: SEALS 2011-104-06-01
Purity/Composition: 96.75%
Appearance: white solid

Method

Target gene:

Histidine and tryptophan

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Additional strain / cell type characteristics:

other:

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

other:

Cytokinesis block (if used):

rfa : deep rough (defective lipopolysaccharide cellcoat); gal : mutation in the galactose metabolism; chl : mutation in nitrate reductase; bio : defective biotin synthesis; uvrB : loss of the excision repair system (deletion of the ultraviolet-repair B gene)

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9-mix induced Aroclor 1254

Test concentrations with justification for top dose:

0.55, 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate were tested in triplicate (based on range finding dose study and cytotoxicity).
The highest concentration of the test susbtance used in the mutation assays was 5000 μg/plate or the level at which the test substance inhibited bacterial growth.

Vehicle / solvent:

Ethanol for the test substance (and DMSO or saline for positive controls)

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

ethanol, DMSO or saline

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: ICR-191 (without metabolic activation) and 2-aminoanthracene (with metabolic activation)

Details on test system and experimental conditions:

- Following dose range findings studies, the test was performed in two independent experiments, at first a direct plate assay was performed and secondly a pre-incubation assay both in the absence and presence of S9-mix.
- Exposure: 48 ± 4h (+ a pre-incubation of 30 min if needed)

Rationale for test conditions:

- Based on the most recent OECD and EC guidelines.
- Dose range finding studies
- First mutation experiment

Evaluation criteria:

In addition to the criteria stated below, any increase in the total number of revertants should be evaluated for its biological relevance including a comparison of the results with the historical control data range.
- A test substance was considered negative (not mutagenic) in the test if: a) The total number of revertants in tester strain TA100 or WP2uvrA is not greater than two times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three times the concurrent control. b) The negative response should be reproducible in at least one follow up experiment.
- A test substance was considered positive (mutagenic) in the test if: a) The total number of revertants in tester strain TA100 or WP2uvrA is greater than two times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537 or TA98 is greater than three times the concurrent control. b) In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow up experiment.

Statistics:

No formal hypothesis testing was done.
- Revertant colonies (histidine independent (His+) for Salmonella typhimurium bacteria and tryptophan independent (Trp+) for Escherichia coli) were counted.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

other: Salmonella typhimurium TA 100, TA 1535, TA 1537, TA 98 and Escherichia coli WP2uvrA

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

in Salmonella typhimurium TA 100, TA 1535, TA 1537, TA 98

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

other: Salmonella typhimurium TA 100, TA 1535, TA 1537, TA 98 and Escherichia coli WP2uvrA

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Salmonella typhimurium TA 100, TA 1535, TA 1537, TA 98

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

- In the dose range finding study, the test substance was initially tested up to concentrations of 5000 µg/plate in the tester strains TA100 and WP2uvrA in the direct plate assay. The test substance precipitated on the plates at the dose level of 5000 μg/plate. Cytotoxicity, as evidenced by a decrease in the number of revertants was observed in tester strain TA100 in the absence and presence of S9-mix. In tester strain WP2uvrA, no toxicity was observed at any of the dose levels tested.
- In the first mutation experiment, the test substance was tested up to concentrations of 1600 and 5000 µg/plate (absence and presence of S9-mix, respectively) in the tester strains TA1535, TA1537 and TA98. The test substance precipitated on the plates at the dose level of 5000 μg/plate. Cytotoxicity, as evidenced by a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in all tester strains in the absence and presence of S9-mix.
- Since the test substance was severely cytotoxic in the first mutation experiment, an additional dose range finding test was performed with strains TA100 and WP2uvrA, both with and without S9-mix according to the pre-incubation method. In this dose range finding study, the test susbstance was initially tested up to concentrations of 512 and 5000 µg/plate in the tester strains TA100 and WP2uvrA, respectively. The test substance precipitated on the plates at dose levels of 1600 and 5000 μg/plate. Since the test substance precipitated heavily on the plates at the concentration of 5000 μg/plate, the number of revertants of this dose level could not be determined. Cytotoxicity was observed in tester strain TA100 in the absence and presence of S9-mix. In tester strain WP2uvrA, no toxicity was observed up to the dose level of 1600 μg/plate. Results of this dose range finding test were reported as part of the second mutation assay.
- In the second mutation experiment, the test substance was tested up to concentrations of 164 and 512 µg/plate (absence and presence of S9-mix, respectively) in the tester strains TA1535, TA1537 and TA98 in the pre-incubation assay. The test substance did not precipitate on the plates at this dose level. Cytotoxicity was observed in all three tester strains in the absence and presence of S9-mix. (Due to precipitate, in the second dose range finding test, no revertant colonies could be determined at the highest dose level tested in strain WP2uvrA, the test substance was tested up to a dose level which showed precipitation on the plates. Therefore, the validity of the test was not considered to be affected.)
- The test substance did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in an independently repeated experiment.
- The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Remarks on result:

other: First experiment: direct plate assay

Applicant's summary and conclusion

Conclusions:

Under the study conditions, the test substance was not mutagenic in the Salmonella typhimurium and Escherichia coli reverse mutation assay.

Executive summary:

A study was conducted to determine the in vitro genetic toxicity of the test substance according to OECD Guideline 471 and EU Method B.13/14 (mutagenicity - reverse mutation test using bacteria), in compliance with GLP. Dose range finding tests as well as direct plate and pre-incubation assays both in the absence and presence of S9-mix were performed. Salmonella typhimurium strains TA1535, TA1537, TA100 and TA98 and Escherichia coli strain WP2uvrA were exposed to the test substance at concentration levels of 0.55, 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate, to negative or positive control substances for 48 ± 4h (plus a pre-incubation of 30 min if needed). In the dose range finding study, the test substance was initially tested up to concentrations of 5000 µg/plate in the tester strains TA100 and WP2uvrA in the direct plate assay. The test substance precipitated on the plates at the dose level of 5000 μg/plate. Cytotoxicity, as evidenced by a decrease in the number of revertants was observed in tester strain TA100 in the absence and presence of S9-mix. In tester strain WP2uvrA, no toxicity was observed at any of the dose levels tested. In the first mutation experiment, the test substance was tested up to concentrations of 1600 and 5000 µg/plate (absence and presence of S9-mix, respectively) in the tester strains TA1535, TA1537 and TA98. The test substance precipitated on the plates at the dose level of 5000 μg/plate. Cytotoxicity, as evidenced by a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in all tester strains in the absence and presence of S9-mix. Since the test substance was severely cytotoxic in the first mutation experiment, an additional dose range finding test was performed with strains TA100 and WP2uvrA, both with and without S9-mix according to the pre-incubation method. In this dose range finding study, the test susbstance was initially tested up to concentrations of 512 and 5000 µg/plate in the tester strains TA100 and WP2uvrA, respectively. The test substance precipitated on the plates at dose levels of 1600 and 5000 μg/plate. Since the test substance precipitated heavily on the plates at the concentration of 5000 μg/plate, the number of revertants of this dose level could not be determined. Cytotoxicity was observed in tester strain TA100 in the absence and presence of S9-mix. In tester strain WP2uvrA, no toxicity was observed up to the dose level of 1600 μg/plate. In the second mutation experiment, the test substance was tested up to concentrations of 164 and 512 µg/plate (absence and presence of S9-mix, respectively) in the tester strains TA1535, TA1537 and TA98 in the pre-incubation assay. The test substance did not precipitate on the plates at this dose level. Cytotoxicity was observed in all three tester strains in the absence and presence of S9-mix. The test substance did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in an independently repeated experiment. The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly. Under the study conditions, the test substance was not mutagenic in bacteria (Verspeek-Rip, 2006).