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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Based on the results of the available in vitro tests, the test substance is considered to be non-genotoxic.

Link to relevant study records

Reference

Reference 1

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

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

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

Specific details on test material used for the study:

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

Target gene:

Histidine and tryptophan

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other:

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)

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.

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

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

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).

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From September 04, 2011 to February 02, 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

Appearance: semi-solid (amorphous): gel

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: MEM
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes

Metabolic activation:

with and without

Metabolic activation system:

Liver S9 of Wistar phenobarbital and ß-naphthoflavone-induced rat liver S9 mix

Test concentrations with justification for top dose:

- Pre-experiment: with and without metabolic activation: 7.8, 15.6, 31.3, 62.5, 125, 250, 500, 1000, 2500 and 5000 µg/mL
- Experiment I: without metabolic activation: 15.6, 250, 500 and 1000 µg/mL and with metabolic activation: 15.6, 500, 1000 and 1500 µg/mL
- Experiment II: without metabolic activation: 15.6, 31.3, 62.5 and 125.0 µg/mL and with metabolic activation: 900, 1600 and 1800 µg/mL

Vehicle / solvent:

- Vehicle (s)/solvent(s) used: cell culture medium (MEM)
- Justification for choice of solvent/vehicle: The test substance was prepared in cell culture medium followed by ultrasound for around 5 minutes prior to treatment. After that the test substance was well suspended. The solvent was compatible with the survival of the cells and the S9 activity.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

- Treatment time:
4 hours (Experiment I with and without metabolic activation, experiment II with metabolic activation)
20 hours (Experiment II without metabolic activation)
- Fixation interval: 20 hours (Experiment I and II with and without metabolic activation)
- Number of replicates: 2 independent experiments
- Number of cells seeded: 1.0E04 - 5.0E04 cells
- Number of cultures: two cultures per concentration
- Number of cells scored: 200 cells per concentration (100 cells per culture) except for 15.6 µg/mL (experiment I with metabolic activation) and 31.3 µg/mL (experiment II without metabolic activation): 300 cells
- Determination of cytotoxicity: Mitotic index, cell density

Rationale for test conditions:

Pre-experiment data

Evaluation criteria:

There are several criteria for determining a positive result:
- a clear and dose-related increase in the number of cells with aberrations,
- a biologically relevant response for at least one of the dose groups, which is higher than the laboratory negative control range (0.0% - 4.0% aberrant cells (with and without metabolic activation)).

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.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

In both experiments, no biologically relevant increase of the aberration rates was noted after treatment with the test substance with and without metabolic activation. The aberration rates of all dose groups treated with the test substance were within the historical control data of the negative control. In the experiments I and II with and without metabolic activation no biologically relevant increase in the frequencies of polyploid cells was found after treatment with the test substance as compared to the controls.
Toxic effects of the test substance were observed in experiment I without metabolic activation at concentrations of 500 µg/mL and higher, with metabolic activation at concentrations of 1000 µg/mL and higher. In experiment II without metabolic activation (long time exposure) toxic effects of the test substance were observed at concentrations of 62.5 µg/mL and higher, with metabolic activation at concentrations of 1600 µg/mL and higher.
EMS (400 and 900 µg/mL) and CPA (0.83 µg/mL) were used as positive controls and induced distinct and biologically relevant increases in cells with structural chromosomal aberrations. The positive controls induced the appropriate responses. There was no evidence of test substance induced over background.
Precipitation of the test substance was observed with and without metabolic activation in both experiments.

Conclusions:

Under the study conditions, the test substance was considered to be non-clastogenic in V79 cells (in vitro cytogenicity / chromosome aberration test in mammalian cells).

Executive summary:

A study was conducted to determine the in vitro genetic toxicity of the test substance according to OECD Guideline 473, EU Method B.10 and EPA OPPTS 870.5375 (in vitro cytogenicity / chromosome aberration test in mammalian cells), in compliance with GLP. Chinese hamster lung fibroblasts (V79 cells; 1.0 - 5.0E04 cells) were exposed to the test substance at concentrations ranging from 0 to 1800 with or without metabolic activation (liver S9 of Wistar phenobarbital and ß-naphthoflavone-induced rat liver S9 mix). The chromosomes were prepared 20 h after start of treatment with the test substance. The treatment interval was 4 h with and without metabolic activation in experiment I. In experiment II, the treatment interval was 4 h with and 20 h without metabolic activation. In both experiments, no biologically relevant increase of the aberration rates was noted after treatment with the test substance with and without metabolic activation. The aberration rates of all dose groups treated with the test substance were within the historical control data of the negative control. In the experiments I and II with and without metabolic activation no biologically relevant increase in the frequencies of polyploid cells was found after treatment with the test substance as compared to the controls. Toxic effects of the test substance were observed in experiment I without metabolic activation at concentrations of 500 µg/mL and higher, with metabolic activation at concentrations of 1000 µg/mL and higher. In experiment II without metabolic activation (long time exposure) toxic effects of the test substance were observed at concentrations of 62.5 µg/mL and higher, with metabolic activation at concentrations of 1600 µg/mL and higher. EMS (400 and 900 µg/mL) and CPA (0.83 µg/mL) were used as positive controls and induced distinct and biologically relevant increases in cells with structural chromosomal aberrations. The positive controls induced the appropriate responses. There was no evidence of test substance induced over background. Precipitation of the test substance was observed with and without metabolic activation in both experiments. Under the study conditions, the test substance was considered to be non-clastogenic in V79 cells (2012).

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From September 26, 2011 to February 14, 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: mammalian cell gene mutation assay

Target gene:

Hypoxanthine-guanine-phosphoribosyl-transferase (HPRT)

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: MEM
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

Liver S9 of Wistar Phenobarbital and ß-Naphthoflavone-induced rat liver S9 mix

Test concentrations with justification for top dose:

- Pre-experiment for experiment I (without metabolic activation): 15.6, 31.3, 62.5, 125, 500, 1000 and 2500 µg/mL
- Pre-experiment for experiment I (with metabolic activation): 31.3, 62.5, 125, 250, 500, 1000, 1750 and 2500 µg/mL
- Pre-experiment for experiment II (only without metabolic activation, 20 h long-term exposure assay): 125, 250, 500, 750, 1000, 1200, 1400, 1600, 1800, 2000, 2200, 2400, 2600 and 2800 µg/mL

- Experiment I: without metabolic activation: 50, 100, 250, 500, 750, 1000, 1250, 1500 and 2000 µg/mL and with metabolic activation: 50, 100, 250, 500, 750, 1000, 1250 and 1500 µg/mL

- Experiment II: without metabolic activation: 7.5, 10, 25, 50, 75, 100, 125, 150 and 175 µg/mL and with metabolic activation: 150, 200, 300, 600, 800, 1000, 1200 and 1400 µg/mL

Vehicle / solvent:

Vehicle (Solvent) used: cell culture medium (MEM + 0% FBS 4h treatment; MEM + 10% FBS 20h treatment). The test substance was supended in cell culture medium and processed by ultrasound for 5 min.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: suspended in medium
DURATION: 4 h (short-term exposure), 20 h (long-term exposure)
Expression time (cells in growth medium): 48-72 h
Selection time (if incubation with selection agent): about one week

SELECTION AGENT ( mutation assay) 11 µg/mL 6-thioguanine (TG)
NUMBER OF REPLICATIONS: two separate experiments (I+II) with single exposure; 5 individual flasks were seeded and evaluated
NUMBER OF CELLS EVALUATED: 400000 cells per flask
DETERMINATION OF CYTOTOXICITY: Method: relative growth; cloning efficiency

Evaluation criteria:

A test is considered to be negative if there is no biologically relevant increase in the number of mutants.
There are several criteria for determining a positive result:
-a reproducible three times higher mutation frequency than the solvent control for at least one of the concentrations;
-a concentration related increase of the mutation frequency; such an evaluation may be considered also in the case that a three-fold increase of
the mutant frequency is not observed;
-if there is by chance a low spontaneous mutation rate in the corresponding negative and solvent controls a concentration related increase of the mutations within their range has to be discussed.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

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

Additional information on results:

Biologically relevant growth inhibition was observed in experiment I and II with and without metabolic activation. In experiment I without metabolic activation the relative growth was 24.6% for the highest concentration evaluated (2000 µg/mL). The highest biologically relevant concentration evaluated with metabolic activation was 1500 µg/mL with a relative growth of 23.9%. In experiment II without metabolic activation the relative growth was 15.7% for the highest concentration (175 µg/mL) evaluated. The highest concentration evaluated with metabolic activation was 1400 µg/mL with a relative growth of 20.0%.

In experiment I without metabolic activation the highest mutation rate (compared to the negative control values) of 1.00 was found at a concentration of 500 µg/mL with a relative growth of 96.5%. In experiment I with metabolic activation the highest mutation rate (compared to the negative control values) of 1.75 was found at a concentration of 1500 µg/mL with a relative growth of 23.9%. In experiment II without metabolic activation the highest mutation rate (compared to the negative control values) of 2.43 was found at a concentration of 175 µg/mL with a relative growth of 15.7%. In experiment II with metabolic activation the highest mutation rate (compared to the negative control values) of 1.21 was found at a concentration of 150 µg/mL with a relative growth of 101.3%.

The positive controls (EMS and DBA) did induce the appropriate response. There was no evidence(or)evidence of a concentration related positive response of induced mutant colonies over background.

Remarks on result:

other: tested up to cytotoxic concentrations

Conclusions:

Under the study conditions, the test substance was considered to be non-mutagenic in the HPRT locus using V79 cells of the Chinese Hamster (in vitro gene mutation test in mammalian cells).

Executive summary:

A study was conducted to determine the in vitro genetic toxicity of the test substance according to OECD Guideline , EU Method B. and EPA OPPTS 870.5300 (in vitro gene mutation test in mammalian cells), in compliance with GLP. Chinese hamster lung fibroblasts V79 cells were exposed to the test substance suspended in cell culture medium (MEM + 0% FBS 4h treatment; MEM + 10% FBS 20h treatment) at concentrations of 50, 100, 250, 500, 750, 1000, 1250, 1500 and 2000 µg/mL (without metabolic activation, Experiment I); 50, 100, 250, 500, 750, 1000, 1250 and 1500 µg/mL (with metabolic activation, Experiment I), 7.5, 10, 25, 50, 75, 100, 125, 150 and 175 µg/mL (without metabolic activation, Experiment II), and 150, 200, 300, 600, 800, 1000, 1200 and 1400 µg/mL (with metabolic activation, Experiment II). The test substance was tested up to cytotoxic concentrations. Metabolic activation consisted in liver S9 of Wistar Phenobarbital and ß-Naphthoflavone-induced rat liver S9 mix. Biologically relevant growth inhibition was observed in experiment I and II with and without metabolic activation. In experiment I without metabolic activation the relative growth was 24.6% for the highest concentration evaluated (2000 µg/mL). The highest biologically relevant concentration evaluated with metabolic activation was 1500 µg/mL with a relative growth of 23.9%. In experiment II without metabolic activation the relative growth was 15.7% for the highest concentration (175 µg/mL) evaluated. The highest concentration evaluated with metabolic activation was 1400 µg/mL with a relative growth of 20.0%.  In experiment I without metabolic activation the highest mutation rate (compared to the negative control values) of 1.00 was found at a concentration of 500 µg/mL with a relative growth of 96.5%. In both the experiments no biologically relevant increase of mutants was found after treatment with the test substance (with and without metabolic activation). No dose response was observed. EMS and DBA were used as positive controls and showed distinct and biologically relevant effects in mutation frequency. Under the study conditions, the test substance was considered to be non-mutagenic in the HPRT locus using V79 cells of the Chinese Hamster (2012).

Reference 4

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Study period:

From September 04, 2011 to February 02, 2012

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

due to RA

Justification for type of information:

Refer to the section 13 for details on the read across justification. The study with the read across substance is considered sufficient to fulfil the information requirements as further explained in the provided endpoint summary.

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: MEM
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes

Metabolic activation:

with and without

Metabolic activation system:

Liver S9 of Wistar phenobarbital and ß-naphthoflavone-induced rat liver S9 mix

Test concentrations with justification for top dose:

- Pre-experiment: with and without metabolic activation: 7.8, 15.6, 31.3, 62.5, 125, 250, 500, 1000, 2500 and 5000 µg/mL
- Experiment I: without metabolic activation: 15.6, 250, 500 and 1000 µg/mL and with metabolic activation: 15.6, 500, 1000 and 1500 µg/mL
- Experiment II: without metabolic activation: 15.6, 31.3, 62.5 and 125.0 µg/mL and with metabolic activation: 900, 1600 and 1800 µg/mL

Vehicle / solvent:

- Vehicle (s)/solvent(s) used: cell culture medium (MEM)
- Justification for choice of solvent/vehicle: The test substance was prepared in cell culture medium followed by ultrasound for around 5 minutes prior to treatment. After that the test substance was well suspended. The solvent was compatible with the survival of the cells and the S9 activity.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

- Treatment time:
4 hours (Experiment I with and without metabolic activation, experiment II with metabolic activation)
20 hours (Experiment II without metabolic activation)
- Fixation interval: 20 hours (Experiment I and II with and without metabolic activation)
- Number of replicates: 2 independent experiments
- Number of cells seeded: 1.0E04 - 5.0E04 cells
- Number of cultures: two cultures per concentration
- Number of cells scored: 200 cells per concentration (100 cells per culture) except for 15.6 µg/mL (experiment I with metabolic activation) and 31.3 µg/mL (experiment II without metabolic activation): 300 cells
- Determination of cytotoxicity: Mitotic index, cell density

Rationale for test conditions:

Pre-experiment data

Evaluation criteria:

There are several criteria for determining a positive result:
- a clear and dose-related increase in the number of cells with aberrations,
- a biologically relevant response for at least one of the dose groups, which is higher than the laboratory negative control range (0.0% - 4.0% aberrant cells (with and without metabolic activation)).

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.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

In both experiments, no biologically relevant increase of the aberration rates was noted after treatment with the test substance with and without metabolic activation. The aberration rates of all dose groups treated with the test substance were within the historical control data of the negative control. In the experiments I and II with and without metabolic activation no biologically relevant increase in the frequencies of polyploid cells was found after treatment with the test substance as compared to the controls.
Toxic effects of the test substance were observed in experiment I without metabolic activation at concentrations of 500 µg/mL and higher, with metabolic activation at concentrations of 1000 µg/mL and higher. In experiment II without metabolic activation (long time exposure) toxic effects of the test substance were observed at concentrations of 62.5 µg/mL and higher, with metabolic activation at concentrations of 1600 µg/mL and higher.
EMS (400 and 900 µg/mL) and CPA (0.83 µg/mL) were used as positive controls and induced distinct and biologically relevant increases in cells with structural chromosomal aberrations. The positive controls induced the appropriate responses. There was no evidence of test substance induced over background.
Precipitation of the test substance was observed with and without metabolic activation in both experiments.

Conclusions:

Based on the results of the read across study, the test substance was considered to be non-clastogenic in V79 cells (in vitro cytogenicity / chromosome aberration test in mammalian cells).

Executive summary:

A study was conducted to determine the in vitro genetic toxicity of the read across substance according to OECD Guideline 473, EU Method B.10 and EPA OPPTS 870.5375 (in vitro cytogenicity / chromosome aberration test in mammalian cells), in compliance with GLP. Chinese hamster lung fibroblasts (V79 cells; 1.0 - 5.0E04 cells) were exposed to the test substance at concentrations ranging from 0 to 1800 with or without metabolic activation (liver S9 of Wistar phenobarbital and ß-naphthoflavone-induced rat liver S9 mix). The chromosomes were prepared 20 h after start of treatment with the test substance. The treatment interval was 4 h with and without metabolic activation in experiment I. In experiment II, the treatment interval was 4 h with and 20 h without metabolic activation. In both experiments, no biologically relevant increase of the aberration rates was noted after treatment with the test substance with and without metabolic activation. The aberration rates of all dose groups treated with the test substance were within the historical control data of the negative control. In the experiments I and II with and without metabolic activation no biologically relevant increase in the frequencies of polyploid cells was found after treatment with the test substance as compared to the controls. Toxic effects of the test substance were observed in experiment I without metabolic activation at concentrations of 500 µg/mL and higher, with metabolic activation at concentrations of 1000 µg/mL and higher. In experiment II without metabolic activation (long time exposure) toxic effects of the test substance were observed at concentrations of 62.5 µg/mL and higher, with metabolic activation at concentrations of 1600 µg/mL and higher. EMS (400 and 900 µg/mL) and CPA (0.83 µg/mL) were used as positive controls and induced distinct and biologically relevant increases in cells with structural chromosomal aberrations. The positive controls induced the appropriate responses. There was no evidence of test substance induced over background. Precipitation of the test substance was observed with and without metabolic activation in both experiments. Based on the results of the read across study, the test substance was considered to be non-clastogenic in V79 cells (2012).

Reference 5

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

From September 26, 2011 to February 14, 2012

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

due to RA

Justification for type of information:

Refer to the section 13 for details on the read across justification. The study with the read across substance is considered sufficient to fulfil the information requirements as further explained in the provided endpoint summary.

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: mammalian cell gene mutation assay

Target gene:

Hypoxanthine-guanine-phosphoribosyl-transferase (HPRT)

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: MEM
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

Liver S9 of Wistar Phenobarbital and ß-Naphthoflavone-induced rat liver S9 mix

Test concentrations with justification for top dose:

- Pre-experiment for experiment I (without metabolic activation): 15.6, 31.3, 62.5, 125, 500, 1000 and 2500 µg/mL
- Pre-experiment for experiment I (with metabolic activation): 31.3, 62.5, 125, 250, 500, 1000, 1750 and 2500 µg/mL
- Pre-experiment for experiment II (only without metabolic activation, 20 h long-term exposure assay): 125, 250, 500, 750, 1000, 1200, 1400, 1600, 1800, 2000, 2200, 2400, 2600 and 2800 µg/mL

- Experiment I: without metabolic activation: 50, 100, 250, 500, 750, 1000, 1250, 1500 and 2000 µg/mL and with metabolic activation: 50, 100, 250, 500, 750, 1000, 1250 and 1500 µg/mL

- Experiment II: without metabolic activation: 7.5, 10, 25, 50, 75, 100, 125, 150 and 175 µg/mL and with metabolic activation: 150, 200, 300, 600, 800, 1000, 1200 and 1400 µg/mL

Vehicle / solvent:

Vehicle (Solvent) used: cell culture medium (MEM + 0% FBS 4h treatment; MEM + 10% FBS 20h treatment). The test substance was supended in cell culture medium and processed by ultrasound for 5 min.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: suspended in medium
DURATION: 4 h (short-term exposure), 20 h (long-term exposure)
Expression time (cells in growth medium): 48-72 h
Selection time (if incubation with selection agent): about one week

SELECTION AGENT ( mutation assay) 11 µg/mL 6-thioguanine (TG)
NUMBER OF REPLICATIONS: two separate experiments (I+II) with single exposure; 5 individual flasks were seeded and evaluated
NUMBER OF CELLS EVALUATED: 400000 cells per flask
DETERMINATION OF CYTOTOXICITY: Method: relative growth; cloning efficiency

Evaluation criteria:

A test is considered to be negative if there is no biologically relevant increase in the number of mutants.
There are several criteria for determining a positive result:
-a reproducible three times higher mutation frequency than the solvent control for at least one of the concentrations;
-a concentration related increase of the mutation frequency; such an evaluation may be considered also in the case that a three-fold increase of
the mutant frequency is not observed;
-if there is by chance a low spontaneous mutation rate in the corresponding negative and solvent controls a concentration related increase of the mutations within their range has to be discussed.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

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

Additional information on results:

Biologically relevant growth inhibition was observed in experiment I and II with and without metabolic activation. In experiment I without metabolic activation the relative growth was 24.6% for the highest concentration evaluated (2000 µg/mL). The highest biologically relevant concentration evaluated with metabolic activation was 1500 µg/mL with a relative growth of 23.9%. In experiment II without metabolic activation the relative growth was 15.7% for the highest concentration (175 µg/mL) evaluated. The highest concentration evaluated with metabolic activation was 1400 µg/mL with a relative growth of 20.0%.

In experiment I without metabolic activation the highest mutation rate (compared to the negative control values) of 1.00 was found at a concentration of 500 µg/mL with a relative growth of 96.5%. In experiment I with metabolic activation the highest mutation rate (compared to the negative control values) of 1.75 was found at a concentration of 1500 µg/mL with a relative growth of 23.9%. In experiment II without metabolic activation the highest mutation rate (compared to the negative control values) of 2.43 was found at a concentration of 175 µg/mL with a relative growth of 15.7%. In experiment II with metabolic activation the highest mutation rate (compared to the negative control values) of 1.21 was found at a concentration of 150 µg/mL with a relative growth of 101.3%.

The positive controls (EMS and DBA) did induce the appropriate response. There was no evidence(or)evidence of a concentration related positive response of induced mutant colonies over background.

Remarks on result:

other: tested up to cytotoxic concentrations

Conclusions:

Based on the results of the read across study, the test substance was considered to be non-mutagenic in the HPRT locus using V79 cells of the Chinese Hamster (in vitro gene mutation test in mammalian cells).

Executive summary:

An in vitro mammalian cell gene mutation assay was conducted to determine the genetic toxicity of the read across substance, mono- and di- C12 PSE, K+, according to OECD Guideline , EU Method B. and EPA OPPTS 870.5300 (in vitrogene mutation test in mammalian cells), in compliance with GLP. Chinese hamster lung fibroblasts V79 cells were exposed to the test substance suspended in cell culture medium (MEM + 0% FBS 4h treatment; MEM + 10% FBS 20h treatment) at concentrations of 50, 100, 250, 500, 750, 1000, 1250, 1500 and 2000 µg/mL (without metabolic activation, Experiment I); 50, 100, 250, 500, 750, 1000, 1250 and 1500 µg/mL (with metabolic activation, Experiment I), 7.5, 10, 25, 50, 75, 100, 125, 150 and 175 µg/mL (without metabolic activation, Experiment II), and 150, 200, 300, 600, 800, 1000, 1200 and 1400 µg/mL (with metabolic activation, Experiment II). The test substance was tested up to cytotoxic concentrations. Metabolic activation consisted in liver S9 of Wistar Phenobarbital and ß-Naphthoflavone-induced rat liver S9 mix. Biologically relevant growth inhibition was observed in experiment I and II with and without metabolic activation. In experiment I without metabolic activation, the relative growth was 24.6% for the highest concentration evaluated (2000 µg/mL). The highest biologically relevant concentration evaluated with metabolic activation was 1500 µg/mL with a relative growth of 23.9%. In experiment II without metabolic activation, the relative growth was 15.7% for the highest concentration (175 µg/mL) evaluated. The highest concentration evaluated with metabolic activation was 1400 µg/mL with a relative growth of 20.0%. In both the experiments no biologically relevant increase of mutants was found after treatment with the test substance (with and without metabolic activation). No dose response was observed. EMS and DBA were used as positive controls and showed distinct and biologically relevant effects in mutation frequency. In conclusion, based on the results of the read across study, the test substance can considered to be non-mutagenic in the HPRT locus using V79 cells of the Chinese Hamster (Wallner, 2012).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Study 1:

A bacterial reverse mutation assay was conducted to determine the in vitro genetic toxicity of the test substance, mono- and di- C12 -18 PSE, Na+, 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 typhimuriumstrains 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 substance 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 independent 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. Based on the results of the read across study, the test substance is considered to be non-mutagenic in bacteria (Verspeek-Rip, 2006).

Study 2:

An in vitro chromosomal aberration assay was conducted to determine the genetic toxicity of the read across substance, mono- and di- C12 PSE, K+, according to OECD Guideline 473, EU Method B.10 and EPA OPPTS 870.5375 (in vitrocytogenicity / chromosome aberration test in mammalian cells), in compliance with GLP. Chinese hamster lung fibroblasts (V79 cells; 1.0 - 5.0E04 cells) were exposed to the test substance at concentrations ranging from 0 to 1800 with or without metabolic activation (liver S9 of Wistar phenobarbital and ß-naphthoflavone-induced rat liver S9 mix). The chromosomes were prepared 20 h after start of treatment with the test substance. The treatment interval was 4 h with and without metabolic activation in experiment I. In experiment II, the treatment interval was 4 h with and 20 h without metabolic activation. In both experiments, no biologically relevant increase of the aberration rates was noted after treatment with the test substance with and without metabolic activation. The aberration rates of all dose groups treated with the test substance were within the historical control data of the negative control. In the experiments I and II with and without metabolic activation no biologically relevant increase in the frequencies of polyploid cells was found after treatment with the test substance as compared to the controls. Toxic effects of the test substance were observed in experiment I without metabolic activation at concentrations of 500 µg/mL and higher, with metabolic activation at concentrations of 1000 µg/mL and higher. In experiment II without metabolic activation (long time exposure), toxic effects of the test substance were observed at concentrations of 62.5 µg/mL and higher, with metabolic activation, at concentrations of 1600 µg/mL and higher. EMS (400 and 900 µg/mL) and CPA (0.83 µg/mL) were used as positive controls and induced distinct and biologically relevant increases in cells with structural chromosomal aberrations. The positive controls induced the appropriate responses. There was no evidence of test substance-induced over background. Precipitation of the test substance was observed with and without metabolic activation in both experiments. Based on the results of the read across study, the test substance is considered to be non-clastogenic in V79 cells (Oppong-Nketiah, 2012).

Study 3:

An in vitro mammalian cell gene mutation assay was conducted to determine the genetic toxicity of the read across substance, mono- and di- C12 PSE, K+, according to OECD Guideline , EU Method B. and EPA OPPTS 870.5300 (in vitrogene mutation test in mammalian cells), in compliance with GLP. Chinese hamster lung fibroblasts V79 cells were exposed to the test substance suspended in cell culture medium (MEM + 0% FBS 4h treatment; MEM + 10% FBS 20h treatment) at concentrations of 50, 100, 250, 500, 750, 1000, 1250, 1500 and 2000 µg/mL (without metabolic activation, Experiment I); 50, 100, 250, 500, 750, 1000, 1250 and 1500 µg/mL (with metabolic activation, Experiment I), 7.5, 10, 25, 50, 75, 100, 125, 150 and 175 µg/mL (without metabolic activation, Experiment II), and 150, 200, 300, 600, 800, 1000, 1200 and 1400 µg/mL (with metabolic activation, Experiment II). The test substance was tested up to cytotoxic concentrations. Metabolic activation consisted in liver S9 of Wistar Phenobarbital and ß-Naphthoflavone-induced rat liver S9 mix. Biologically relevant growth inhibition was observed in experiment I and II with and without metabolic activation. In experiment I without metabolic activation, the relative growth was 24.6% for the highest concentration evaluated (2000 µg/mL). The highest biologically relevant concentration evaluated with metabolic activation was 1500 µg/mL with a relative growth of 23.9%. In experiment II without metabolic activation, the relative growth was 15.7% for the highest concentration (175 µg/mL) evaluated. The highest concentration evaluated with metabolic activation was 1400 µg/mL with a relative growth of 20.0%. In both the experiments no biologically relevant increase of mutants was found after treatment with the test substance (with and without metabolic activation). No dose response was observed. EMS and DBA were used as positive controls and showed distinct and biologically relevant effects in mutation frequency. In conclusion, based on the results of the read across study, the test substance can considered to be non-mutagenic in the HPRT locus using V79 cells of the Chinese Hamster (Wallner, 2012).

Based on the available data from the Ames test and read across studies, the test substance can be considered to be non-genotoxic. The higher fatty alcohol content in the test substance is not expected to have a differential impact on the genotoxicity potential, as available in vitro/in vivo genotoxicity assays with C10-16 alcohol (in vitro chromosomal aberration assay) or dodecanol (in vivo micronucleus assay) as well as 1 -docosanol (in vitro mammalian cell gene mutation assay) were negative (OECD SIDS, 2006).

Justification for classification or non-classification

Based on the results of available Ames test along with other in vitro read across studies, the test substance is assessed not to meet the criteria for classification for genotoxicity according to CLP criteria (Regulation 1272/2008/EC).