Salts of phosphoric acid, 2-ethylhexyl esters with C16-18 (even numbered) and C18-unsaturated-alkylamines

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:

20 June 2019 - 15 July 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

July 21, 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

31 May 2008

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Salts of phosphoric acid, 2-ethylhexyl esters with C16-18 (even numbered) and C18-unsaturated-alkylamines

EC Number:

949-859-1

Molecular formula:

not applicable

IUPAC Name:

Salts of phosphoric acid, 2-ethylhexyl esters with C16-18 (even numbered) and C18-unsaturated-alkylamines

Test material form:

liquid

Details on test material:

- Appearance: Clear yellow liquid
- Storage condition: At room temperature

Method

Target gene:

- S. typhimurium: Histidine gene
- E. coli: Tryptophan gene

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : Rat liver microsomal enzymes (S9 homogenate) were obtained from Trinova Biochem GmbH, Giessen, Germany and were prepared from male Sprague Dawley rats that had been injected intraperitoneally with Aroclor 1254 (500 mg/kg body weight).
- method of preparation of S9 mix: S9-mix was prepared immediately before use and kept refrigerated. S9-mix contained per 10 mL: 30 mg NADP (Randox Laboratories Ltd., Crumlin, United Kingdom) and 15.2 mg
glucose-6-phosphate (Roche Diagnostics, Mannheim, Germany) in 5.5 mL Milli-Q water (Millipore Corp., Bedford, MA., USA); 2 mL 0.5 M sodium phosphate buffer pH 7.4; 1 mL 0.08 M MgCl2 solution (Merck); 1 mL 0.33 M KCl solution (Merck). The above solution was filter (0.22 µm)-sterilized. To 9.5 mL of S9-mix components 0.5 mL S9-fraction was added (5% (v/v) S9-fraction) to complete the S9-mix.
- quality controls of S9: Each S9 batch was characterized with the mutagens benzo-(a)-pyrene (Sigma) and 2-aminoanthracene, which require metabolic activation, in tester strain TA100 at concentrations of 5 µg/plate and 2.5 µg/plate, respectively.

Test concentrations with justification for top dose:

Direct plate assay:
Experiment 1
Preliminary test (without and with S9) TA100 and WP2uvrA: 0.18, 0.55, 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate
Main study:
TA1535, without S9-mix: 0.55, 1.7, 5.4, 17, 52 and 164 μg/plate
TA1537 and TA98, without S9-mix: 0.056, 0.18, 0.55, 1.7, 5.4, 17, 52 and 164 μg/plate
TA1535, TA1537 and TA98, with S9-mix: 1.7, 5.4, 17, 52, 164 and 512 μg/plate

Pre-incubation assay:
Experiment 1
Preliminary test, without S9, TA100 and WP2uvrA: 0.056, 0.18, 0.55, 1.7, 5.4, 17, 52 and 164 μg/plate
Preliminary test, with S9, TA100: 0.18, 0.55, 1.7, 5.4, 17, 52, 164 and 512 μg/plate
Preliminary test, with S9, WP2uvrA: 0.55, 1.7, 5.4, 17, 52, 164, 512 and 1600 μg/plate
Main study:
TA1535, TA1537 and TA98, without S9-mix: 0.056, 0.18, 0.55, 1.7, 5.4 and 17 μg/plate
TA1535, TA1537 and TA98, with S9-mix: 0.55, 1.7, 5.4, 17, 52 and 164 μg/plate

Vehicle / solvent:

- Vehicle used: ethanol
- Justification for choice of vehicle: A solubility test was performed based on visual assessment. The test item formed a clear (light yellow to colourless) solution in ethanol.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

methylmethanesulfonate

other:

Remarks:

Without S9

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene (2AA) in DMSO, 1 μg for TA98 and TA100 (Direct plate assay), 2.5 μg for TA1535 and TA1537, 5 μg for TA100 (Pre-incubation assay), 15 μg for WP2uvrA

Remarks:

With S9

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Doses of the test substance were tested in triplicate in each strain. Two independent experiments were conducted.

METHOD OF TREATMENT/ EXPOSURE:
in agar (plate incorporation); preincubation

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period: 30 minutes
- Exposure duration: 48 hours

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: The reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies.

METHODS FOR MEASUREMENTS OF GENOTOXICIY
- Amount of revertant colonies.

OTHER:
- The presence of precipitation of the test compound on the plates was determined.

Evaluation criteria:

A test item is 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 item is 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:

Not performed.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation and time of the determination: Precipitation of the substance on the plates was not observed at the end of the incubation period in any tester strain.

RANGE-FINDING/SCREENING STUDIES (if applicable): 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 both TA100 and WP2uvrA tester strains in the absence and presence of S9-mix. Since the test item was cytotoxic in the first mutation experiment, an additional dose range finding test was performed with the tester strains TA100 and WP2uvrA, both with and without S9-mix according to the pre-incubation method. In this dose range finding study, the test item was tested up to concentrations of 164 μg/plate in the absence of S9-mix in both tester strains and up to 512 and 1600 μg/plate in tester strains TA100 and WP2uvrA, respectively, in the presence of S9-mix. The substance did not precipitate on the plates at any dose level tested. 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 both tester strains in the absence and presence of S9-mix.

STUDY RESULTS
- Concurrent vehicle negative and positive control data : 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.

In the first mutation experiment, the test item was tested up to concentrations of 164 and 512 μg/plate in the absence and presence of S9-mix, respectively, in the strains TA1535, TA1537 and TA98. The substance did not precipitate on the plates at this dose level. 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 three tester strains in the absence and presence of S9-mix.
In the second mutation experiment, the test item was tested up to concentrations of 17 and 164 μg/plate in the absence and presence of S9-mix, respectively, in the tester strains TA1535, TA1537 and TA98 in the pre-incubation assay. The substance did not precipitate on the plates at any dose level tested. Cytotoxicity, as evidenced by a reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in all three tester strains in the absence and presence of S9-mix.
The test item 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.

HISTORICAL CONTROL DATA
- Positive historical control data:
TA1535 TA1537 TA98
S9-mix - + - + - +
Range 128 – 1530 73 – 1481 58 – 1422 54 – 1239 365 – 1978 250 – 2018
Mean 919 256 802 328 1305 910
SD 172 122 362 154 236 355
n 3215 3122 2777 3187 3202 3216

TA100 WP2uvrA
S9-mix - + - +
Range 439 – 1993 408 - 2379 93 – 1999 109 - 1968
Mean 907 1308 1073 437
SD 167 386 537 158
n 3231 3179 2923 2987

SD = Standard deviation
n = Number of observations
Historical control data from experiments performed between Apr 2016 and Apr 2019.

- Negative (solvent/vehicle) historical control data:
TA1535 TA1537 TA98 TA100 WP2uvrA
S9-mix - + - + - + - + - +
Range 3 – 29 3 – 27 3 – 20 3 – 23 8 - 61 8 – 60 61 – 176 60 - 176 10 – 61 9 - 68
Mean 10 11 6 6 16 22 112 108 27 33
SD 3 3 2 3 5 7 18 21 8 9
n 3303 3265 3232 3212 3251 3326 3336 3246 3021 2993

SD = Standard deviation
n = Number of observations
Historical control data from experiments performed between Apr 2016 and Apr 2019.

Applicant's summary and conclusion

Conclusions:

In an Ames test, performed according to OECD 471 guideline and GLP principles, the substance was found not to be mutagenic with or without metabolic activation.

Executive summary:

An Ames test was performed according to OECD 471 guideline and GLP principles.

In the dose-range finding study, the test item was initially tested up to concentrations of 5000 μg/plate in the strains TA100 and WP2uvrA in the direct plate assay. The substance did not precipitate on the plates at this dose level. 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 both tester strains in the absence and presence of S9-mix. Results of this dose-range finding test were reported as part of the first mutation assay.

In the first mutation experiment, the test item was tested up to concentrations of 164 and 512 μg/plate in the absence and presence of S9-mix, respectively, in the strains TA1535, TA1537 and TA98. The substance did not precipitate on the plates at this dose level. 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 three tester strains in the absence and presence of S9-mix.

Since the test item was cytotoxic in the first mutation experiment, an additional dose range finding test was performed with the tester strains TA100 and WP2uvrA, both with and without S9-mix according to the pre-incubation method. In this dose range finding study, the test item was tested up to concentrations of 164 μg/plate in the absence of S9-mix in both tester strains and up to 512 and 1600 μg/plate in tester strains TA100 and WP2uvrA, respectively, in the presence of S9-mix. The substance did not precipitate on the plates at any dose level tested.

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 both tester strains in the absence and presence of S9-mix.

In the second mutation experiment, the test item was tested up to concentrations of 17 and 164 μg/plate in the absence and presence of S9-mix, respectively, in the tester strains TA1535, TA1537 and TA98 in the pre-incubation assay. The substance did not precipitate on the plates at any dose level tested. Cytotoxicity, as evidenced by a reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in all three tester strains in the absence and presence of S9-mix.

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.

The test item 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 a follow-up experiment.

In conclusion, based on the results of this study it is concluded that the substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.