Phosphoric acid, C4 and C12-14(even-numbered) Alkyl Esters, Ammonium 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:

24th May - 21 October, 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

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

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, mono(C8-12 (even-numbered) linear alkyl) ester

Molecular formula:

Not applicable (UVCB)

IUPAC Name:

Phosphoric acid, mono(C8-12 (even-numbered) linear alkyl) ester

Constituent 2

Reference substance name:

Phosphoric acid, di(C8-12 (even-numbered) linear alkyl) ester

Molecular formula:

Not applicable (UVCB)

IUPAC Name:

Phosphoric acid, di(C8-12 (even-numbered) linear alkyl) ester

Specific details on test material used for the study:

Source and lot/batch No.of test material: 7703200
- Expiration date of the lot/batch: 2019-02-28

Method

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:

phenobarbital/β-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

Based on the results of a Preliminary Concentration Range Finding Test, the test item concentrations
in the Initial Mutation Test (5 strains) were, 1581, 500, 158.1, 50, 15.81, 5, 1.581 and 0.5 μg/plate,
the test item concentrations in the Confirmatory Mutation Test (5 strains) in all Salmonella typhimuriu
m strains were 1581, 500, 158.1, 50, 15.81, 5, 1.581, 0.5 and 0.1581 μg/plate and in Escherichia coli
WP2 uvrA strain were 5000, 1581, 500, 158.1, 50, 15.81, 5 and 1.581 μg test item/plate, the test item
concentrations in the Complementary Initial Mutation Test in Escherichia coli WP2 strain were 5000,
1581, 500, 158.1, 50, 15.81 and 5 μg test item/plate. Examined concentrations in the Complementary
Confirmatory Mutation Test in Salmonella typhimurium TA100 strain without metabolic activation
were 158.1, 50, 15.81, 5, 1.581, 0.5 and 0.1581 μg test item/plate.

Vehicle / solvent:

Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solubility of the test item was examined using distilled
water, dimethyl sulfoxide (DMSO), N,N-dimethylformamide (DMF) and acetone. At 100 mg/mL con
centration opalescence, dense emulsion was observed using distilled water as vehicle and clear solu
tion was observed using DMSO, DMF and acetone as vehicles.

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

untreated

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

methylmethanesulfonate

other: 4-nitro-1,2-phenylenediamine

Remarks:

-S9

Controls 2

Untreated negative controls:

yes

Remarks:

untreated

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene;

Remarks:

+S9

Controls 3

Untreated negative controls:

yes

Remarks:

untreated

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-amino anthracene;

Remarks:

+S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: Initial Mutation Test - Plate Incorporation Method; Mutation Test - Pre-Incubation Method; Confirmatory Mutation Test - Pre-Incubation Method; Complementary Initial Mutation Test - Plate Incorporation Method; Complementary Confirmatory Mutation Test - Pre-Incubation Method. Procedure for Exposure in the Initial Mutation Test and in the Complementary Initial Mutation Test:
A standard plate incorporation procedure was performed as an Initial Mutation Test. The same meth
od was used in the Complementary Initial Mutation Test. Bacteria (cultured in Nutrient Broth No.2 as described in Section 5.3.6.) were exposed to the test item both in the presence and absence of an
appropriate metabolic activation system.
Molten top agar was prepared and kept at 45°C. The equivalent number of minimal glucose agar plates (three plates per concentration and for each control) was properly labelled. The test item and
other components were prepared freshly and added to the overlay (45°C). The content of the tubes:
top agar: 2000 μL
vehicle (solvent) or test item solution (or reference controls): 50 μL
overnight culture of test strain: 100 μL phosphate buffer (pH 7.4) or S9 mix: 500 μL
This solution was mixed and poured on the surface of minimal agar plates. For activation studies, inst
ead of phosphate buffer, 0.5 mL of the S9 mix was added to each overlay tube. The entire test consi
sted of non-activated and activated test conditions, with the addition of untreated, negative (solvent)
and positive controls. After preparation, the plates were incubated at 37°C for 48±1 hours.
Procedure for Exposure in the Confirmatory Mutation Test and Complementary Confirmatory Mutation
Test:
A pre-incubation procedure was performed as a Confirmatory Mutation Test since in the Initial Mu
tation Test no positive effect was observed. The same method was used in the Complementary
Confirmatory Mutation Test.
For the pre-incubation method, bacteria (cultured in Nutrient Broth No.2. as described in 5.3.6.) were
exposed to the test item both in the presence and absence of an appropriate metabolic activation
system. The equivalent number of minimal glucose agar plates was properly labelled. Molten top
agar was prepared and kept at 45°C.
Before the overlaying, 50 μL of test item formulations or its vehicle (or positive reference controls or
their solvent), 100 μL of the overnight culture of bacterial cells and the 0.5 mL of S9 mix (activated
test conditions) or phosphate buffer pH 7.4 (nonactivated test conditions) were added into the appropriate tubes to provide direct contact between bacteria and the test item. These tubes (3 tubes per
control and 3 tubes for each concentration level) were gently mixed and incubated for 20 min at 37ºC
in a shaking incubator.
After the incubation period, 2 mL of molten top agar were added to the tubes, and then the content
mixed and poured on the surface of minimal glucose agar plates. The entire test consisted of nonactivated
and activated test conditions, with the addition of untreated, negative and positive controls.
After preparation, the plates were incubated at 37°C for 48±1 hour.

Evaluation criteria:

The colony numbers on the untreated / negative (vehicle/solvent) / positive control and test item
treated plates were determined by manual counting. Visual examination of the plates was also
performed; precipitation or signs of growth inhibition (if any) were recorded and reported. The mean number of revertants per plate, the standard deviation and the mutation factor* values were calculated for each concentration level of the test item and for the controls using Microsoft ExcelTM s
oftware.
* Mutation factor (MF): mean number of revertants on the test item plate / mean number of revertants
on the vehicle control plate.
Criteria for Validity:
The study was considered valid if:
- the number of revertant colonies of the negative (solvent) and positive controls were in the historical
control range in all strains of the main tests;
- at least five analyzable concentrations were presented in all strains of the main tests.
Criteria for a Positive Response:
A test item was considered mutagenic if:
- a dose–related increase in the number of revertants occurred and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurred in
at least one strain with or without metabolic activation.
An increase was considered biologically relevant if:
- the number of reversions was more than two times higher than the reversion rate of the negative
(solvent) control in Salmonella typhimurium TA98, TA100 and Escherichia coli WP2 uvrA bacterial
strains;
- the number of reversions was more than three times higher than the reversion rate of the negative
(solvent) control in Salmonella typhimurium TA1535 and TA1537 bacterial strains.
Criteria for a Negative Response:
The test item was considered to have shown no mutagenic activity in this study if it produces neither
a dose-related increase in the number of revertants nor a reproduciblevbiologically relevant positive
response at any of the dose groups, with or without metabolic activation.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Initial mutation test: 1581 μg/plate (+/- S9) Confirmatory mutation test: 1581 μg/plate (+S9); 1581 and 500 μg/plate (-S9)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Initial mutation test: 1581 and 500 μg/plate (-S9); 1581 μg/plate (+S9) Confirmatory mutation te st: 1581, 500, 158.1 and 50 μg/plate (-S9); 1581 and 500 μg/plate (+S9)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Initial mutation test: 1581 and 500 μg/plate (-S9); 1581 μg/plate (+S9) Confirmatory mutation te st: 1581, 500 and 158.1 μg/plate (-S9); 1581 μg/plate (+S9)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Initial mutation test: 1581, 500 and 158.1 μg/plate (-S9); 1581 μg/plate (+S9) Confirmatory muta tion test: 1581, 500, 158.1 and 50 μg/plate (-S9); 1581 and 500 μg/plate (+S9) Complementary confirmatory mutation test: 158.1 and 50 μg/plate (-S9)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

but tested up to limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

No precipitate was detected on the plates in the main tests in all examined bacterial strains with and without metabolic activation.
Lower numbers of revertant colonies may indicate inhibitory, cytotoxic effect of the test item in Escherichia coli WP2 uvrA without metabolic activation at 5000 μg test item/plate.

Any other information on results incl. tables

In the Initial Mutation Test and in the Complementary Initial Mutation Test (using the plate incorporation

method), the highest revertant rate was observed in Salmonella typhimurium TA1535 bacterial strain

at 50 μg/plate and 5 μg/plate concentrations without metabolic activation (the observed mutation factor

value was 1.64). However, there was no clear dose-response relationship, the observed mutation factor

values were below the biologically relevant threshold limit and the numbers of revertant colonies were

within the historical control range.

In the Confirmatory Mutation Test and in the Complementary Confirmatory Mutation Test (using the

pre-incubation method), the highest revertant rate was observed in Salmonella typhimurium TA1535 at

0.5 μg/plate concentration without metabolic activation (the observed mutation factor value was 2.14).

However, there was no clear dose-response relationship, the observed mutation factor values were

below the biologically relevant threshold limit and the numbers of revertant colonies were within the

historical control range.

Higher numbers of revertant colonies compared to the vehicle (solvent) control were detected in the

main tests in some other sporadic cases. However, no dose-dependence was observed in those cases

and they were below the biologically relevant threshold value. The numbers of revertant colonies were

within the historical control range in each case, so they were considered as reflecting the biological

variability of the test.

Sporadically, lower revertant counts compared to the vehicle (solvent) control were observed in the main

tests at some non-cytotoxic concentrations. However, no background inhibition was recorded and the

mean numbers of revertant colonies were in the historical control range in all cases, thus they were

considered as biological variability of the test system.

Applicant's summary and conclusion

Conclusions:

The test item had no mutagenic activity in the applied bacterium tester strains under the test conditions used in this study.

Executive summary:

The test item was tested for potential mutagenic activity using the Bacterial Reverse Mutation Assay.

The experiments were carried out using histidine-requiring auxotroph strains of Salmonella

typhimurium (Salmonella typhimurium TA98, TA100, TA1535 and TA1537) and the tryptophanrequiring

auxotroph strain of Escherichia coli (Escherichia coli WP2 uvrA) in the presence and

absence of a post mitochondrial supernatant (S9 fraction) prepared from the livers of phenobarbital/β-

naphthoflavoneinduced rats.

The study included a Preliminary Compatibility Test, a Preliminary Concentration Range Finding

Test (Informatory Toxicity Test), an Initial Mutation Test (Plate Incorporation Method), a Confirmatory

Mutation Test (Pre-Incubation Method), a Complementary Initial Mutation Test (Plate Incorporation

Method, during the Experimental Period II) and a Complementary Confirmatory Mutation Test (Pre-

Incubation Method).

Based on the results of a solubility test, the test item was formulated in Dimethyl sulfoxide (DMSO).

Concentrations of 5000; 2500; 1000; 316; 100, 31.6 and 10 μg/plate were examined in the Preliminary

Concentration Range Finding Test. Based on the results of the Range Finding Test, the test item

concentrations in the Initial Mutation Test (5 strains) were, 1581, 500, 158.1, 50, 15.81, 5, 1.581 and

0.5 μg/plate, the test item concentrations in the Confirmatory Mutation Test (5 strains) in all Salmonella

typhimurium strains were 1581, 500, 158.1, 50, 15.81, 5, 1.581, 0.5 and 0.1581 μg/plate and in

Escherichia coli WP2 uvrA strain were 5000, 1581, 500, 158.1, 50, 15.81, 5 and 1.581 μg test item/

plate, the test item concentrations in the Complementary Initial Mutation Test in Escherichia coli WP2

strain were 5000, 1581, 500, 158.1, 50, 15.81 and 5 μg test item/plate. Examined concentrations in the

Complementary Confirmatory Mutation Test in Salmonella typhimurium TA100 strain without metabolic

activation were 158.1, 50, 15.81, 5, 1.581, 0.5 and 0.1581 μg test item/plate.

In the Preliminary Concentration Range Finding Test and in the main tests, none of the observed

revertant colony numbers were above the respective biological threshold value. There were no doserelated

trends and no indication of any treatment effect. In all test item treated groups, the numbers of

revertant colonies did not exceed the biological relevance when compared to the vehicle control and

were within the normal biological variability of the test system.

No precipitate was detected on the plates in the Preliminary Concentration Range Finding Test and in

the main tests in all examined bacterial strains with and without metabolic activation.

Inhibitory, cytotoxic effect of the test item was observed in the Preliminary Concentration Range Finding

Test and in the main tests in all Salmonella typhimurium strains with and without metabolic activation at

several concentrations. Furthermore in the Confirmatory Mutation Test the lower numbers of revertant

colonies may indicate inhibitory, cytotoxic effect of the test item in Escherichia coli WP2 uvrA without

metabolic activation at 5000 μg test item/plate. The mean values of revertant colonies of the negative

(vehicle/solvent) control plates were within the historical control range, the reference mutagens showed

the expected increase in the number of revertant colonies, the viability of the bacterial cells was checked

by a plating experiment in each test. At least five analyzable concentrations were presented in all strains

of the main tests, the examined concentration range was considered to be adequate. The study was

considered to be valid.

The reported data of this mutagenicity assay show that under the experimental conditions applied the

test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains

used. In conclusion, the test item had no