Phosphoric acid, mono and bis-linear butyl esters, potassium salts

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 May 1997 to 22 May 1997

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1997

Report date:

1997

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: US EPA 40 CFR 798.5265 and 798.5100

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 5

Qualifier:

according to guideline

Guideline:

other: MAFF Japan (59 NohSan No 4200)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Phosphoric acid, butyl ester, potassium salt

EC Number:

258-379-5

EC Name:

Phosphoric acid, butyl ester, potassium salt

Cas Number:

53126-06-0

Molecular formula:

KO4P

IUPAC Name:

Potassium butyl hydrogen phosphate

Test material form:

solid - liquid: aqueous solution

Details on test material:

Phosphoric acid, n-butyl ester, potassium salt (1.37 ester groups per molecule)

Specific details on test material used for the study:

- Physical characteristics: Water white liquid
- Purity: 50% active ingredient in water
- Haskell number: 22586
- Stability: Test substance appeared to be stable under the conditions of the study; no evidence of instability was observed.

Method

Target gene:

Histidine and tryptophan

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium, other: TA100, TA1535, TA97a and TA98

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Metabolic activation system:

Aroclor-induced S9

Test concentrations with justification for top dose:

RANGE-FINDING STUDY
- Nominal test item concentrations of 10, 25, 50, 100, 250, 500, 1000, 2500 and 5000 μg/plate

DEFINITIVE STUDY
- Nominal test item concentrations of 25, 100, 500, 1000, 2500 and 5000 μg/plate

Vehicle / solvent:

Sterile water

Controls

Controls 1

Negative solvent / vehicle controls:

yes

Remarks:

sterile water

Controls 2

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

NAAZ (2 μg/plate for TA100 without metabolic activation

Controls 3

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

2AA (1 μg/plate for TA100 with metabolic activation)

Controls 4

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

NAAZ (2 μg/plate for TA1535 without metabolic activation)

Controls 5

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

2AA (2 μg/plate for TA1535 with metabolic activation)

Controls 6

Positive controls:

yes

Positive control substance:

other: ICR 191 Acridine

Remarks:

ICR 191 (2 μg/plate for TA97a without metabolic activation)

Controls 7

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

2AA (1 μg/plate for TA97a with metabolic activation)

Controls 8

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

2NF (25 μg/plate for TA98 without metabolic activation)

Controls 9

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

2AA (2 μg/plate for TA98 with metabolic activation)

Controls 10

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

MMS (1000 μg/plate for WP2 uvrA (pKM101) without metabolic activation)

Controls 11

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

2AA (25 μg/plate for WP2 uvrA (pKM101) with methabolic activation)

Details on test system and experimental conditions:

TEST SUBSTANCE AND NEGATIVE CONTROL
- Preparation of the stock solution of the test substance at 50 mg/mL resulted in a homogeneous, colourless, solution.
- Additional dilutions of the test substance were performed at room temperature in sterile water.
- The negative control was assumed to be stable during the study and no evidence of instability was observed.
- Any impurities were not expected to have interfered with the study.

POSITIVE INDICATORS
- Deionised water was used as the solvent for NAAZ, ICR 191 and MMS.
- The solvent for other positive indicators was DMSO.
- Positive indicators were assumed to be stable in this study and no evidence of instability was observed.
- Any impurities were not expected to have interfered with the study.

TESTER STRAIN CHARACTERISATION, STORAGE AND CULTURE
- S typhimurium tester strains were obtained from Dr Bruce Ames, Berkeley, CA.
- The phenotypic characteristics and mutational sensitivites of the S typhimurium strains are described in the attached document.
- E coli WP2 uvrA (pKM101) was obtained from the National Collection of Industrial Bacteria, Torrey Research Station, Scotland. Because tryptophan biosynthesis is blocked by an ochre nonsense mutation, revertants arise as a result of base pair substitution. A second class of mutants may arise as a result of nonsense suppressor mutations in genes coding for tRNAs. Frameshift mutagens are not generally expected to be detected by this strain.

TESTER STRAIN STORAGE AND CULTURE
- All bacterial strains were stored frozen in 8 % DMSO in Oxoid nutrient broth at approximately -70 °C.
- Overnight cultures were prepared by inoculating 20 mL of Oxoid nutrient broth with 0.1 mL of thawed bacterial suspension and incubating at 37 °C with shaking.
- Overnight cultures were then stored on ice until used for mutagenesis assays.
- Bacterial strain phenotypes were confirmed concurrently with each trial. Results demonstrated the appropriate responses (Table 1).

METABOLIC ACTIVATION SYSTEM
- Because the tester strains lack many of the enzymes required to convert various promutagens to a reactive state, the assay was performed with and without a rat liver homogenate activation system (S9 mix) similar to the method of Maron and Ames (1983).
- The S9 mix consisted of MgCl2 (8 mM), KCl (33 mM), glucose-6-phosphate (5 mM), NADP (4 mM), sodium phosphate pH 7.4 (100 mM) and S9 protein/1.0 mL S9 mix.
- The S9 purcahsed from MOLTOX was the 9000 x g supernatant of liver homogenate (1 g wet liver; 3.0 mL KCl).
- Livers were from young male Sprague Dawley rats injected i.p. with Aroclor 1254 (500 mg/kg) five days before sacrifice.

DOSE SELECTION
- In accordance with EPA, OECD and MAFF Japan test guidelines, the highest concentration evaluated in the study was a suspension of the test substance at a nominal concentration of 5000 μg/plate.
- Solubility information was confirmed empirically as needed during the study.
- Concentrations were calculated with the assumption that addition of the test substance to the solvent did not change the volume of the resulting solution.

STABILITY AND CONCENTRATION VERIFICATION
- Solutions of the test substance were prepared immediately prior to treatment and were presumed to be stable under the conditions of the study.
- Treatment and control dosing solutions were not analysed for concentration, uniformity or stability.
- Top agar was not assayed for stability or concentration of the test substance or control articles since this assessment was not considered necessary to achieve the objectives of the study.

BACTERIAL MUTAGENICITY ASSAYS
- The study consisted of one trial with and without activation.
- Three replicates were plated for each tester strain, test concentration and condition.
- Positive indicators and negative controls were included in all assays.
- Treatments with activation were conducted by adding 0.1 mL of an overnight culture containing at least 1 x 10E08 bacteria to 2 mL of top agar (0.6 % agar w/v and 0.6 % NaCl w/v) supplemented with 0.05 mM L-histidine and 0.05 mmM D-biotin for S typhimurium strains or 0.05 mM L-tryptophan for the E coli strain.
- The components were mixed and poured onto a plate containing 25 mL of Davis minimal agar with dextrose (minimal agar plates purchased from MOLTOX).
- Treatments without activation were identical to those with activation with the exception that the S9 mix was replaced with 0.5 mL of sterile water.
- Revertant colonies were counted after the individually labelled plates were incubated at approximately 37 °C for about 48 hours.
- When necessary, plates were refrigerated prior to counting.

Evaluation criteria:

- Acceptability criteria for the study are attached.
- Classification guidelines relating to the study are attached.

Statistics:

STATISTICAL ANALYSIS
- For each tester strain, the average number of revertants and the standard deviation at each concentration (with and without S9 activation) were calculated.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

other: Salmonella typhimurium TA100, TA1535, TA97a and TA98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

- Mutagenic activity of the tester strains is shown in Tables 2 to 6 (attached) together with a list of abbreviations used in the tables.

Applicant's summary and conclusion

Conclusions:

No evidence of mutagenic activity was detected using Salmonella typhimurium strains TA100, TA1535, TA97a and TA98 plus Escherichia coli strain WP2 uvrA (pKM101).

Executive summary:

The test substance was evaluated for mutagenicity in accordance with OECD 471 and other appropriate test guidelines using Salmonella typhimurium strains TA100, TA1535, TA97a and TA98 and in Escherichia coli strain WP2 uvrA (pKM101) with and without an exogenous metabolic activation system (S9). Concentrations of 10, 25, 50, 100, 250. 500, 1000 and 5000 μg/plate were evaluated in comparison to negative (solvent) controls in a range-finding study using Salmonella typhimurium strains TA97a and TA100 plus Escherichia coli strain WP2 uvrA (pKM101). Concentrations of 25, 100, 500, 1000, 2500 and 5000 μg/plate were subsequently used in Salmonella typhimurium strains TA100, TA1535, TA97a and TA98 plus Escherichia coli strain WP2 uvrA (pKM101). No evidence of mutagenic activity was detected under the conditions of the study. The substance was considered negative.