Phosphoric acid, mono- and di-C12-14-alkyl esters

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The substance was negative in the Ames test (bacterial reverse mutation assay) employing Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100, and Escherichia coli strain WP2 uvrA, both in the presence and absence of a metabolic activation system (rat liver S9).

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12th June 2019 - 1st July 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 July 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

30 May 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Version / remarks:

August 1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

The Japanese Ministry of Health, Labour and Welfare (MHLW), Ministry of Economy, Trade and Industry (METI), and Ministry of the Environment (MOE) Guidelines of 31 March 2011

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: ICH S2(R1) guideline adopted June 2012 (ICH S2(R1) Federal Register.

Version / remarks:

Adopted 2012; 77:33748-33749

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

CAS Number: 68412-59-9
Batch Number: 1023S17201
Purity: 100%
Expiry Date: 09 July 2020
Storage Conditions: Room temperature in the dark

Target gene:

Strains Genotype Type of mutations indicated
TA1537 his C 3076; rfa-; uvrB-: frame shift mutations
TA98 his D 3052; rfa-; uvrB-;R-factor
TA1535 his G 46; rfa-; uvrB-: base-pair substitutions
TA100 his G 46; rfa-; uvrB-;R-factor
E.coli WP2uvrA trp-; uvrA-: base-pair substitution

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

Rat liver homogenate metabolizing system (10% liver S9 in standard co-factors).

Test concentrations with justification for top dose:

Experiment 1: 0, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate. Experiment 2: 0, 0.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate. The maximum concentration was 5000 μg/plate, which is the maximum recommended dose according to the OECD TG 471.

Vehicle / solvent:

Tetrahydrofuran (THF)

Untreated negative controls:

yes

Remarks:

THF

Negative solvent / vehicle controls:

yes

Remarks:

THF

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

Without S9 activation

Untreated negative controls:

yes

Remarks:

THF

Negative solvent / vehicle controls:

yes

Remarks:

THF

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

other: 2-Aminoanthracene

Remarks:

With S9 activation

Details on test system and experimental conditions:

Test System - Bacteria:
All of the Salmonella strains are histidine dependent by virtue of a mutation through the histidine operon and are derived from S. typhimurium strain LT2 through mutations in the histidine locus. Additionally due to the "deep rough" (rfa-) mutation they possess a faulty lipopolysaccharide coat to the bacterial cell surface thus increasing the cell permeability to larger molecules. A further mutation, through the deletion of the uvrB- bio gene, causes an inactivation of the excision repair system and a dependence on exogenous biotin. In the strains TA98 and TA100, the R-factor plasmid pKM101 enhances chemical and UV-induced mutagenesis via an increase in the error-prone repair pathway. The plasmid also confers ampicillin resistance which acts as a convenient marker (Mortelmans and Zeiger, 2000). In addition to a mutation in the tryptophan operon, the E. coli tester strain contains a uvrA- DNA repair deficiency which enhances its sensitivity to some mutagenic compounds. This deficiency allows the strain to show enhanced mutability as the uvrA repair system would normally act to remove and repair the damaged section of the DNA molecule (Green and Muriel, 1976 and Mortelmans and Riccio, 2000).

Experimental Design and Study Conduct.
Substance Preparation and Analysis.
The substance was insoluble in sterile distilled water, dimethyl sulphoxide, dimethyl formamide and acetonitrile at 50 mg/mL, and acetone at 100 mg/mL but was fully soluble in tetrahydrofuran at 200 mg/mL in solubility checks performed in–house. Tetrahydrofuran was, therefore, selected as the vehicle.
The substance was accurately weighed and, on the day of each experiment, approximate half-log dilutions prepared in dried tetrahydrofuran by mixing on a vortex mixer and sonication for 30 minutes at 40 °C. No correction for purity was required. Tetrahydrofuran is toxic to the bacterial cells at and above 50 μL (0.05 mL), therefore all of the formulations were prepared at concentrations four times greater than required on Vogel-Bonner agar plates. To compensate, each formulation was dosed using 25 μL (0.025 mL) aliquots. Tetrahydrofuran is considered an acceptable vehicle for use in this test system (Maron et al., 1981).
All formulations were used within four hours of preparation and were assumed to be stable for this period. Analysis for concentration, homogeneity and stability of the substance formulations is not a requirement of the test guidelines and was, therefore, not determined. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.

Experiment 1 – Plate Incorporation Method.

Dose selection
The substance was tested using the following method. The maximum concentration was 5000 μg/plate (the OECD TG 471 maximum recommended dose level). The following substance concentrations 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate were assayed in triplicate against each tester strain, using the direct plate incorporation method.

Without Metabolic Activation
A 0.025 mL aliquot of the appropriate concentration of substance, solvent vehicle or 0.1 mL of the appropriate positive control was added together with 0.1 mL of the bacterial strain culture, 0.5 mL of phosphate buffer and 2 mL of molten, trace amino-acid supplemented media. These were then mixed and overlayed onto a Vogel-Bonner agar plate. Negative (untreated) controls were also performed on the same day as the mutation test. Each concentration of the substance, appropriate positive, vehicle and negative controls, and each bacterial strain, was assayed using triplicate plates.

With Metabolic Activation
The procedure was the same as for the 'Without Metabolic Activation' except that following the addition of the substance formulation and bacterial culture, 0.5 mL of S9-mix was added to the molten, trace amino-acid supplemented media instead of phosphate buffer.

Incubation and Scoring
All of the plates were incubated at 37 ± 3 °C for between 48 and 72 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity). Manual counts were performed intermittently at 1500 μg/plate and for all plates at 5000 μg/plate because of substance precipitation. A number of further manual counts were also performed due to colonies spreading, light background contamination and artefacts on the plates, thus distorting the actual plate count.

Experiment 2 – Pre-Incubation Method
As the result of Experiment 1 was considered negative, Experiment 2 was performed using the pre-incubation method in the presence and absence of metabolic activation (S9-mix).

Dose selection
The dose range used for Experiment 2 was determined by the results of Experiment 1 and was 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate.
Nine substance concentrations per bacterial strain were selected in Experiment 2 in order to achieve both four non-toxic dose levels and to allow for the potential change in toxicity of the substance following the change in test methodology from plate incorporation to pre-incubation.

Without Metabolic Activation.
A 0.1 mL aliquot of the appropriate bacterial strain culture, 0.5 mL of phosphate buffer and 0.025 mL of the appropriate concentration of substance formulation, solvent vehicle or 0.1 mL of appropriate positive control were incubated at 37 ± 3 °C for 20 minutes (with shaking) prior to addition of 2 mL of molten, trace amino-acid supplemented media and subsequent plating onto Vogel-Bonner plates. Negative (untreated) controls were also performed on the same day as the mutation test employing the plate incorporation method. All testing for this experiment was performed in triplicate.

With Metabolic Activation.
The procedure was the same as described above in 'Without Metabolic Activation' except that following the addition of the test item formulation and bacterial strain culture, 0.5 mL of S9-mix was added to the tube instead of phosphate buffer, prior to incubation at 37 ± 3 °C for 20 minutes (with shaking) and addition of molten, trace amino-acid supplemented media. All testing for this experiment was performed in triplicate.

Incubation and Scoring.
All of the plates were incubated at 37 ± 3 °C for between 48 and 72 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity). Manual counts were performedintermittently at 1500 and 5000 μg/plate because of substance precipitation. Further manual counts were also performed due to colonies spreading, thus distorting the actual plate count.

Rationale for test conditions:

Recommended test system in international guidelines (OECD, EC).

Evaluation criteria:

There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested.
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. A fold increase greater than two times the concurrent solvent control for TA100, TA98 and WP2uvrA or a three-fold increase for TA1535 and TA1537 (especially if accompanied by an out-of-historical range response.
5. Statistical analysis of data as determined by UKEMS.
A substance will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit making a definite judgment about substance activity. Results of this type will be reported as equivocal.

Statistics:

Statistical significance was confirmed by using Dunnetts Regression Analysis (* = p < 0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent control. Values that are statistically significant but are within the in-house historical vehicle/untreated control range are not reported.

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Exp. 2: From concentrations of 150 ug/plate onwards (Sparse bacterial background lawn); Exp. 1 From concentrations of 1500 ug/plate onwards (Sparse bacterial background lawn and substance precipitate).

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Exp. 2: From concentrations of 150 ug/plate onwards (Sparse bacterial background lawn); Exp. 1 From concentrations of 1500 ug/plate onwards (Sparse bacterial background lawn and substance precipitate);

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Exp. 2: From concentrations of 150 ug/plate onwards (Sparse bacterial background lawn); Exp. 1 From concentrations of 1500 ug/plate onwards (substance precipitate).

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Exp. 2, from concentration 150 ug/plate onwards (Very weak bacterial background lawn); Exp. 1 From concentrations of 1500 ug/plate onwards (substance precipitate).

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Exp 2, From concentration 1500 ug/plate onwards (Sparse bacterial background lawn and substance precipitate); Exp. 1 From concentrations of 1500 ug/plate onwards (substance precipitate).

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Negative

Remarks:

Up to 5000 ug/plate

Experiment 1: Mutagenic response of the substance in the S. typhimurium and E.coli Reverse Mutation Assay (Plate Incorporation).

Without metabolic activation.

Dose (µg/plate)	TA100 (± SD)	TA1535 (± SD)	WP2urvA (± SD)	TA98 (± SD)	TA1537 (± SD)
Solvent control (THF)	137 (21.1)	13 (1)	27 (3.1)	23 (6.1)	10 (1.7)
Positive control	574 (120.3)	446 (47.8)	846 (51.2)	139 (9.7)	130 (25.3)
1.5	141 (15.9)	14 (4.0)	25 (6.4)	30 (10.3)	11 (2.1)
5	139 (8.0)	17 (4.0)	25 (7.8)	23 (4.6)	11 (2.6)
15	113 (2.6)	18 (0.0)	25 (6.2)	26 (5.0)	13 (1.5)
50	128 (8.1)	14 (7.2)	33 (6.6)	19 (6.8)	11 (3.2)
150	100 (9.3)	13 (3.8)	33 (2.1)	21 (3.0)	12 (3.5)
500	70 (8.1)	10 (1.5)	29 (3.5)	13 (5.6)	9 (1.5)
1500	44 - P (4.4)	5 - P (1.5)	29 - P (3.5)	3 - PS (1.2)	1- P (0.6)
5000	9 - PS (2.5)	0 - PS (0.6)	22 - PS (4.7)	2 - PS (1.2)	0 - PS (0.0)

Mean number of revertant colonies/3 replicate plates (±S.D.)

P Substance precipitate

S Sparse bacterial background lawn

With metabolic activation.

Dose (µg/plate)	TA100 (± SD)	TA1535 (± SD)	WP2urvA (± SD)	TA98 (± SD)	TA1537 (± SD)
Solvent control (THF)	109 (8.1)	13 (1.5)	32 (1.5)	24 (4.6)	11 (3.5)
Positive control	2224 (49.6)	286 (29.5)	188 (41.9)	139 (7.8)	288 (9.2)
1.5	121 (16.5)	14 (2.5)	36 (6.4)	18 (1.5)	13 (4.0)
5	132 (12.1)	15  (5.9)	34 (5.2)	22 (3.2)	14 (2.9)
15	121 (13.4)	9 (0.6)	38 (1.0)	24 (1.2)	10 (2.5)
50	126 (9.8)	11 (3.2)	33 (9.1)	19 (1.5)	12 (3.5)
150	92 (9.5)	7 (2.5)	33 (3.5)	19 (2.5)	4 (0)
500	77 (8.5)	7 (2.5)	28 (8.2)	10 (1.5)	4 (0.6)
1500	68 - PS (27)	4 - P (2.6)	24 - P  (6.4)	10 - PS (1.5)	0 - P (0)
5000	24 - PS (1.7)	2 - PS (2.1)	18 - PS (1.5)	4 - PS (1.7)	0 - PS (0)

Mean number of revertant colonies/3 replicate plates (±S.D.)

P Substance precipitate

S Sparse bacterial background lawn

Experiment 2: Mutagenic response of the substance in the S. typhimurium and E.coli Reverse Mutation Assay (Pre-Incubation).

Without metabolic activation.

Dose (µg/plate)	TA100 (± SD)	TA1535 (± SD)	WP2urvA (± SD)	TA98 (± SD)	TA1537 (± SD)
Solvent control (THF)	99 (7.1)	14 (5.1)	39 (4.7)	22 (4.6)	13 (1.2)
Positive control	568 (17.0)	534 (65.4)	586 (18.7)	205 (9.6)	224 (42.8)
0.5	117 (13.1)	16 (3.6)	30 (7.2)	18 (2.3)	14 (0.6)
1.5	121 (4.7)	18 (1.5)	33 (2.1)	17 (2.1)	11 (1)
5	112 (12.5)	21 (4.6)	31 (1.2)	14 (3.6)	7 (1.2)
15	96 (10.3)	19 (2.1)	28 (8.7)	22 (3.2)	12 (2.1)
50	83 (7.0)	12 (0.6)	30 (3.5)	20 (4.5)	7 (5.3)
150	66 - S (16.6)	14 - S (2.9)	26 (6.9)	8 - S (2.6)	0 - V (0.0)
500	56 - S (9.1)	0 - T (0.0)	32 (2.1)	0 - T (0.0)	0 - V (0.0)
1500	0 - PV (0.0)	0 - PT (0.0)	24 - PS (2.6)	0 - PT (0.0)	0 - PV (0.0)
5000	0 - PV (0.0)	0 - PT (0.0)	24 - PS (1.5)	0 - PT (0.0)	0 - PT (0.0)

Mean number of revertant colonies/3 replicate plates (±S.D.)

P Substance precipitate

S Sparse bacterial background lawn

T Toxic, no bacterial background lawn

V Very weak bacterial background lawn

With tmetabolic activation.

Dose (µg/plate)	TA100 (± SD)	TA1535 (± SD)	WP2urvA (± SD)	TA98 (± SD)	TA1537 (± SD)
Solvent control (THF)	130 (9)	12 (1.7)	41 (8.7)	28 (12.5)	15 (4.0)
Positive control	1534 (40.7)	254 (16.3)	164 (24.4)	136 (19.2)	260 (1.7)
0.5	127 (5.0)	18 (3.8)	36 (5.5)	25 (4.0)	17 (2.6)
1.5	132 (6.8)	13 (1.5)	40 (5.9)	27 (2.1)	15 (3.2)
5	121 (12.3)	17 (2.1)	37 (11.8)	18 (2.0)	18 (4.0)
15	126 (6.6)	14 (4.0)	39 (4.2)	28 (11.8)	18 (4.0)
50	109 (8.3)	13 (3.2)	38 (2.8)	28 (4.5)	11 (6.7)
150	100 (5.9)	13 (3.5)	40 (7.2)	24 (5.5)	5 - S (1.5)
500	0 - V (0.0)	8 (3.5)	30 (2.5)	18 (3.6)	0 - V (0.0)
1500	0 - PV (0.0)	5 - PS (4.9)	31 - P (3.5)	8- PS (3.2)	0 - V (0.0)
5000	0 - PV (0.0)	0 - PV (0.0)	71 - PV (5.5)	0 - PV (0.0)	0 - V (0.0)

Mean number of revertant colonies/3 replicate plates (±S.D.)

P Substance precipitate

S Sparse bacterial background lawn

T Toxic, no bacterial background lawn

V Very weak bacterial background lawn

Conclusions:

The substance is not mutagenic in the S. typhimurium and E. coli reverse mutation assay in the presence and absence of metabolic activation.  

Executive summary:

The potential of the substance to induce reverse mutations at the histidine locus in several strains of Salmonella typhimurium and at the tryptophan locus of Escherichia coli evaluated in the presence or absence of an exogenous mammalian metabolic activation system (S9). The study was conducted in accordance with OECD TG 471 ‘Bacterial Reverse Mutation Assay’.

Two experiments were conducted employing the plate incorporation and the preincubation test methods. Both experiments were conducted on S. typhimurium: TA98, TA100, TA1535, and TA1537 and E. coli WP2uvrA strains; with and without metabolic activation system (10 % v/v liver S9 in standard co-factors). The dose range for exp.1 was based on OECD TG 471.

The plate incorporation test employed the following concentration of substance: 0, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate (in triplicate). The preincubation test employed the following concentration of substance: 0, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate (in triplicate); chosen on the basis of exp. 1 results.

In the plate incorporation experiment the substance induced a visible reduction in the growth of the bacterial background lawn and/or a significant reduction in revertant colony frequency (below 0.5 compared to the concurrent vehicle control) initially from 1500 and 150 μg plate in the absence and presence of metabolic activation (S9-mix), respectively. In the preincubation experiment the substance induced a stronger toxic response with visible reductions in the growth of the bacterial background lawn and/or a significant reduction in revertant colony frequency (below 0.5 compared to the concurrent vehicle control) initially noted from 150 μg plate in both the absence and presence of metabolic activation (S9-mix).

A substance precipitate was noted under an inverted microscope at 500 μg/plate and by eye at and above 1500 μg/plate in both the presence and absence of S9-mix in exp. 1 and 2. This observation did not prevent the scoring of revertant colonies.

There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the substance, either with or without S9-mix in exp.1 (plate incorporation method).

Similarly, no biologically relevant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, at any substance dose, either with or without S9-mix in exp. 2 (pre-incubation method). One statistically significant value was noted (TA1535 at 5 μg/plate in the absence of S9-mix), however, this response was within the historical vehicle/untreated control, therefore considered of no biological relevance.

Based on the results of this study it is concluded that the substance is not mutagenic in the S. typhimurium and E. coli reverse mutation assay in the presence and absence of metabolic activation.  

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the findings of a reliable in vitro bacterial reverse mutation study (Ames), classification of the substance is not justified.