Phosphonic acid, mixed C12-20-alkyl and C14-18-unsatd. alkyl derivs.

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Between 04 November 2011 and 05 December 2011 (experimental phase)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Valid and conclusive guideline study under GLP

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Commission Regulation (EC) number 440/2008

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

(including METI, MHLW and MAFF guidances)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Department of Health of Government of the U.K., inspection 19-21 July 2011; no analysis carried out to determine the homogeneity, concentration or stability of the test item formulation

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Phosphonic acid, mixed C12-20-alkyl and C14-18-unsatd. alkyl derivs.

EC Number:

300-326-6

EC Name:

Phosphonic acid, mixed C12-20-alkyl and C14-18-unsatd. alkyl derivs.

Cas Number:

93925-25-8

Molecular formula:

Complex

IUPAC Name:

Phosphonic acid, mixed C12-20-alkyl and C14-18-unsatd. alkyl derivs.

Test material form:

liquid: viscous

Details on test material:

- Substance type: Organic
- Physical state: White liquid
- Analytical purity: Considered to be 100 % due to the UVCB character
- Lot/batch No.: 0100823714
- Expiration date of the lot/batch: 28 September 2012
- Storage condition of test material: Room temperature, in the dark
- Other: Date received at the test laboratory: 28 September 2011

Method

Target gene:

Histidine for Salmonella typhimurium, Tryptophan for Escherichia coli

Species / strain

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: Genotypes: TA1535 = his G 46, rfa-, uvr B-; TA1537 = his C 3076, rfa-, uvr B-; TA 98 = his D 3052, rfa-, uvr B-, R-factor; TA 100 = his G 46, rfa-, uvr B-, R-factor; WP2 = trp-, uvr A-

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbitone and ß-Naphthoflavone induced rat liver, S9

Test concentrations with justification for top dose:

Preliminary Toxicity Test: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
Mutation Test - Experiments 1 and 2 (Range-finding Test and Main Test): 50, 150, 500, 1500 and 5000 µg/plate

Vehicle / solvent:

- Vehicle/solvent used: Acetone
- Justification for choice of solvent/vehicle: The test item was immiscible in dimethyl sulphoxide (DMSO) at 50 mg/mL but was fully miscible in acetone at 100 mg/mL in solubility checks performed in house. Following solubility information provided by the Sponsor, sterile distilled water was not evaluated as a potential vehicle in this test system. Acetone was therefore selected as the vehicle.

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of all strains used

Negative solvent / vehicle controls:

yes

Remarks:

Acetone

True negative controls:

no

Positive controls:

yes

Remarks:

4-Nitroquinoline-N-oxide (CAS 56-57-5) at 0.2 µg/plate for TA 98; 9-Aminoacridine (CAS 90-45-9) at 80 µg/plate for TA 1537; N-ethyl-N'-nitro-N-nitrosoguanidine (CAS 4245-77-6) at 2, 3 and 5 µg/plate for WP2, TA 100 and TA 1535, respectively

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

Without metabolic activation (-S9)

Controls 2

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of all strains used

Negative solvent / vehicle controls:

yes

Remarks:

Acetone

True negative controls:

no

Positive controls:

yes

Remarks:

Benzo(a)pyrene (CAS 50-32-8) at 5 µg/plate for TA98 or 2-Aminoanthracene (CAS 613-13-8) at 1, 2, 2 and 10 µg/plate for TA 100, TA 1535, TA 1537 and WP2, respectively

Positive control substance:

benzo(a)pyrene

other: 2-Aminoanthracene

Remarks:

With metabolic activation (+S9)

Details on test system and experimental conditions:

METHOD OF APPLICATION: In agar (plate incorporation)

DURATION
- Preincubation period for bacterial strains: 10 h
- Exposure duration: 48 - 72 h

NUMBER OF REPLICATIONS: Triplicate plating

DETERMINATION OF CYTOTOXICITY
- Method: Plates were assessed for numbers of revertant colonies and examined for effects on the growth of the bacterial background lawn.

Evaluation criteria:

ACCEPTANCE CRITERIA
The reverse mutation assay may be considered valid if the following criteria are met:
- All tester strain cultures exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls.
- The appropriate characteristics for each tester strain have been confirmed, e.g. rfa cell-wall mutation and pKM101 plasmid R-factor etc.
- All tester strain cultures should be in the approximate range of 0.9 to 9 • 10^9 bacteria per mL.
- Each mean positive control value should be at least twice the respective vehicle control value for each strain, thus demonstrating both the intrinsic sensitivity of the tester strains to mutagenic exposure and the integrity of the S9-mix.
- There should be a minimum of four non-toxic test item dose levels.
- There should not be an excessive loss of plates due to contamination.

EVALUTION CRITERIA
There are several criteria for determining a positive result, such as a dose-related increase in revertant frequency over the dose range tested and/or a reproducible increase at one or more concentrations in at least one bacterial strain with or without metabolic activation. Biological relevance of the results will be considered first, statistical methods, as recommended by the UKEMS can also be used as an aid to evaluation, however, statistical significance will not be the only determining factor for a positive response. A test item 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 a definitive judgement about the test item activity. Results of this type will be reported as equivocal.

Statistics:

Standard deviation and Dunnett's linear regression analysis

Results and discussion

Test results

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Remarks:

Tested up to maximum recommended dose of 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: There was no marked change in pH when the test item was dosed into media; pH 7.33 without test item, range from 9.77 to 2500 µg test item/mL was pH 7.29 to 7.32 with no visible trend, these data were taken from the MLA study of Flanders (2012, Harlan Report no. 41103491)
- Effects of osmolality: The osmolality did not increase by more than 50 mOsm when the test item was dosed into media; mOsm 383 without test item; range from 9.77 to 2500 µg test item/mL was 387 to 307 decreasing with increasing test item concentration, these data were taken from the MLA study of Flanders (2012, Harlan Report no. 41103491)
- Evaporation from medium: Unlikely due to the low vapour pressure of the test item (0.00043 Pa at 25 °C, Tremain & Atwal 2011, Harlan Report no. 41103264)
- Water solubility: The test item can be considered water insoluble (water solubility < 0.1 mg/L at 20 °C, Fox & White 2012, Harlan Report no. 41103263), therefore it was dissolved in acetone.
- Precipitation: A test item precipitate (globular in appearance) was noted under an inverted microscope at 1500 µg/plate and by eye at 5000 µg/plate, this observation did not prevent the scoring of revertant colonies.

RANGE-FINDING/SCREENING STUDIES:
Preliminary Toxicity Test: The test item was non-toxic to the strains of bacteria used (TA100 and WP2). The test item formulation and S9-mix used in this experiment were both shown to be sterile.

COMPARISON WITH HISTORICAL CONTROL DATA:
All master strains were found to be satisfactory when were checked for characteristics, viability and spontaneous reversion rate prior to use. The amino acid supplemented top agar and S9 mix used in both experiments was shown to be sterile. There was also no evidence of excessive contamination. The culture density for each bacterial strain used in each experiment was also checked and considered acceptable. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Preliminary Toxicity Test

The following table shows the colony counts and precipitation observations.

Table 1: Numbers of revertant colonies in the preliminary toxicity test and observation of precipitation

Strain	With (+) or without (-) S9-mix	Dose [µg/plate]
		0	0.15	0.5	1.5	5	15	50	150	500	1500	5000
TA 100	-	113	107	96	113	78	75	94	86	113	99	83P
	+	98	81	94	74	95	79	93	86	81	81	99P
WP2	-	25	24	26	22	20	24	22	19	21	21	22P
	+	39	25	40	26	28	38	22	37	35	33	33P

P: Precipitate observed

Mutation Test

The results for the negative controls (spontaneous mutation rates) are presented below in Table 2 and were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.

Table 2: Spontaneous Mutation Rates (Concurrent Negative Controls)

Experiment	Strain		Number of revertants [colonies per plate]
			Replicate			Mean
			1	2	3
Range-finding Test	Base pair substitution type	TA 100	75	73	79	76
		TA 1535	14	13	9	12
		WP2	20	19	13	17
	Frameshift substitution type	TA 98	30	22	22	25
		TA 1537	14	10	13	12
Main Test	Base pair substitution type	TA 100	87	97	98	94
		TA 1535	15	19	21	18
		WP2	25	35	18	26
	Frameshift substitution type	TA 98	24	27	32	28
		TA 1537	14	12	11	12

The mean number (from 3 replicates) of revertant colonies and the standard deviations, for the test item, positive and vehicle controls, both with and without metabolic activation, are presented in Table 3 below.

Table 3: Number of revertants in the treatments and the positive control and observation of precipitation

Experiment	Strain	Mean Number of revertants± Standard Deviation
		Dose [µg/plate]						Positive control (as indicated above)
		0 (Negative control)	50	150	500	1500	5000
-S9 Range-finding Test	TA 100	70±8.4	72±10.8	63±2.0	70±8.5	66±2.5	71P±11.0	539±40.0
	TA 1535	16±1.5	11±0.6	15±3.1	16±1.5	9±1.2	12P±0.6	218±60.9
	WP2	23±6.1	26±0.6	19±3.1	14±5.8	19±2.1	19P±4.7	960±31.5
	TA 98	14±2.1	16±2.5	15±2.3	16±3.8	9±0.6	9P±1.0	120±21.7
	TA 1537	14±2.6	13±4.4	10±4.2	9±3.2	6±1.5	6P±1.2	1014±144.4
+S9 Range-finding Test	TA 100	76±10.5	78±8.1	80±1.0	73±3.2	80±3.5	70P±10.6	623±37.6
	TA 1535	13±3.2	11±1.2	10±1.5	13±3.8	16±5.0	12P±1.7	394±43.7
	WP2	28±3.2	28±3.5	21±4.0	30±4.0	25±4.5	32P±4.7	121±15.1
	TA 98	14±4.7	11±0.6	11±1.7	12±2.1	11±1.2	13P±3.6	311±8.7
	TA 1537	15±2.9	11±3.2	13±4.9	12±3.0	20±0.6	14P±1.5	335±25.1
-S9 Main Test	TA 100	85±7.2	91±2.9	85±6.9	84±9.0	91±4.9	94±6.1	532±3.2
	TA 1535	18±3.2	14±4.4	16±2.3	19±5.5	14±1.5	13±5.0	170±4.0
	WP2	28±4.4	25±5.5	23±4.6	24±5.0	26±5.9	30±8.7	769±54.8
	TA 98	26±6.1	33±5.0	31±7.2	22±6.8	19±5.5	9±2.3	127±20.6
	TA 1537	9±3.5	10±3.6	6±1.2	6±1.7	6±0.6	5±1.2	1319±210.8
+S9 Main Test	TA 100	91±10.1	84±3.5	90±12.9	77±13.1	75±9.2	76±14.5	1354±153.5
	TA 1535	11±2.5	13±4.7	9±1.0	12±1.5	9±2.6	11±2.5	357±6.1
	WP2	33±4.5	28±7.0	27±6.0	29±4.6	31±2.5	25±4.6	147±5.3
	TA 98	13±4.4	13±5.0	10±1.7	12±3.8	10±2.6	11±2.6	286±1.0
	TA 1537	11±4.6	9±1.5	12±3.5	7±2.6	7±2.5	12±1.7	295±1.7

P: Precipitate observed

A graphical presentation of the data is given in the illustration below.

The test item caused no visible reduction in the growth of the bacterial background lawn at any dose level and was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. A test item precipitate (globular in appearance) was noted under an inverted microscope at 1500 µg/plate and by eye at 5000 µg/plate, this observation did not prevent the scoring of revertant colonies. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation in either the range-finding or main tests. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative (no gene mutagenic effect in bacteria with and without metabolic activation)

Non-mutagenic to bacteria (absence of reverse mutation)

Executive summary:

The genetic toxicity (in vitro) of the test item was investigated in a GLP-compliant study by testing for bacterial reverse mutation (Ames test) using the strains TA 1535, TA 1537, TA 98, TA 100 (Salmonella typhimurium) and WP2 (Escherichia coli) according to the EU B.13/14 (2008), OECD TG 471 (1997) and OPPTS 870.5100 (1998) protocols and compatible with the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF. The experiment is deemed valid, conclusive and thus suitable for assessment without restrictions.

The five bacterial strains were treated with the test item, using the Ames plate incorporation method at five dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10 % liver S9 in standard co-factors). The dose range for the range-finding test was determined in a preliminary toxicity assay and was 50 to 5000 µg/plate. The experiment was repeated on a separate day using the same dose range as the range-finding test, fresh cultures of the bacterial strains and fresh test item formulations.

The vehicle (acetone) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test item caused no visible reduction in the growth of the bacterial background lawn at any dose level and was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. A test item precipitate (globular in appearance) was noted under an inverted microscope at 1500 µg/plate and by eye at 5000 µg/plate, this observation did not prevent the scoring of revertant colonies.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation in either the range-finding or main tests.

In conclusion the test item was found non-mutagenic (in vitro) to bacteria in that it did not cause reverse mutation under the conditions of this test.